@article {1811856,
	title = {Structurally Specific Z-DNA Proteolysis Targeting Chimera Enables Targeted Degradation of Adenosine Deaminase Acting on RNA 1},
	journal = {J Am Chem Soc},
	volume = {146},
	number = {11},
	year = {2024},
	month = {2024 Mar 20},
	pages = {7584-7593},
	abstract = {Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus broadening the scope and repertoire of DNA-based PROTACs. Unlike conventional A- or B-form DNA, Z-form DNA is a configuration that exclusively manifests itself under specific stress conditions and with specific target sequences, which can be recognized by specific reader proteins, such as ADAR1 or ZBP1, to exert downstream biological functions. The core of our innovation lies in the strategic engagement of Z-form DNA with ADAR1 and its degradation is achieved by leveraging a VHL ligand conjugated to Z-form DNA to recruit the E3 ligase. This ingenious construct engendered a series of Z-PROTACs, which we utilized to selectively degrade the Z-DNA-binding protein ADAR1, a molecule that is frequently overexpressed in cancer cells. This meticulously orchestrated approach triggers a cascade of PANoptotic events, notably encompassing apoptosis and necroptosis, by mitigating the blocking effect of ADAR1 on ZBP1, particularly in cancer cells compared with normal cells. Moreover, the Z-PROTAC design exhibits a pronounced predilection for ADAR1, as opposed to other Z-DNA readers, such as ZBP1. As such, Z-PROTAC likely elicits a positive immunological response, subsequently leading to a synergistic augmentation of cancer cell death. In summary, the Z-DNA-based PROTAC (Z-PROTAC) approach introduces a modality generated by the conformational change from B- to Z-form DNA, which harnesses the structural specificity intrinsic to potentiate a selective degradation strategy. This methodology is an inspiring conduit for the advancement of PROTAC-based therapeutic modalities, underscoring its potential for selectivity within the therapeutic landscape of PROTACs to target undruggable proteins.},
	keywords = {Adenosine Deaminase, DNA, Z-Form, DNA-Binding Proteins, Proteolysis, Proteolysis Targeting Chimera, RNA, Ubiquitin-Protein Ligases},
	issn = {1520-5126},
	doi = {10.1021/jacs.3c13646},
	author = {Wang, Zhen and Zhang, Dingpeng and Qiu, Xing and Inuzuka, Hiroyuki and Xiong, Yan and Liu, Jing and Chen, Li and Chen, He and Xie, Ling and Kaniskan, H {\"U}mit and Chen, Xian and Jin, Jian and Wei, Wenyi}
}
@article {1803076,
	title = {The lipid globotriaosylceramide promotes germinal center B cell responses and antiviral immunity},
	journal = {Science},
	volume = {383},
	number = {6684},
	year = {2024},
	month = {2024 Feb 16},
	pages = {eadg0564},
	abstract = {Influenza viruses escape immunity owing to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. We found that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 bound and disengaged CD19 from its chaperone CD81, permitting CD19 to translocate to the B cell receptor complex to trigger signaling. Moreover, Gb3 regulated major histocompatibility complex class II expression to increase diversity of T follicular helper and GC B cells reactive with subdominant epitopes. In influenza infection, elevating Gb3, either endogenously or exogenously, promoted broadly reactive antibody responses and cross-protection. These data demonstrate that Gb3 determines the affinity and breadth of B cell immunity and has potential as a vaccine adjuvant.},
	keywords = {Antibody Formation, B-Lymphocytes, Germinal Center, Trihexosylceramides},
	issn = {1095-9203},
	doi = {10.1126/science.adg0564},
	author = {Sharma, Pankaj and Xiaolong Zhang and Kevin Ly and Zhang, Yuxiang and Hu, Yu and Ye, Adam Yongxin and Jianqiao Hu and Ji Hyung Kim and Mumeng Lou and Chong Wang and Quinton Celuzza and Kondo, Yuji and Keiko Furukawa and Bundle, David R and Koichi Furukawa and Alt, Frederick W and Florian Winau}
}
@article {1803086,
	title = {Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining},
	journal = {Nat Commun},
	volume = {15},
	number = {1},
	year = {2024},
	month = {2024 Feb 10},
	pages = {1250},
	abstract = {Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.},
	keywords = {DNA, DNA Breaks, Double-Stranded, DNA End-Joining Repair, DNA Ligase ATP, DNA Ligases, DNA Repair},
	issn = {2041-1723},
	doi = {10.1038/s41467-024-45553-z},
	author = {Stinson, Benjamin M and Carney, Sean M and Walter, Johannes C and Loparo, Joseph J}
}
@article {1803071,
	title = {Heterologous Sarbecovirus Receptor Binding Domains as Scaffolds for SARS-CoV-2 Receptor Binding Motif Presentation},
	journal = {ACS Infect Dis},
	volume = {10},
	number = {2},
	year = {2024},
	month = {2024 Feb 09},
	pages = {553-561},
	abstract = {Structure-guided rational immunogen design can generate optimized immunogens that elicit a desired humoral response. Design strategies often center on targeting conserved sites on viral glycoproteins that will ultimately confer potent neutralization. For SARS-CoV-2 (SARS-2), the surface-exposed spike glycoprotein includes a broadly conserved portion, the receptor binding motif (RBM), that is required to engage the host cellular receptor, ACE2. Expanding humoral responses to this site may result in a more potent neutralizing antibody response against diverse sarbecoviruses. Here, we used a "resurfacing" approach and iterative design cycles to graft the SARS-2 RBM onto heterologous sarbecovirus scaffolds. The scaffolds were selected to vary the antigenic distance relative to SARS-2 to potentially focus responses to RBM. Multimerized versions of these immunogens elicited broad neutralization against sarbecoviruses in the context of preexisting SARS-2 immunity. These validated engineering approaches can help inform future immunogen design efforts for sarbecoviruses and are generally applicable to other viruses.},
	keywords = {Antibodies, Neutralizing, COVID-19, Humans, SARS-CoV-2, Severe acute respiratory syndrome-related coronavirus},
	issn = {2373-8227},
	doi = {10.1021/acsinfecdis.3c00483},
	author = {Hauser, Blake M and Maya Sangesland and Lam, Evan C and St Denis, Kerri J and Sheehan, Maegan L and Vu, Mya L and Cheng, Agnes H and Sordilla, Sophia and Lamson, Dana Thornlow and Almawi, Ahmad W and Balazs, Alejandro B and Daniel Lingwood and Schmidt, Aaron G}
}
@article {1803091,
	title = {Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants},
	journal = {J Biol Chem},
	volume = {300},
	number = {2},
	year = {2024},
	month = {2024 Jan 09},
	pages = {105643},
	abstract = {Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn{\textquoteright}s disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in\&nbsp;vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.},
	issn = {1083-351X},
	doi = {10.1016/j.jbc.2024.105643},
	author = {Chanez-Paredes, Sandra D and Abtahi, Shabnam and Zha, Juanmin and Li, Enkai and Marsischky, Gerald and Zuo, Li and Grey, Michael J and He, Weiqi and Turner, Jerrold R}
}
@article {Lim2023.12.04.570007,
	title = {Tardigrade secretory proteins protect biological structures from desiccation},
	journal = {bioRxiv},
	year = {2023},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focused on the proteins belonging to the secretory family of tardigrade proteins termed secreted-abundant heat soluble ({\textquotedblleft}SAHS{\textquotedblright}) proteins, and investigated their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevented desiccated liposomes from fusion, and enhanced desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the potential application of SAHS proteins as stabilizing molecules for preservation of cells.Competing Interest StatementThe authors have declared no competing interest.},
	doi = {10.1101/2023.12.04.570007},
	url = {https://www.biorxiv.org/content/early/2023/12/05/2023.12.04.570007},
	author = {Lim, Samuel and Charles B. Reilly and Zeina Barghouti and Benedetto Marelli and Jeffrey C. Way and Pamela A. Silver}
}
@article {1785941,
	title = {Identification of a novel, tunable interface in the HP1 protein, Swi6, that underpins epigenetic inheritance},
	journal = {bioRxiv},
	year = {2023},
	month = {2023 Nov 28},
	abstract = {HP1 proteins bind dynamically to H3K9 methylation and are essential for establishing and maintaining transcriptionally silent epigenetic states, known as heterochromatin. HP1 proteins can dimerize, forming a binding interface that interacts with and recruits diverse chromatin-associated factors. HP1 proteins rapidly evolve through sequence changes and gene duplications, but the extent of variation required to achieve functional specialization is unknown. To investigate how changes in amino acid sequence impact epigenetic inheritance, we performed a targeted mutagenesis screen of the dimerization and protein interaction domain of the S.pombe HP1 homolog Swi6. We discovered that substitutions mapping to an auxiliary motif in Swi6 outside the dimerization interface can lead to complete functional divergence. Specifically, we identified point mutations at a single amino acid residue that resulted in either persistent gain or loss of function in epigenetic inheritance without affecting heterochromatin establishment. These substitutions increase Swi6 chromatin occupancy in vivo and alter Swi6-protein interactions that selectively affect H3K9me inheritance. Based on our findings, we propose that relatively minor changes in Swi6 amino acid composition can lead to profound changes in epigenetic inheritance, underscoring the remarkable plasticity associated with HP1 proteins and their ability to evolve new functions.},
	doi = {10.1101/2023.11.28.569027},
	author = {Ames, Amanda and Seman, Melissa and Larkin, Ajay and Raiymbek, Gulzhan and Chen, Ziyuan and Levashkevich, Alex and Biteen, Julie Suzanne and Ragunathan, Kaushik}
}
@article {Sharma2023.09.23.559132,
	title = {The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity},
	journal = {bioRxiv},
	year = {2023},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Influenza viruses escape immunity due to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. Here, we demonstrate that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 binds and disengages CD19 from its chaperone CD81 for subsequent translocation to the B cell receptor (BCR) complex to trigger signaling. Abundance of Gb3 amplifies the PI3-kinase/Akt/Foxo1 pathway to drive affinity maturation. Moreover, this lipid regulates MHC-II expression to increase diversity of T follicular helper (Tfh) and GC B cells reactive with subdominant epitopes. In influenza infection, Gb3 promotes broadly reactive antibody responses and cross-protection. Thus, we show that Gb3 determines affinity as well as breadth in B cell immunity and propose this lipid as novel vaccine adjuvant against viral infection.One Sentence Summary Gb3 abundance on GC B cells selects antibodies with high affinity and broad epitope reactivities, which are cross-protective against heterologous influenza infection.Competing Interest StatementThe authors have declared no competing interest.},
	doi = {10.1101/2023.09.23.559132},
	url = {https://www.biorxiv.org/content/early/2023/09/24/2023.09.23.559132},
	author = {Sharma, Pankaj and Xiaolong Zhang and Kevin Ly and Zhang, Yuxiang and Hu, Yu and Ye, Adam Yongxin and Jianqiao Hu and Ji Hyung Kim and Mumeng Lou and Chong Wang and Quinton Celuzza and Kondo, Yuji and Keiko Furukawa and David R. Bundle and Koichi Furukawa and Frederick W. Alt and Florian Winau}
}
@article {1759926,
	title = {Dynamics of the DYNLL1-MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs},
	journal = {Nat Struct Mol Biol},
	year = {2023},
	month = {2023 Sep 11},
	abstract = {The extent and efficacy of DNA end resection at DNA double-strand breaks (DSB) determine the repair pathway choice. Here we describe how the 53BP1-associated protein DYNLL1 works in tandem with the Shieldin complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1, where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1, predominantly in G1 cells. Shieldin localization to DSBs depends on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be resensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.},
	issn = {1545-9985},
	doi = {10.1038/s41594-023-01074-9},
	author = {Swift, Michelle L and Zhou, Rui and Syed, Aleem and Moreau, Lisa A and Tomasik, Bart{\l}omiej and Tainer, John A and Konstantinopoulos, Panagiotis A and D{\textquoteright}Andrea, Alan D and He, Yizhou Joseph and Chowdhury, Dipanjan}
}
@article {1759916,
	title = {In silico protein interaction screening uncovers DONSON{\textquoteright}s role in replication initiation},
	journal = {Science},
	volume = {381},
	number = {6664},
	year = {2023},
	month = {2023 Sep 22},
	pages = {eadi3448},
	abstract = {CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.},
	keywords = {Animals, Cell Cycle Proteins, Computer Simulation, DNA Replication, DNA-Binding Proteins, Dwarfism, Humans, Mice, Microcephaly, Minichromosome Maintenance Proteins, Nuclear Proteins, Protein Interaction Mapping, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Xenopus laevis},
	issn = {1095-9203},
	doi = {10.1126/science.adi3448},
	author = {Lim, Yang and Tamayo-Orrego, Lukas and Schmid, Ernst and Tarnauskaite, Zygimante and Kochenova, Olga V and Gruar, Rhian and Muramatsu, Sachiko and Lynch, Luke and Schlie, Aitana Verdu and Carroll, Paula L and Chistol, Gheorghe and Reijns, Martin A M and Kanemaki, Masato T and Jackson, Andrew P and Walter, Johannes C}
}
@article {1759921,
	title = {The oncomodulin receptor ArmC10 enables axon regeneration in mice after nerve injury and neurite outgrowth in human iPSC-derived sensory neurons},
	journal = {Sci Transl Med},
	volume = {15},
	number = {708},
	year = {2023},
	month = {2023 Aug 09},
	pages = {eadg6241},
	abstract = {Oncomodulin (Ocm) is a myeloid cell-derived growth factor that enables axon regeneration in mice and rats after optic nerve injury or peripheral nerve injury, yet the mechanisms underlying its activity are unknown. Using proximity biotinylation, coimmunoprecipitation, surface plasmon resonance, and ectopic expression, we have identified armadillo-repeat protein C10 (ArmC10) as a high-affinity receptor for Ocm. ArmC10 deletion suppressed inflammation-induced axon regeneration in the injured optic nerves of mice. ArmC10 deletion also suppressed the ability of lesioned sensory neurons to regenerate peripheral axons rapidly after a second injury and to regenerate their central axons after spinal cord injury in mice (the conditioning lesion effect). Conversely, Ocm acted through ArmC10 to accelerate optic nerve and peripheral nerve regeneration and to enable spinal cord axon regeneration in these mouse nerve injury models. We showed that ArmC10 is highly expressed in human-induced pluripotent stem cell-derived sensory neurons and that exposure to Ocm altered gene expression and enhanced neurite outgrowth. ArmC10 was also expressed in human monocytes, and Ocm increased the expression of immune modulatory genes in these cells. These findings suggest that Ocm acting through its receptor ArmC10 may be a useful therapeutic target for nerve repair and immune modulation.},
	keywords = {Animals, Axons, Ganglia, Spinal, Humans, Induced Pluripotent Stem Cells, Mice, Nerve Regeneration, Neuronal Outgrowth, Sensory Receptor Cells},
	issn = {1946-6242},
	doi = {10.1126/scitranslmed.adg6241},
	author = {Xie, Lili and Yin, Yuqin and Jayakar, Selwyn and Kawaguchi, Riki and Wang, Qing and Peterson, Sheri and Shi, Caleb and Turnes, Bruna Lenfers and Zhang, Zihe and Oses-Prieto, Juan and Li, Jian and Burlingame, Al and Woolf, Clifford J and Geschwind, Daniel and Rasband, Matthew and Benowitz, Larry I}
}
@mastersthesis {1760071,
	title = {Applications and Confounds in Drug Discovery and Repurposing},
	number = {30570041},
	year = {2023},
	type = {PhD Thesis},
	abstract = {
	The process of discovering a new\&nbsp;drug\&nbsp;is always evolving with the knowledge, technologies, and needs of the time. This information should be used to guide your search and to separate legitimate\&nbsp;drug\&nbsp;candidates from artifacts and suboptimal leads.\&nbsp;In\&nbsp;fact, it has been said that a\&nbsp;Drug\&nbsp;Hunter{\textquoteright}s\&nbsp;job is not to find the best molecule, but to find a reason why every molecule is not the best molecule. The focus of this dissertation is firstly the\&nbsp;application\&nbsp;of computational\&nbsp;drug\&nbsp;discovery\&nbsp;and\&nbsp;repurposing\&nbsp;to identify new treatments for diseases. Secondly, it is the mechanistic understanding of two artifacts common\&nbsp;in\&nbsp;early-stage\&nbsp;drug\&nbsp;discovery\&nbsp;and\&nbsp;repurposing\&nbsp;that if used appropriately, should remove potential false-positive screening hits from being pursued as lead candidates.



	Chapter 1\&nbsp;describes the large-scale docking technology developed\&nbsp;in\&nbsp;the lab and how it can be used to discover new\&nbsp;drugs\&nbsp;for protein targets of interest to a particular disease. It further describes the utility of\&nbsp;drug\&nbsp;repurposing\&nbsp;and how it was used during the COVID-19 pandemic to search for novel antivirals. Briefly, it introduces how ligands discovered\&nbsp;in\&nbsp;drug\&nbsp;repurposing\&nbsp;screens were ultimately found to be acting through mechanisms that confounded their antiviral activities.



	Chapter 2\&nbsp;demonstrates how compounds that induce a phenomenon known as\&nbsp;drug-induced phospholipidosis are not legitimate antivirals, and that this effect is a confound\&nbsp;in\&nbsp;cell-based antiviral\&nbsp;repurposing\&nbsp;screens. This shared mechanism underlies the activity of many σ1\&nbsp;and σ2\&nbsp;ligands, among others, that were pursued as potential antivirals early\&nbsp;in\&nbsp;the COVID-19 pandemic. Counter-screening for this activity will help save time, money, and resources from being spent on\&nbsp;drugs\&nbsp;that have no legitimate promise as antiviral\&nbsp;drugs.



	Chapter 3\&nbsp;identifies colloidal aggregation as another mechanism by which many compounds show up as false-positive screening hits\&nbsp;in\&nbsp;biochemical\&nbsp;drug\&nbsp;repurposing\&nbsp;screens. Importantly, we demonstrate that by reducing the formation of colloids\&nbsp;in\&nbsp;screening assays, we can remove false-positive enzymatic activity of multiple ligands that otherwise appear to be inhibitors of viral proteins.



	Chapter 4\&nbsp;demonstrates a legitimate use for σ2\&nbsp;ligands as potential therapeutics, importantly controlling for both phospholipidosis and aggregation as confounding factors\&nbsp;in\&nbsp;their activity. We demonstrate with novel selective ligands that σ2\&nbsp;receptor ligands are antiallodynic\&nbsp;in\&nbsp;neuropathic pain models, and that their effects are time-dependent, replicating similar phenotypes of other σ2\&nbsp;ligands from the literature.



	Chapter 5\&nbsp;applies the large-scale docking technique on the lipid-binding G-protein coupled cannabinoid-1 (CB1) receptor. Here, we demonstrate the concept of {\textquotedblleft}new chemistry for new biology{\textquotedblright} by first identifying a novel CB1 agonist and then finding that it has strongly analgesic properties but lacks two of the major cannabinoid side-effects: sedation and catalepsy.

},
	url = {http://search.proquest.com.ezp-prod1.hul.harvard.edu/dissertations-theses/applications-confounds-drug-discovery-repurposing/docview/2863727834/se-2?accountid=11311},
	author = {Tummino, Tia A}
}
@article {Simpson2023.06.20.545791,
	title = {Structural basis for recognition of unfolded proteins by the ER stress sensor ERN1/IRE1α},
	journal = {bioRxiv},
	year = {2023},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {IRE1α is an endoplasmic reticulum sensor that recognizes misfolded proteins to activate the unfolded protein response (UPR). We used cholera toxin (CTx), which activates IRE1α in cells, to understand how unfolded proteins are recognized. In vitro, the A1 subunit of CTx (CTxA1) bound IRE1α lumenal domain (IRE1αLD). Global unfolding was not required. Instead, IRE1αLD recognized a 7-residue motif within a metastable region of CTxA1 that was also found in microbial and host proteins involved in IRE1α activation. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α regulation. Mutation of the recognition motif blocked CTx-induced IRE1α activation in live cells. These findings describe a mechanism for substrate recognition by IRE1α that induces the UPR.Competing Interest StatementThe authors have declared no competing interest.},
	doi = {10.1101/2023.06.20.545791},
	url = {https://www.biorxiv.org/content/early/2023/06/22/2023.06.20.545791},
	author = {Mariska S. Simpson and De Luca, Heidi and Sarah Cauthorn and Luong, Phi and Namrata D. Udeshi and Svinkina, Tanya and Stefanie S. Schmeider and Steven A. Carr and Michael J. Grey and Wayne I. Lencer}
}
@article {1803081,
	title = {Allosteric activation of cell wall synthesis during bacterial growth},
	journal = {Nat Commun},
	volume = {14},
	number = {1},
	year = {2023},
	month = {2023 Jun 10},
	pages = {3439},
	abstract = {The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls the activation of PG synthesis during other cellular processes, including cell division.},
	keywords = {Allosteric Regulation, Bacterial Proteins, Cell Wall, Penicillin-Binding Proteins, Peptidoglycan, Polysaccharides},
	issn = {2041-1723},
	doi = {10.1038/s41467-023-39037-9},
	author = {Shlosman, Irina and Fivenson, Elayne M and Gilman, Morgan S A and Sisley, Tyler A and Walker, Suzanne and Bernhardt, Thomas G and Kruse, Andrew C and Loparo, Joseph J}
}
@article {1702016,
	title = {Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α},
	journal = {Mol Cell},
	volume = {83},
	number = {11},
	year = {2023},
	month = {2023 Jun 01},
	pages = {1903-1920.e12},
	abstract = {Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVβ5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/β5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98\&nbsp;{\r A} RMSD irisin/αVβ5 complex docking model. Irisin binds very tightly to an alternative interface on αVβ5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.},
	keywords = {Cell Communication, Fibronectins, Humans, Signal Transduction},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2023.05.008},
	author = {A, Mu and Wales, Thomas E and Zhou, Haixia and Draga-Colet{\u a}, Sorin-Valeriu and Gorgulla, Christoph and Blackmore, Katherine A and Mittenb{\"u}hler, Melanie J and Kim, Caroline R and Bogoslavski, Dina and Zhang, Qiuyang and Wang, Zi-Fu and Jedrychowski, Mark P and Seo, Hyuk-Soo and Song, Kijun and Xu, Andrew Z and Sebastian, Luke and Gygi, Steven P and Arthanari, Haribabu and Dhe-Paganon, Sirano and Griffin, Patrick R and Engen, John R and Spiegelman, Bruce M}
}
@article {1702011,
	title = {Simultaneous enhancement of multiple functional properties using evolution-informed protein design},
	journal = {bioRxiv},
	year = {2023},
	month = {2023 May 09},
	abstract = {Designing optimized proteins is important for a range of practical applications. Protein design is a rapidly developing field that would benefit from approaches that enable many changes in the amino acid primary sequence, rather than a small number of mutations, while maintaining structure and enhancing function. Homologous protein sequences contain extensive information about various protein properties and activities that have emerged over billions of years of evolution. Evolutionary models of sequence co-variation, derived from a set of homologous sequences, have proven effective in a range of applications including structure determination and mutation effect prediction. In this work we apply one of these models (EVcouplings) to computationally design highly divergent variants of the model protein TEM-1 β-lactamase, and characterize these designs experimentally using multiple biochemical and biophysical assays. Nearly all designed variants were functional, including one with 84 mutations from the nearest natural homolog. Surprisingly, all functional designs had large increases in thermostability and most had a broadening of available substrates. These property enhancements occurred while maintaining a nearly identical structure to the wild type enzyme. Collectively, this work demonstrates that evolutionary models of sequence co-variation (1) are able to capture complex epistatic interactions that successfully guide large sequence departures from natural contexts, and (2) can be applied to generate functional diversity useful for many applications in protein design.},
	doi = {10.1101/2023.05.09.539914},
	author = {Fram, Benjamin and Truebridge, Ian and Su, Yang and Riesselman, Adam J and Ingraham, John B and Passera, Alessandro and Napier, Eve and Thadani, Nicole N and Lim, Samuel and Roberts, Kristen and Kaur, Gurleen and Stiffler, Michael and Marks, Debora S and Bahl, Christopher D and Khan, Amir R and Sander, Chris and Gauthier, Nicholas P}
}
@mastersthesis {1688161,
	title = {Disordered Proteins Regulate Stress- and Damage-Dependent DNA Binding},
	year = {2023},
	type = {PhD Thesis},
	abstract = {Three-dimensional structure has been used as a starting point to characterize\&nbsp;protein\&nbsp;function since the advent of X-ray crystallography in the 1950s. However, not all\&nbsp;proteins\&nbsp;bear stable tertiary or even secondary structures. Referred to as intrinsically\&nbsp;disordered\&nbsp;protein\&nbsp;regions, or IDRs, how then do we relate these structures (or lack thereof) to their biochemical function? The electrostatically charged and, counterintuitively, selectively inflexible nature of many IDRs point to a key macromolecular interaction partner with similar physical properties--DNA. In this dissertation, we explore the functions of three different IDRs: the C-terminus of\&nbsp;E. coli\&nbsp;single-stranded\&nbsp;binding\&nbsp;protein\&nbsp;(SSB), the C-terminus of\&nbsp;X. laevis\&nbsp;XRCC4-like factor (XLF), and the N-terminus of the human estrogen receptor α (ESR1). In each of these cases, these IDRs are crucial for regulating the access of other factors or domains to\&nbsp;DNA\&nbsp;during times of cellular\&nbsp;stress\&nbsp;and\&nbsp;DNA\&nbsp;damage. For SSB, we use single-molecule imaging in live cells to show that the stable exposure of its C-terminus during replication fork stalling facilitates the recruitment of stall-resolution factors. From site-directed mutagenesis and ensemble biochemical end-joining assays, we demonstrate that the C-terminal extension of double-strand break repair factor XLF enables ligation machinery to fluidly access breaks while maintaining a bridge between\&nbsp;DNA\&nbsp;ends. Finally, preliminary NMR and\&nbsp;DNA\&nbsp;binding\&nbsp;data show that phosphorylations to the ESR1\&nbsp;N-terminus induced by oxidative stress pathways during cancer progression reduce its propensity to form any discernible structure as well as its surprisingly sequence-specific\&nbsp;DNA\&nbsp;affinity. In each of these cases, the ability of the IDR to assume an extended conformation in an activated state allows for the procession of key cellular processes, including replication progression,\&nbsp;DNA\&nbsp;repair, and the transcription of genes in response to\&nbsp;DNA\&nbsp;damage.},
	url = {http://search.proquest.com.ezp-prod1.hul.harvard.edu/dissertations-theses/disordered-proteins-regulate-stress-damage/docview/2787955682/se-2},
	author = {Piatt, Sadie Claire}
}
@article {1702006,
	title = {A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW},
	journal = {Angew Chem Int Ed Engl},
	volume = {62},
	number = {25},
	year = {2023},
	month = {2023 Jun 19},
	pages = {e202301522},
	abstract = {The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved F{\"o}rster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.},
	keywords = {Bacterial Proteins, Cell Wall, Fluorescence Resonance Energy Transfer, Membrane Proteins, Penicillin-Binding Proteins, Peptidoglycan, Peptidoglycan Glycosyltransferase, Staphylococcus aureus},
	issn = {1521-3773},
	doi = {10.1002/anie.202301522},
	author = {Park, Youngseon and Taguchi, Atsushi and Baidin, Vadim and Kahne, Daniel and Walker, Suzanne}
}
@article {1682171,
	title = {A Cdk5-derived peptide inhibits Cdk5/p25 activity and improves neurodegenerative phenotypes},
	journal = {Proc Natl Acad Sci U S A},
	volume = {120},
	number = {16},
	year = {2023},
	month = {2023 Apr 18},
	pages = {e2217864120},
	abstract = {Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This deleterious effect is mediated by pathological cleavage of the Cdk5 activator p35 into the truncated product p25, leading to prolonged Cdk5 activation and altered substrate specificity. Elevated p25 levels have been reported in humans and rodents with neurodegeneration, and the benefit of genetically blocking p25 production has been demonstrated previously in rodent and human neurodegenerative models. Here, we report a 12-amino-acid-long peptide fragment derived from Cdk5 (Cdk5i) that is considerably smaller than existing peptide inhibitors of Cdk5 (P5 and CIP) but shows high binding affinity toward the Cdk5/p25 complex, disrupts the interaction of Cdk5 with p25, and lowers Cdk5/p25 kinase activity. When tagged with a fluorophore (FITC) and the cell-penetrating transactivator of transcription (TAT) sequence, the Cdk5i-FT peptide exhibits cell- and brain-penetrant properties and confers protection against neurodegenerative phenotypes associated with Cdk5 hyperactivity in cell and mouse models of neurodegeneration, highlighting Cdk5i{\textquoteright}s therapeutic potential.},
	issn = {1091-6490},
	doi = {10.1073/pnas.2217864120},
	author = {Pao, Ping-Chieh and Seo, Jinsoo and Lee, Audrey and Kritskiy, Oleg and Patnaik, Debasis and Penney, Jay and Raju, Ravikiran M and Geigenmuller, Ute and Silva, M Catarina and Lucente, Diane E and Gusella, James F and Dickerson, Bradford C and Loon, Anjanet and Yu, Margaret X and Bula, Michael and Yu, Melody and Haggarty, Stephen J and Tsai, Li-Huei}
}
@article {Chanez-Paredes2022.10.13.512159,
	title = {Distinct steady-state properties and TNF responses of epithelial long myosin light chain kinase (MLCK) splice variants},
	journal = {bioRxiv},
	year = {2023},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Intestinal epithelia express two long myosin light chain kinase (MLCK) splice variants, MLCK1 and MLCK2. Unlike MLCK2, MLCK1 is concentrated at the perijunctional actomyosin ring and this localization is enhanced by tumor necrosis factor (TNF) signaling. Here we sought to identify and characterize the domain(s) that direct basal and TNF-induced MLCK1 subcellular localization. Quantitative morphometry demonstrated specific increases in MLCK1 expression and perijunctional localization in Crohn{\textquoteright}s disease patient biopsies, relative to controls. TNF induced perijunctional recruitment of MLCK1-EGFP but did not affect localization of MLCK2-EGFP, which was predominantly associated with basal stress fibers. Recombinant N-terminal MLCK1 and MLCK2 regions accelerated actin polymerization in vitro but were not different from one another. In contrast, the affinity of N-terminal MLCK1 binding to F-actin was greater than that of MLCK2. Perijunctional MLCK1 and MLCK2 domain recruitment in intestinal epithelial cells paralleled in vitro F-actin binding. The unique MLCK1 Ig3 domain was necessary, but not sufficient, for both F-actin binding and perijunctional recruitment, but, nevertheless, displaced perijunctional MLCK1, enhanced steady-state barrier function, and limited TNF-induced MLCK1 recruitment and barrier loss. These data demonstrate selective perijunctional MLCK1 recruitment in Crohn{\textquoteright}s disease, suggest that F-actin binding contributes to perijunctional recruitment, and show that Ig3 can act as a dominant negative effector that limits TNF-induced MLCK1 recruitment and barrier loss. These results data provide key mechanistic detail that will enable development of therapeutics that target Ig3, or its intercellular binding partners, to reverse inflammation-induced barrier loss and limit disease progression.Competing Interest StatementThe authors have declared no competing interest.},
	doi = {10.1101/2022.10.13.512159},
	url = {https://www.biorxiv.org/content/early/2023/04/10/2022.10.13.512159},
	author = {Chanez-Paredes, Sandra D. and Abtahi, Shabnam and Zha, Juanmin and Zuo, Li and He, Weiqi and Turner, Jerrold R.}
}
@article {1676106,
	title = {Activity and Structural Dynamics of Human ABCA1 in a Lipid Membrane},
	journal = {J Mol Biol},
	volume = {435},
	number = {8},
	year = {2023},
	month = {2023 Apr 15},
	pages = {168038},
	abstract = {The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer{\textquoteright}s disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing. In this work, we established a stable expression system for both a human cell-based sterol export assay and protein purification for in vitro biochemical and structural studies. ABCA1 produced in this system was active in sterol export and displayed enhanced ATPase activity after reconstitution into a lipid bilayer. Our single-particle cryo-EM study of ABCA1 in nanodiscs showed protein induced membrane curvature, revealed multiple distinct conformations, and generated a structure of nanodisc-embedded ABCA1 at 4.0-{\r A} resolution representing a previously unknown conformation. Comparison of different ABCA1 structures and molecular dynamics simulations demonstrates both concerted domain movements and conformational variations within each domain. Taken together, our platform for producing and characterizing ABCA1 in a lipid membrane enabled us to gain important mechanistic and structural insights and paves the way for investigating modulators that target the functions of ABCA1.},
	keywords = {Apolipoprotein A-I, ATP Binding Cassette Transporter 1, ATP-Binding Cassette Transporters, Cell Membrane, Humans, Phospholipids, Sterols},
	issn = {1089-8638},
	doi = {10.1016/j.jmb.2023.168038},
	author = {Plummer-Medeiros, Ashlee M and Culbertson, Alan T and Morales-Perez, Claudio L and Maofu Liao}
}
@article {1665958,
	title = {MUC1-C is a master regulator of MICA/B NKG2D ligand and exosome secretion in human cancer cells},
	journal = {J Immunother Cancer},
	volume = {11},
	number = {2},
	year = {2023},
	month = {2023 Feb},
	abstract = {BACKGROUND: The MUC1-C protein evolved in mammals to protect barrier tissues from loss of homeostasis; however, MUC1-C promotes oncogenesis in association with chronic inflammation. Aberrant expression of MUC1-C in cancers has been linked to depletion and dysfunction of T cells in the tumor microenvironment. In contrast, there is no known involvement of MUC1-C in the regulation of natural killer (NK) cell function.

METHODS: Targeting MUC1-C genetically and pharmacologically in cancer cells was performed to assess effects on intracellular and cell surface expression of the MHC class I chain-related polypeptide A (MICA) and MICB ligands. The MICA/B promoters were analyzed for H3K27 and DNA methylation. Shedding of MICA/B was determined by ELISA. MUC1-C interactions with ERp5 and RAB27A were assessed by coimmunoprecipitation and direct binding studies. Exosomes were isolated for analysis of secretion. Purified NK cells were assayed for killing of cancer cell targets.

RESULTS: Our studies demonstrate that MUC1-C represses expression of the MICA and MICB ligands that activate the NK group 2D receptor. We show that the inflammatory MUC1-C{\textrightarrow}NF-κB pathway drives enhancer of zeste homolog 2-mediated and DNMT-mediated methylation of the MICA and MICB promoter regions. Targeting MUC1-C genetically and pharmacologically with the GO-203 inhibitor induced intracellular and cell surface MICA/B expression but not MICA/B cleavage. Mechanistically, MUC1-C regulates the ERp5 thiol oxidoreductase that is necessary for MICA/B protease digestion and shedding. In addition, MUC1-C interacts with the RAB27A protein, which is required for exosome formation and secretion. As a result, targeting MUC1-C markedly inhibited secretion of exosomes expressing MICA/B. In concert with these results, we show that targeting MUC1-C promotes NK cell-mediated killing.

CONCLUSIONS: These findings uncover pleotropic mechanisms by which MUC1-C confers evasion of cancer cells to NK cell recognition and destruction.},
	keywords = {Exosomes, Histocompatibility Antigens Class I, Humans, Killer Cells, Natural, Ligands, Mucin-1, Neoplasms, NK Cell Lectin-Like Receptor Subfamily K, Tumor Microenvironment},
	issn = {2051-1426},
	doi = {10.1136/jitc-2022-006238},
	author = {Morimoto, Yoshihiro and Yamashita, Nami and Daimon, Tatsuaki and Hirose, Haruka and Yamano, Shizuka and Haratake, Naoki and Ishikawa, Satoshi and Bhattacharya, Atrayee and Fushimi, Atsushi and Ahmad, Rehan and Takahashi, Hidekazu and Dashevsky, Olga and Mitsiades, Constantine and Kufe, Donald}
}
@article {1664100,
	title = {Deep Eutectic Solvents for Subcutaneous Delivery of Protein Therapeutics},
	journal = {Adv Sci (Weinh)},
	year = {2023},
	month = {2023 Jan 15},
	pages = {e2205389},
	abstract = {Proteins are among the most common therapeutics for the treatment of diabetes, autoimmune diseases, cancer, and metabolic diseases, among others. Despite their common use, current protein therapies, most of which are injectables, have several limitations. Large proteins such as monoclonal antibodies (mAbs) suffer from poor absorption after subcutaneous injections, thus forcing their administration by intravenous injections. Even small proteins such as insulin suffer from slow pharmacokinetics which poses limitations in effective management of diabetes. Here, a deep eutectic-based delivery strategy is used to offer a generalized approach for improving protein absorption after subcutaneous injections. The lead formulation enhances absorption of mAbs after subcutaneous injections by ≈200\%. The same composition also improves systemic absorption of subcutaneously injected insulin faster than Humalog, the current gold-standard of rapid acting insulin. Mechanistic studies reveal that the beneficial effect of deep eutectics on subcutaneous absorption is mediated by their ability to reduce the interactions of proteins with the subcutaneous matrix, especially collagen. Studies also confirm that these deep eutectics are safe for subcutaneous injections. Deep eutectic-based formulations described here open new possibilities for subcutaneous injections of therapeutic proteins.},
	issn = {2198-3844},
	doi = {10.1002/advs.202205389},
	author = {Curreri, Alexander M and Kim, Jayoung and Dunne, Michael and Angsantikul, Pavimol and Goetz, Morgan and Gao, Yongsheng and Mitragotri, Samir}
}
@article {1640025,
	title = {Tacrolimus-binding protein FKBP8 directs myosin light chain kinase-dependent barrier regulation and is a potential therapeutic target in Crohn{\textquoteright}s disease},
	journal = {Gut},
	volume = {72},
	number = {5},
	year = {2023},
	month = {2023 May},
	pages = {870-881},
	abstract = {OBJECTIVE: Intestinal barrier loss is a Crohn{\textquoteright}s disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically.

DESIGN: Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects.

RESULTS: MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects.

CONCLUSION: Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.},
	keywords = {Animals, Caco-2 Cells, Crohn Disease, Humans, Intestinal Mucosa, Mice, Mice, Knockout, Myosin-Light-Chain Kinase, Tacrolimus, Tacrolimus Binding Proteins, Tight Junctions, Tumor Necrosis Factor-alpha},
	issn = {1468-3288},
	doi = {10.1136/gutjnl-2021-326534},
	author = {Zuo, Li and Kuo, Wei-Ting and Cao, Feng and Chanez-Paredes, Sandra D and Zeve, Daniel and Mannam, Prabhath and Jean-Fran{\c c}ois, L{\'e}a and Day, Anne and Vallen Graham, W and Sweat, Yan Y and Shashikanth, Nitesh and Breault, David T and Turner, Jerrold R}
}
@mastersthesis {1681726,
	title = {Biochemical, Biophysical and Structural Characterization of the Conserved Herpesviral Nuclear Egress Complex},
	year = {2022},
	type = {PhD Thesis},
	abstract = {To exit infected cells, progeny virions must pass through host cell membrane barriers. Herpesviruses are masters of membrane manipulation as they deform membranes at multiple stages while exiting a cell. The first instance occurs when the virally encoded nuclear egress complex (NEC) bends the nuclear membrane around nucleocapsids, assembled entirely in the nucleus and too large to fit through nuclear pores, resulting in their envelopment and trafficking through the nuclear membrane. Despite being essential for herpesvirus replication, how the NEC bends the nuclear membrane around capsids is not well understood. In this work, we used biochemical, biophysical, and structural approaches to further our mechanistic understanding of NEC-mediated membrane deformation. Here, we report that electrostatics govern NEC-membrane interactions leading to membrane budding. We propose that the virus uses phosphorylation to control NEC-mediated membrane budding during infection as phosphomimicking mutations block budding in vivo and in vitro. Furthermore, we establish that the membrane-proximal regions (MPRs) of the NEC order lipid headgroups and acyl chains to generate local areas of negative membrane curvature, important for formation of the body of the bud, and negative Gaussian curvature (NGC), important for scission of the budding membrane. We propose that NEC oligomerization, known to occur and required for budding, creates a rigid, closely packed scaffold on the membrane increasing the area of local negative membrane curvature thereby forming the body of the bud. At the edges of the scaffold, non-oligomerized NEC generates NGC to form the neck of the bud. We also report the crystal structure of the NEC from Epstein-Barr virus (EBV), a gammaherpesvirus. Comparison to NECs from alpha- and betaherpesviruses show that the overall structure is well conserved across all three herpesvirus subfamilies. We show, experimentally, that the EBV NEC is conformationally dynamic which we hypothesize is important for function. Furthermore, we establish that the EBV NEC is a self-contained membrane budding machine which also requires oligomerization for function. We propose that MPR-mediated membrane ordering and NEC oligomerization function in concert to mediate nuclear egress for all herpesviruses.},
	author = {Thorsen, Michael K.}
}
@article {1665959,
	title = {Viruses inhibit TIR gcADPR signalling to overcome bacterial defence},
	journal = {Nature},
	volume = {611},
	number = {7935},
	year = {2022},
	month = {2022 Nov},
	pages = {326-331},
	abstract = {The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are {\textquoteright}sponges{\textquoteright} that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 {\textquoteright}{\textquoteright}-2{\textquoteright} glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1{\textquoteright}{\textquoteright}-3{\textquoteright} gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1{\textquoteright}{\textquoteright}-2{\textquoteright} gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.},
	keywords = {Bacteria, Bacterial Proteins, Bacteriophages, Crystallography, X-Ray, Plant Proteins, Protein Domains, Receptors, Interleukin-1, Signal Transduction, Toll-Like Receptors, Viral Proteins},
	issn = {1476-4687},
	doi = {10.1038/s41586-022-05375-9},
	author = {Leavitt, Azita and Erez Yirmiya and Gil Amitai and Lu, Allen and Garb, Jeremy and Herbst, Ehud and Morehouse, Benjamin R and Hobbs, Samuel J and Antine, Sadie P and Sun, Zhen-Yu J and Kranzusch, Philip J and Sorek, Rotem}
}
@article {Shlosman2022.11.07.515454,
	title = {Allosteric activation of cell wall synthesis during bacterial growth},
	journal = {bioRxiv},
	year = {2022},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls activation of PG synthesis during other cellular processes, including cell division.Competing Interest StatementA.C.K. is a co-founder and consultant for Tectonic Therapeutic and Seismic Therapeutic and for the Institute for Protein Innovation, a non-profit research institute.},
	doi = {10.1101/2022.11.07.515454},
	url = {https://www.biorxiv.org/content/early/2022/11/07/2022.11.07.515454},
	author = {Shlosman, Irina and Fivenson, Elayne M. and Gilman, Morgan S.A. and Sisley, Tyler A. and Walker, Suzanne and Bernhardt, Thomas G. and Kruse, Andrew C. and Loparo, Joseph J.}
}
@article {Chanez-Paredes2022.10.13.512159,
	title = {Selective perijunctional MLCK1 recruitment in Crohn{\textquoteright}s disease: Identification of essential structural domains},
	journal = {bioRxiv},
	year = {2022},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Intestinal epithelia express two long MLCK splice variants, MLCK1 and MLCK2. We have previously shown that disruption of inflammation-induced MLCK1 recruitment to the perijunctional actomyosin ring prevents barrier loss and attenuates disease progression. Here we sought to define the domains responsible for distinct MLCK1 and MLCK2 behaviors. Quantitative analysis of human biopsies demonstrated specific increases in MLCK1 expression and perijunctional localization in Crohn{\textquoteright}s disease. When expressed in cultured intestinal epithelial cells, we found, as expected, that MLCK1 is most concentrated at the perijunctional actomyosin ring. In contrast, MLCK2 is predominantly associated with basal F-actin stress fibers. Immunoglobulin-cell adhesion molecule domain 3 (IgCAM3) must be critical for MLCK1 recruitment, as that domain is incomplete in MLCK2. Consistent with this, truncation mutants consisting of N-terminal IgCAM domains 1-4, without C-terminal catalytic domains, localized similarly to full-length MLCK1 and MLCK2, respectively. Further mutagenesis allowed identification of IgCAM2 and IgCAM3 domains as the minimal region required for MLCK1 recruitment. Although IgCAM3 does not concentrate perijunctionally, it can act as a dominant negative effector that limits steady-state and TNF-induced MLCK1 recruitment and barrier loss. Together, the demonstration of selective MLCK1 upregulation and perijunctional recruitment in Crohn{\textquoteright}s disease and identification of domains required for perijunctional MLCK1 recruitment provide a conceptual understanding and structural data needed for development of therapeutic means of blocking MLCK1-mediated barrier loss without the toxicity of enzymatic MLCK inhibition.SIGNIFICANCE STATEMENT Recent work has demonstrated that long myosin light chain kinase isoform 1 (MLCK1) recruitment to the perijunctional actomyosin ring is a critical component of inflammation-induced intestinal barrier loss. Chanez-Paredes et al. show that this occurs in Crohn{\textquoteright}s disease and define the essential structural elements that direct MLCK1 recruitment, thereby creating a foundation for therapeutic interruption of MLCK1 recruitment in disease.Competing Interest StatementJRT is a founder of Thelium Therapeutics and a consultant for Entrinsic and Kallyope.},
	doi = {10.1101/2022.10.13.512159},
	url = {https://www.biorxiv.org/content/early/2022/10/14/2022.10.13.512159},
	author = {Chanez-Paredes, Sandra D. and Abtahi, Shabnam and Zha, Juanmin and Zuo, Li and He, Weiqi and Turner, Jerrold R.}
}
@article {1653394,
	title = {Compartmentalization of the replication fork by single-stranded DNA-binding protein regulates translesion synthesis},
	journal = {Nat Struct Mol Biol},
	volume = {29},
	number = {9},
	year = {2022},
	month = {2022 Sep},
	pages = {932-941},
	abstract = {Processivity clamps tether DNA polymerases to DNA, allowing their access to the primer-template junction. In addition to DNA replication, DNA polymerases also participate in various genome maintenance activities, including translesion synthesis (TLS). However, owing to the error-prone nature of TLS polymerases, their association with clamps must be tightly regulated. Here we show that fork-associated ssDNA-binding protein (SSB) selectively enriches the bacterial TLS polymerase Pol IV at stalled replication forks. This enrichment enables Pol IV to associate with the processivity clamp and is required for TLS on both the leading and lagging strands. In contrast, clamp-interacting proteins (CLIPs) lacking SSB binding are spatially segregated from the replication fork, minimally interfering with Pol IV-mediated TLS. We propose that stalling-dependent structural changes within clusters of fork-associated SSB establish hierarchical access to the processivity clamp. This mechanism prioritizes a subset of CLIPs with SSB-binding activity and facilitates their exchange at the replication fork.},
	keywords = {DNA, DNA Replication, DNA-Binding Proteins, DNA-Directed DNA Polymerase, Escherichia coli},
	issn = {1545-9985},
	doi = {10.1038/s41594-022-00827-2},
	author = {Chang, Seungwoo and Thrall, Elizabeth S and Laureti, Luisa and Piatt, Sadie C and Pag{\`e}s, Vincent and Loparo, Joseph J}
}
@mastersthesis {1664098,
	title = {Characterizing mVenus adsorption to photodegraded polyethylene using circular dichroism and fluorescence spectroscopy},
	year = {2022},
	type = {Master{\textquoteright}s Thesis},
	author = {Amaris, Althea}
}
@article {Park2022.07.14.500081,
	title = {Cryo-EM structure of a RAS/RAF recruitment complex},
	journal = {bioRxiv},
	year = {2022},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Cryo-EM structures of a KRAS/BRAF/MEK1/14-3-3 complex reveal KRAS bound to the flexible Ras-binding domain of BRAF, captured in two orientations. Autoinhibitory interactions are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by Ras and suggest stabilization of this pre-activation intermediate as an alternative to blocking binding of KRAS.Competing Interest StatementThe authors have declared no competing interest.},
	doi = {10.1101/2022.07.14.500081},
	url = {https://www.biorxiv.org/content/early/2022/07/14/2022.07.14.500081},
	author = {Park, Eun Young and Rawson, Shaun and Schmoker, Anna and Kim, Byeong-Won and Oh, Sehee and Song, KangKang and Jeon, Hyesung and Eck, Michael}
}
@article {1650268,
	title = {Monomeric prefusion structure of an extremophile gamete fusogen and stepwise formation of the postfusion trimeric state},
	journal = {Nat Commun},
	volume = {13},
	number = {1},
	year = {2022},
	month = {2022 07 13},
	pages = {4064},
	abstract = {Here, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 {\textdegree}C. HAP2 has a trimeric postfusion structure with similarity to viral class II fusion proteins, but its prefusion structure has been elusive. The crystal structure of a monomeric prefusion state of Cyani HAP2 shows it is highly extended with three domains in the order D2, D1, and D3. Three hydrophobic fusion loops at the tip of D2 are each required for postfusion state formation. We followed by negative stain electron microscopy steps in the process of detergent micelle-stimulated postfusion state formation. In an intermediate state, two or three linear HAP2 monomers associate at the end of D2 bearing its fusion loops. Subsequently, D2 and D1 line the core of a trimer and D3 folds back over the exterior of D1 and D2. D3 is not required for formation of intermediate or postfusion-like states.},
	keywords = {Extremophiles, Germ Cells, Protein Conformation, Viral Envelope Proteins, Viral Fusion Proteins},
	issn = {2041-1723},
	doi = {10.1038/s41467-022-31744-z},
	author = {Feng, Juan and Dong, Xianchi and Su, Yang and Lu, Chafen and Springer, Timothy A.}
}
@article {1645408,
	title = {Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its -glycosylated N- and C-linker},
	journal = {Elife},
	volume = {11},
	year = {2022},
	month = {2022 May 09},
	abstract = {Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.},
	keywords = {Blood Platelets, Polysaccharides, Protein Binding, Urea, von Willebrand Factor},
	issn = {2050-084X},
	doi = {10.7554/eLife.75760},
	author = {Bonazza, Klaus and Iacob, Roxana E and Hudson, Nathan E and Li, Jing and Lu, Chafen and Engen, John R and Springer, Timothy A.}
}
@article {1652749,
	title = {Enzymatic analysis of WWP2 E3 ubiquitin ligase using protein microarrays identifies autophagy-related substrates},
	journal = {J Biol Chem},
	volume = {298},
	number = {5},
	year = {2022},
	month = {2022 05},
	pages = {101854},
	abstract = {WWP2 is a HECT E3 ligase that targets protein Lys residues for ubiquitination and is comprised of an N-terminal C2 domain, four central WW domains, and a C-terminal catalytic HECT domain. The peptide segment between the middle WW domains, the 2,3-linker, is known to autoinhibit the catalytic domain, and this autoinhibition can be relieved by phosphorylation at Tyr369. Several protein substrates of WWP2 have been identified, including the tumor suppressor lipid phosphatase PTEN, but the full substrate landscape and biological functions of WWP2 remain to be elucidated. Here, we used protein microarray technology and the activated enzyme phosphomimetic mutant WWP2Y369E to identify potential WWP2 substrates. We identified 31 substrate hits for WWP2Y369E using protein microarrays, of which three were known autophagy receptors (NDP52, OPTN, and SQSTM1). These three hits were validated with in\&nbsp;vitro and cell-based transfection assays and the Lys ubiquitination sites on these proteins were mapped by mass spectrometry. Among the mapped ubiquitin sites on these autophagy receptors, many had been previously identified in the endogenous proteins. Finally, we observed that WWP2 KO SH-SH5Y neuroblastoma cells using CRISPR-Cas9 showed a defect in mitophagy, which could be rescued by WWP2Y369E transfection. These studies suggest that WWP2-mediated ubiquitination of the autophagy receptors NDP52, OPTN, and SQSTM1 may positively contribute to the regulation of autophagy.},
	keywords = {Autophagy, Cell Cycle Proteins, Humans, Membrane Transport Proteins, Nuclear Proteins, Protein Array Analysis, Proteins, Sequestosome-1 Protein, Ubiquitin-Protein Ligases, Ubiquitination},
	issn = {1083-351X},
	doi = {10.1016/j.jbc.2022.101854},
	author = {Jiang, Hanjie and Chiang, Claire Y and Chen, Zan and Nathan, Sara and D{\textquoteright}Agostino, Gabriel and Paulo, Joao A and Song, Guang and Zhu, Heng and Gabelli, Sandra B and Cole, Philip A}
}
@article {Erlandson2022.04.19.488796,
	title = {Engineering and characterization of a long half-life relaxin receptor RXFP1 agonist},
	journal = {bioRxiv},
	year = {2022},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Relaxin-2 is a peptide hormone with important roles in human cardiovascular and reproductive biology. Its ability to activate cellular responses such as vasodilation, angiogenesis, and anti-inflammatory and anti-fibrotic effects have led to significant interest in using relaxin-2 as a therapeutic for heart failure and several fibrotic conditions. However, recombinant relaxin-2 has a very short serum half-life, limiting its clinical applications. Here we present protein engineering efforts targeting the relaxin-2 hormone in order to increase its serum half-life, while maintaining its ability to activate the G protein-coupled receptor RXFP1. To achieve this, we optimized a fusion between relaxin-2 and an antibody Fc fragment, generating a version of the hormone with a circulating half-life of up to five days in mice while retaining potent agonist activity at the RXFP1 receptor both in vitro and in vivo.Competing Interest StatementA.C.K. and S.C.E are inventors on a patent application for engineered single-chain relaxin proteins. A.C.K. is a co-founder and consultant for Tectonic Therapeutic and Seismic Therapeutic and for the Institute for Protein Innovation, a non-profit research institute.},
	doi = {10.1101/2022.04.19.488796},
	url = {https://www.biorxiv.org/content/early/2022/04/19/2022.04.19.488796},
	author = {Erlandson, Sarah C. and Wang, Jialu and Jiang, Haoran and Rockman, Howard A. and Kruse, Andrew C.}
}
@mastersthesis {Duncan-LoweyBrianna2022EoCD,
	title = {Effectors of Cell Death in Bacterial Antiphage Defense},
	year = {2022},
	school = {ProQuest Dissertations Publishing},
	type = {PhD Thesis},
	abstract = {Bacteria encode many systems to detect and respond to infection with bacteriophages. Cyclic-oligonucleotide based antiphage signaling systems (CBASS) are widespread antiviral systems encoded in approximately 10\% of bacterial genomes. Upon infection of CBASS-encoding bacteria, nucleotide second messengers are synthesized and can diffuse throughout the cell to bind diverse effector proteins. We use structural and biochemical methods to characterize effector proteins that specifically recognize nucleotide second messengers and are activated to induce cell death. Once this abortive infection system is activated, the effector proteins kill the bacterial host before the phage is able to replicate, thereby halting phage infection. We characterize one family of effectors, Cap4 proteins, that use a SAVED domain to specifically recognize nucleotide second messengers and are then activated to kill the cell through the indiscriminate cleavage of double-stranded DNA. These data highlight SAVED domains as widespread ligand-binding domains found in many CBASS effectors and revealed an evolutionary connection between CBASS and CRISPR immunity. We also characterized effector proteins that contain transmembrane domains, revealing that they target the inner membrane to induce cell death after phage infection. These data highlight membrane disruption as a widespread strategy to induce cell death in CBASS immunity. We further characterize one family of transmembrane effectors, Cap15 proteins, which we show use a minimal β-barrel domain to recognize nucleotide second messengers. Together, these studies begin to characterize diverse CBASS effectors that fulfill two requirements: 1. specific recognition of nucleotide second messengers, either by SAVED or β-barrel domains and 2. the induction of cell death, either through destruction of nucleic acids by Cap4 nucleases or inner membrane disruption by Cap15 transmembrane effectors. These data support an emerging model where CBASS effectors use a modular domain architecture to sense second messengers and induce cell death to halt phage replication.},
	keywords = {Biochemistry, microbiology, Virology},
	isbn = {9798209894797},
	author = {Brianna Duncan-Lowey}
}
@article {1636646,
	title = {Structure of the Streptococcus pyogenes NAD Glycohydrolase Translocation Domain and Its Essential Role in Toxin Binding to Oropharyngeal Keratinocytes},
	journal = {J Bacteriol},
	volume = {204},
	number = {1},
	year = {2022},
	month = {2022 01 18},
	pages = {e0036621},
	abstract = {The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus, GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of cotoxins streptolysin O (SLO) and NAD+-glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a β-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro, suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. IMPORTANCE NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus, the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.},
	keywords = {Amino Acid Substitution, Bacterial Adhesion, Bacterial Proteins, Bacterial Toxins, Cell Line, Humans, Keratinocytes, Models, Molecular, NAD+ Nucleosidase, Oropharynx, Protein Binding, Protein Conformation, Protein Domains, Protein Transport, Streptococcus pyogenes, Streptolysins},
	issn = {1098-5530},
	doi = {10.1128/JB.00366-21},
	author = {Velarde, Jorge J and Piai, Alessandro and Lichtenstein, Ian J and Lynskey, Nicola N and Chou, James J and Wessels, Michael R}
}
@article {1650281,
	title = {Structural basis for catalyzed assembly of the Sonic hedgehog-Patched1 signaling complex},
	journal = {Dev Cell},
	volume = {57},
	number = {5},
	year = {2022},
	month = {2022 03 14},
	pages = {670-685.e8},
	abstract = {The dually lipidated Sonic hedgehog (SHH) morphogen signals through the tumor suppressor membrane protein Patched1 (PTCH1) to activate the Hedgehog pathway, which is fundamental in development and cancer. SHH engagement with PTCH1 requires the GAS1 coreceptor, but the mechanism is unknown. We demonstrate a unique role for GAS1, catalyzing SHH-PTCH1 complex assembly in vertebrate cells by direct SHH transfer from the extracellular SCUBE2 carrier to PTCH1. Structure of the GAS1-SHH-PTCH1 transition state identifies how GAS1 recognizes the SHH palmitate and cholesterol modifications in modular fashion and how it facilitates lipid-dependent SHH handoff to PTCH1. Structure-guided experiments elucidate SHH movement from SCUBE2 to PTCH1, explain disease mutations, and demonstrate that SHH-induced PTCH1 dimerization causes its internalization from the cell surface. These results define how the signaling-competent SHH-PTCH1 complex assembles, the key step triggering the Hedgehog pathway, and provide a paradigm for understanding morphogen reception and its regulation.},
	keywords = {Catalysis, Cholesterol, Hedgehog Proteins, Patched-1 Receptor, Signal Transduction, Structure-Activity Relationship},
	issn = {1878-1551},
	doi = {10.1016/j.devcel.2022.02.008},
	author = {Huang, Pengxiang and Wierbowski, Bradley M and Lian, Tengfei and Chan, Charlene and Garc{\'\i}a-Linares, Sara and Jiang, Jiansen and Salic, Adrian}
}
@mastersthesis {1654407,
	title = {Mechanistic Studies of NEDD4 Family HECT E3 Ubiquitin Ligases},
	year = {2022},
	type = {PhD Thesis},
	abstract = {
	
		
			
				
					The NEDD4 family HECT (homologous to E6AP C terminus) E3 ligases contain nine enzymes that catalyze the ubiquitination of numerous important proteins including tumor suppressor PTEN and transcription factors Oct4 and EGR2. The NEDD4 ubiquitination enzymes therefore impact many cellular events and need to be tightly controlled. Dysregulation of NEDD4 enzymes is associated with many pathologies like cancer and neurodegenerative diseases. NEDD4 protein architecture includes an N-terminal C2 domain, followed by two to four WW domains and culminating in a C-terminal HECT domain. Previous work from our lab has demonstrated that a linker region between two of the WW domains can undergo an intra-molecular interaction with catalytic HECT domain, serving an auto-inhibitory role. In this thesis, we investigated the molecular mechanisms of this auto-regulatory linker and found that the linker auto-inhibition can be relieved by either linker phosphorylation, which disrupts the linker-HECT interaction. Moreover, we found that linker autoinhibition can be overcome by the NEDD4 family member binding with allosteric activators like NDFIP1 and ubiquitin variants. We have also demonstrated that the NEDD4 family member WWP1 K740N/N745S germline variants, associated with cancer, do not relieve auto-inhibition as suggested in a recent clinical study. Furthermore, we performed substrate screening with a phosphor-mimetic/activated form of the NEDD4 family member WWP2 using protein microarrays. We have identified a group of new potential WWP2 protein substrates from this screening and followed up on three of these, the autophagy receptors NDP52, OPTN and SQSTM1. In vitro and in vivo, we demonstrated that WWP2 ubiquitinates these autophagy receptors and plays an important role in regulating mitophagy. In addition, in this thesis work, we developed two chemical strategies to site-specifically modify proteins of interest for biochemical studies. Firstly, we developed an N-terminal labeling method with NHS ester compounds, which shows high specificity. We applied this strategy to study WWP2 autoubiquitination and E2-E3 molecular interaction. Secondly, we adapted a recently developed technique for chemically-ubiquitinating protein of interest using ubiquitin hydrazide mimics. We expanded its application to other proteins, including ubiquitin-conjugating enzyme E2 and HECT E3 ligases. We applied the generated E2-Ub or E3-Ub as enzyme intermediate state mimics to study the HECT E3 catalytic mechanisms. Our data support a model in which the HECT-Ub intermediate undergoes a conformational switch from an inverted T shape to an L shape, which reduces the E3 affinity for the E2. This model accounts for an efficient turnover process that allows for rapid E2 recycling and enhanced substrate ubiquitination.
				

				
					http://jhir.library.jhu.edu/handle/1774.2/67218
				
			
		
	

},
	author = {Jiang, Hanjie}
}
@article {Harvey2022.01.12.476085,
	title = {An in silico method to assess antibody fragment polyreactivity},
	journal = {bioRxiv},
	year = {2022},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. We designed a set of experiments using a diverse na{\"{\i}ve synthetic camelid antibody fragment ({\textquoteleft}nanobody{\textquoteright}) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC \&gt; 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our model{\textquoteright}s performance on three independent nanobody scaffolds, where over 90\% of predicted substitutions successfully reduced polyreactivity. Importantly, the model allowed us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its pharmacological properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.Competing Interest StatementC.C.L is a co-founder of K2 Biotechnologies Inc., which applies continuous evolution technologies to antibody engineering. D.S.M. is an advisor for Dyno Therapeutics, Octant, Jura Bio, Tectonic Therapeutic and Genentech, and is a co-founder of Seismic Therapeutic. A.C.K. is a co-founder and consultant for Tectonic Therapeutic and Seismic Therapeutic and for the Institute for Protein Innovation, a non-profit research institute.},
	doi = {10.1101/2022.01.12.476085},
	url = {https://www.biorxiv.org/content/early/2022/01/13/2022.01.12.476085},
	author = {Harvey, Edward P. and Shin, Jung-Eun and Skiba, Meredith A. and Nemeth, Genevieve R. and Hurley, Joseph D. and Wellner, Alon and Shaw, Ada Y. and Miranda, Victor G. and Min, Joseph K. and Liu, Chang C. and Marks, Debora S. and Kruse, Andrew C.}
}
@article {1645407,
	title = {Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs},
	journal = {Nat Struct Mol Biol},
	volume = {28},
	number = {3},
	year = {2021},
	month = {2021 03},
	pages = {258-267},
	abstract = {G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30\% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1β1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 {\r A}. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.},
	keywords = {Allosteric Regulation, Cryoelectron Microscopy, GTP-Binding Protein alpha Subunits, Gi-Go, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Guanosine Diphosphate, Heterotrimeric GTP-Binding Proteins, Humans, Lipid Bilayers, Micelles, Models, Molecular, Nanostructures, Neurotensin, Protein Conformation, Receptors, Neurotensin, Signal Transduction},
	issn = {1545-9985},
	doi = {10.1038/s41594-020-00554-6},
	author = {Zhang, Meng and Gui, Miao and Wang, Zi-Fu and Gorgulla, Christoph and Yu, James J and Wu, Hao and Sun, Zhen-Yu J and Klenk, Christoph and Merklinger, Lisa and Morstein, Lena and Hagn, Franz and Pl{\"u}ckthun, Andreas and Brown, Alan and Nasr, Mahmoud L and Wagner, Gerhard}
}
@article {1636645,
	title = {Allosteric MEK inhibitors act on BRAF/MEK complexes to block MEK activation},
	journal = {Proc Natl Acad Sci U S A},
	volume = {118},
	number = {36},
	year = {2021},
	month = {2021 09 07},
	abstract = {The RAF/MEK/ERK pathway is central to the control of cell physiology, and its dysregulation is associated with many cancers. Accordingly, the proteins constituting this pathway, including MEK1/2 (MEK), have been subject to intense drug discovery and development efforts. Allosteric MEK inhibitors (MEKi) exert complex effects on RAF/MEK/ERK pathway signaling and are employed clinically in combination with BRAF inhibitors in malignant melanoma. Although mechanisms and structures of MEKi bound to MEK have been described for many of these compounds, recent studies suggest that RAF/MEK complexes, rather than free MEK, should be evaluated as the target of MEKi. Here, we describe structural and biochemical studies of eight structurally diverse, clinical-stage MEKi to better understand their mechanism of action on BRAF/MEK complexes. We find that all of these agents bind in the MEK allosteric site in BRAF/MEK complexes, in which they stabilize the MEK activation loop in a conformation that is resistant to BRAF-mediated dual phosphorylation required for full activation of MEK. We also show that allosteric MEK inhibitors act most potently on BRAF/MEK complexes rather than on free active MEK, further supporting the notion that a BRAF/MEK complex is the physiologically relevant pharmacologic target for this class of compounds. Our findings provide a conceptual and structural framework for rational development of RAF-selective MEK inhibitors as an avenue to more effective and better-tolerated agents targeting this pathway.},
	keywords = {Allosteric Regulation, Enzyme Activation, Enzyme Stability, Humans, MAP Kinase Kinase Kinases, Phosphorylation, Protein Conformation, Protein Kinase Inhibitors, Proto-Oncogene Proteins B-raf, Signal Transduction},
	issn = {1091-6490},
	doi = {10.1073/pnas.2107207118},
	author = {Gonzalez-Del Pino, Gonzalo L and Li, Kunhua and Park, Eun Young and Schmoker, Anna M and Ha, Byung Hak and Eck, Michael J}
}
@article {1636640,
	title = {Antibodies That Engage the Hemagglutinin Receptor-Binding Site of Influenza B Viruses},
	journal = {ACS Infect Dis},
	volume = {7},
	number = {1},
	year = {2021},
	month = {2021 01 08},
	pages = {1-5},
	abstract = {We describe cross-reactive human antibodies recognizing influenza B viruses spanning nearly 80 years of antigenic drift. Structures show that they engage the receptor-binding site (RBS) of the viral hemagglutinin with strong similarities to their influenza A counterparts, despite structural differences between the RBS of influenza A and B. Our data show that these antibodies readily cross-react with both influenza B Victoria and Yamagata lineages. We also note that all antibodies are encoded by IGHV3-9/IGK1-33. Future research will provide insight into the prevalence of these antibodies in the human population.},
	keywords = {Binding Sites, Hemagglutinin Glycoproteins, Influenza Virus, Hemagglutinins, Humans, Influenza B virus, Influenza, Human},
	issn = {2373-8227},
	doi = {10.1021/acsinfecdis.0c00726},
	author = {Bajic, Goran and Harrison, Stephen C}
}
@article {1636647,
	title = {Effector-mediated membrane disruption controls cell death in CBASS antiphage defense},
	journal = {Mol Cell},
	volume = {81},
	number = {24},
	year = {2021},
	month = {2021 12 16},
	pages = {5039-5051.e5},
	abstract = {Cyclic oligonucleotide-based antiphage signaling systems (CBASS) are antiviral defense operons that protect bacteria from phage replication. Here, we discover a widespread class of CBASS transmembrane (TM) effector proteins that respond to antiviral nucleotide signals and limit phage propagation through direct membrane disruption. Crystal structures of the Yersinia TM effector Cap15 reveal a compact 8-stranded β-barrel scaffold that forms a cyclic dinucleotide receptor domain that oligomerizes upon activation. We demonstrate that activated Cap15 relocalizes throughout the cell and specifically induces rupture of the inner membrane. Screening for active effectors, we identify the function of distinct families of CBASS TM effectors and demonstrate that cell death via disruption of inner-membrane integrity is a common mechanism of defense. Our results reveal the function of the most prominent class of effector protein in CBASS immunity and define disruption of the inner membrane as a widespread strategy of abortive infection in bacterial phage defense.},
	keywords = {Bacterial Proteins, Bacteriophages, Cell Death, Cell Membrane, Escherichia coli, Host-Pathogen Interactions, Ligands, Protein Conformation, Protein Multimerization, Protein Transport, Signal Transduction, Structure-Activity Relationship, Yersinia},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2021.10.020},
	author = {Brianna Duncan-Lowey and McNamara-Bordewick, Nora K and Tal, Nitzan and Sorek, Rotem and Kranzusch, Philip J}
}
@article {1636648,
	title = {Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state},
	journal = {Elife},
	volume = {10},
	year = {2021},
	month = {2021 12 02},
	abstract = {Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On- and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation{\textquoteright}s on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low-affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high-affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by {\textquoteright}inside-out signaling.{\textquoteright}},
	keywords = {Binding Sites, Cell Adhesion, Cytoskeleton, Humans, Integrins, Jurkat Cells, K562 Cells, Kinetics, Ligands, Models, Molecular},
	issn = {2050-084X},
	doi = {10.7554/eLife.73359},
	author = {Li, Jing and Yan, Jiabin and Springer, Timothy A.}
}
@article {1636642,
	title = {Mechanism of membrane-curvature generation by ER-tubule shaping proteins},
	journal = {Nat Commun},
	volume = {12},
	number = {1},
	year = {2021},
	month = {2021 01 25},
	pages = {568},
	abstract = {The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. These proteins have two pairs of trans-membrane (TM) segments, followed by an amphipathic helix (APH), but how they induce curvature is poorly understood. Here, we show that REEPs form homodimers by interaction within the membrane. When overexpressed or reconstituted at high concentrations with phospholipids, REEPs cause extreme curvature through their TMs, generating lipoprotein particles instead of vesicles. The APH facilitates curvature generation, as its mutation prevents ER network formation of reconstituted proteoliposomes, and synthetic L- or D-amino acid peptides abolish ER network formation in Xenopus egg extracts. In Schizosaccharomyces japonicus, the APH is required for reticulon{\textquoteright}s exclusive ER-tubule localization and restricted mobility. Thus, the TMs and APH cooperate to generate high membrane curvature. We propose that the formation of splayed REEP/reticulon dimers is responsible for ER tubule formation.},
	keywords = {Animals, Cell Membrane, Endoplasmic Reticulum, Fungal Proteins, Lipoproteins, Membrane Proteins, Microscopy, Electron, Mutation, Protein Multimerization, Schizosaccharomyces, Xenopus Proteins},
	issn = {2041-1723},
	doi = {10.1038/s41467-020-20625-y},
	author = {Wang, Ning and Clark, Lindsay D and Gao, Yuan and Kozlov, Michael M and Shemesh, Tom and Rapoport, Tom A}
}
@article {Arellano-Santoyo2021.10.12.464151,
	title = {Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization},
	journal = {bioRxiv},
	year = {2021},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.Competing Interest StatementThe authors have declared no competing interest.},
	doi = {10.1101/2021.10.12.464151},
	url = {https://www.biorxiv.org/content/early/2021/10/13/2021.10.12.464151},
	author = {Arellano-Santoyo, Hugo and Hernandez-Lopez, Rogelio A. and Stokasimov, Emma and Wang, Ray Yu-Ruei and Pellman, David and Leschziner, Andres E.}
}
@article {1636643,
	title = {Rapid generation of potent antibodies by autonomous hypermutation in yeast},
	journal = {Nat Chem Biol},
	volume = {17},
	number = {10},
	year = {2021},
	month = {2021 10},
	pages = {1057-1064},
	abstract = {The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe {\textquoteright}autonomous hypermutation yeast surface display{\textquoteright} (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.},
	keywords = {Antibodies, Antibody Formation, Antigens, COVID-19, Humans, Peptide Library, Protein Engineering, Recombinant Proteins, Saccharomyces cerevisiae, SARS-CoV-2, Single-Domain Antibodies, Spike Glycoprotein, Coronavirus},
	issn = {1552-4469},
	doi = {10.1038/s41589-021-00832-4},
	author = {Wellner, Alon and McMahon, Conor and Gilman, Morgan S A and Clements, Jonathan R and Clark, Sarah and Nguyen, Kianna M and Ho, Ming H and Hu, Vincent J and Shin, Jung-Eun and Feldman, Jared and Hauser, Blake M and Caradonna, Timothy M and Wingler, Laura M and Schmidt, Aaron G and Marks, Debora S and Jonathan Abraham and Kruse, Andrew C and Liu, Chang C}
}
@article {1654411,
	title = {Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2},
	journal = {Elife},
	volume = {10},
	year = {2021},
	month = {2021 12 23},
	abstract = {HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.},
	keywords = {Animals, Antibodies, Monoclonal, Binding Sites, Antibody, Biophysical Phenomena, Culicidae, Germ Cells, Malaria, Membrane Fusion, Plasmodium berghei, Protein Binding, Protozoan Proteins},
	issn = {2050-084X},
	doi = {10.7554/eLife.74707},
	author = {Feng, Juan and Dong, Xianchi and Adam DeCosta and Su, Yang and Fiona Angrisano and Sala, Katarzyna A and Blagborough, Andrew M and Lu, Chafen and Springer, Timothy A.}
}
@mastersthesis {Wierbowski2021,
	title = {Molecular Mechanisms of Sonic Hedgehog Release and Delivery},
	year = {2021},
	pages = {258},
	school = {Harvard University},
	type = {PhD Thesis},
	address = {Ann Arbor},
	abstract = {The Hedgehog signaling pathway numbers among the five highly conserved cell{\textendash}cell signaling pathways that operate early in metazoan development. The pathway{\textquoteright}s activating ligand, the Sonic hedgehog (SHH) morphogen, is posttranslationally modified by palmitate and cholesterol, two lipids that tether SHH strongly to the membrane of producing cells. To effect many of its patterning roles, however, SHH must signal to faraway target cells, raising the question of how lipidated SHH is mobilized to act at a distance. In this dissertation, I investigate the biochemical basis for SHH movement between producing and responding cells by addressing two related questions: (1) how is lipidated SHH released, in a soluble form, from the membrane of producing cells and (2) how is SHH delivered to its receptor, Patched1 (PTCH1), on target cells?In Chapter 2, we address the first question by employing a quantitative cell-based assay to measure SHH release kinetics as a function of two known SHH release factors: the transporter-like membrane protein Dispatched1 (DISP1) and a secreted chaperone SCUBE2, which binds the lipidated termini of SHH. We determine that transfer of SHH from the producing cell membrane to exogenous SCUBE2 is rate-limited by DISP1 transporter activity, which we find is powered by the plasma membrane Na+ gradient.In Chapter 3, we address the second question by following the fate of the soluble SCUBE2{\textendash}SHH complex. Using cell-based experiments and biochemical reconstitution, we discover that SHH transfer from SCUBE2 to PTCH1 is accomplished by the sequential activities of the SHH coreceptors CDON/BOC and GAS1, factors necessary for Hedgehog signaling in vivo but with yet-unclear molecular functions. We find that, first, CDON/BOC bind both SCUBE2 and SHH, to recruit SCUBE2{\textendash}SHH to the cell surface. Next, GAS1, which we find binds SHH lipid moieties, accepts SHH from SCUBE2. Finally, GAS1 coordinates transfer of SHH lipids to PTCH1, activating the pathway.Collectively, these studies establish a biochemical framework for the movement of lipidated SHH from the producing cell membrane to the PTCH1 receptor, in which successive handoffs of the SHH lipid appendages along an extracellular chaperone cascade ultimately dictate its tissue distribution and cell-autonomous activity.},
	keywords = {0379:Cellular biology, 0487:Biochemistry, 0758:Developmental biology, Biochemistry, Cellular biology, Chaperone, Coreceptor, development, Developmental Biology, Hedgehog signaling, Lipid, Transporter},
	isbn = {9798582571513},
	author = {Wierbowski, Bradley Michael and Salic, Adrian}
}
@mastersthesis {1654401,
	title = {CryoET Reveals Structures Essential to Infections by Pathogens from Viruses to Protozoa},
	year = {2021},
	pages = {245},
	type = {PhD Thesis},
	abstract = {
	Infections by viruses and flagellated protozoa lead to many human diseases, ranging from mild cold sores to devastating cancers. These pathogens infect billions of people worldwide and afflict wild and domestic animals, placing an enormous burden on global public health and economy. To resolve their in situ structures essential for infection, we have firstly established the validity of our approach of cryoET with sub-tomogram averaging using virus as a gold standard organism, then applied the same approach to investigate the structural details of flagellum in more complex protozoa Trypanosoma brucei.In this thesis work, we firstly established the validity of our cryoET approach by obtaining in situ structures of the vesicular stomatitis virus glycoprotein G trimer in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. Later for herpesvirus HCMV, we resolved 3D structures of gB trimers in two distinct conformations at up to 21 {\r A} resolution. We further captured the prefusion gB in complex with an {\textquotedblleft}L{\textquotedblright}-shaped density attributed to the gH/gL complex. Our results deepen the knowledge of membrane fusion mechanism during HCMV infection.How does nuclear egress complex (NEC){\textemdash}the mediator of viral capsid budding into the cytoplasm{\textemdash}interacts with the viral capsid and how proper curvature of the coat is achieved to enable budding? We applied our cryoET approach to report that binding of a capsid protein, UL25, promotes the formation of a pentagonal rather than hexagonal NEC arrangement. Our results suggest that during nuclear budding, interactions between UL25 bound to the pentagonal capsid vertices and NEC introduce pentagonal insertions into the hexagonal NEC array to yield an NEC coat of the appropriate size and curvature, leading to productive budding and egress of UL25decorated capsids.iii Furthermore, to understand structural foundations of eukaryotic flagella supporting motility and signaling necessary for infection, transmission, and pathogenesis, we then applied our cryoET approach to study T. brucei, a protozoan parasite in the Excavata lineage that causes African trypanosomiasis. From our resolved structure of its 96-nm axonemal repeats, we discovered several lineage-specific structures, including novel inter-doublet linkages and microtubule inner proteins. We also determined structures of the paraflagellar rod (PFR), PFR-axoneme connectors, and the axonemal central pair complex. Together, our findings fill the previously critical gap in understanding structural foundations of eukaryotic flagella, provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology to engineering principles in bionics.



	Copyright - Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works; Last updated - 2021-11-06



	\&nbsp;

},
	keywords = {0307:Molecular biology, 0571:Pathology, 0720:Virology, CryoET, Infection, Molecular Biology, pathogen, Pathology, Trypanosoma brucei, Virology, Virus},
	isbn = {9798460436378},
	author = {Zhang, Jiayan}
}
@article {1654473,
	title = {NEDD9 Is a Novel and Modifiable Mediator of Platelet-Endothelial Adhesion in the Pulmonary Circulation},
	journal = {Am J Respir Crit Care Med},
	volume = {203},
	number = {12},
	year = {2021},
	month = {2021 06 15},
	pages = {1533-1545},
	abstract = {Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications. Objectives: To identify a hypoxia-sensitive, modifiable mediator of platelet-pulmonary artery endothelial cell adhesion and thrombotic remodeling. Methods: Network medicine was used to profile protein-protein interactions in hypoxia-treated human pulmonary artery endothelial cells. Data from liquid chromatography-mass spectrometry and microscale thermophoresis informed the development of a novel antibody (Ab) to inhibit platelet-endothelial adhesion, which was tested in cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH) and three animal models in\&nbsp;vivo. Measurements and Main Results: The protein NEDD9 was identified in the hypoxia thrombosome network in silico. Compared with normoxia, hypoxia (0.2\% O2) for 24 hours increased HIF-1α (hypoxia-inducible factor-1α)-dependent NEDD9 upregulation in\&nbsp;vitro. Increased NEDD9 was localized to the plasma-membrane surface of cells from control donors and patients with CTEPH. In endarterectomy specimens, NEDD9 colocalized with the platelet surface adhesion molecule P-selectin. Our custom-made anti-NEDD9 Ab targeted the NEDD9-P-selectin interaction and inhibited the adhesion of activated platelets to pulmonary artery endothelial cells from control donors in\&nbsp;vitro and from patients with CTEPH ex vivo. Compared with control mice, platelet-pulmonary endothelial aggregates and pulmonary hypertension induced by ADP were decreased in NEDD9-/- mice or wild-type mice treated with the anti-NEDD9 Ab, which also decreased chronic pulmonary thromboembolic remodeling in\&nbsp;vivo. Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.},
	keywords = {Adaptor Proteins, Signal Transducing, Animals, Blood Platelets, Cell Adhesion, Cells, Cultured, Endothelial Cells, Female, Humans, Hypoxia, Male, Mice, Middle Aged, Models, Animal, Pulmonary Circulation, Pulmonary Embolism, Signal Transduction},
	issn = {1535-4970},
	doi = {10.1164/rccm.202003-0719OC},
	author = {Alba, George A and Samokhin, Andriy O and Wang, Rui-Sheng and Zhang, Ying-Yi and Wertheim, Bradley M and Arons, Elena and Greenfield, Edward A and Lundberg Slingsby, Martina H and Ceglowski, Julia R and Haley, Kathleen J and Bowman, Frederick P and Yu, Yen-Rei and Haney, John C and Eng, George and Mitchell, Richard N and Sheets, Anthony and Vargas, Sara O and Seo, Sachiko and Channick, Richard N and Leary, Peter J and Rajagopal, Sudarshan and Loscalzo, Joseph and Battinelli, Elisabeth M and Maron, Bradley A}
}
@inbook {1660966,
	title = {Biophysical methods},
	booktitle = {The Design \& Development of Novel Drugs and Vaccines},
	year = {2021},
	pages = {211-221},
	publisher = {Elsevier},
	organization = {Elsevier},
	author = {Rai, Praveen and Singh, Sujeet and Nimesh, Surendra}
}
@article {1654410,
	title = {Virus-derived peptide inhibitors of the herpes simplex virus type 1 nuclear egress complex},
	journal = {Sci Rep},
	volume = {11},
	number = {1},
	year = {2021},
	month = {2021 02 18},
	pages = {4206},
	abstract = {Herpesviruses infect a majority of the human population, establishing lifelong latent infections for which there is no cure. Periodic viral reactivation spreads infection to new hosts while causing various disease states particularly detrimental in the immunocompromised. Efficient viral replication, and ultimately the spread of infection, is dependent on the nuclear egress complex (NEC), a conserved viral heterodimer that helps translocate viral capsids from the nucleus to the cytoplasm where they mature into infectious virions. Here, we have identified peptides, derived from the capsid protein UL25, that are capable of inhibiting the membrane-budding activity of the NEC from herpes simplex virus type 1 in vitro. We show that the inhibitory ability of the peptides depends on their length and the propensity to form an α-helix but not on the exact amino acid sequence. Current therapeutics that target viral DNA replication machinery are rendered ineffective by drug resistance due to viral mutations. Our results establish a basis for the development of an alternative class of inhibitors against nuclear egress, an essential step in herpesvirus replication, potentially expanding the current repertoire of available therapeutics.},
	keywords = {Amino Acid Sequence, Capsid Proteins, Cell Nucleus, Cytoplasm, DNA Replication, DNA, Viral, Herpesvirus 1, Human, Mutation, Nuclear Envelope, Nuclear Proteins, Peptides, Protein Conformation, alpha-Helical, Viral Proteins, Virus Replication},
	issn = {2045-2322},
	doi = {10.1038/s41598-021-83402-x},
	author = {Draganova, Elizabeth B and Heldwein, Ekaterina E}
}
@article {1572170,
	title = {N-Terminal Protein Labeling with N-Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein-Protein Interactions Using Labeled Protein},
	journal = {Curr Protoc},
	volume = {1},
	number = {1},
	year = {2021},
	month = {2021 Jan},
	pages = {e14},
	abstract = {Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein{\textquoteright}s thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. {\textcopyright} 2021 Wiley Periodicals LLC. Basic Protocol 1: Labeling a protein of interest at its N-terminus with NHS esters through stepwise reaction Alternate Protocol: Labeling a protein of interest at its N-terminus with NHS esters through a one-pot reaction Basic Protocol 2: Purifying the N-terminal fluorescent-labeled protein and determining its concentration and labeling efficiency Basic Protocol 3: Using MST to determine the binding affinity of an N-terminal fluorescent-labeled protein to a binding partner. Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay.},
	issn = {2691-1299},
	doi = {10.1002/cpz1.14},
	author = {Jiang, Hanjie and Cole, Philip A}
}
@article {1572172,
	title = {Exifone Is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration},
	journal = {ACS Chem Neurosci},
	volume = {12},
	number = {2},
	year = {2021},
	month = {2021 Jan 20},
	pages = {271-284},
	abstract = {Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer{\textquoteright}s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson{\textquoteright}s disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.},
	issn = {1948-7193},
	doi = {10.1021/acschemneuro.0c00308},
	author = {Patnaik, Debasis and Pao, Ping-Chieh and Zhao, Wen-Ning and Silva, M Catarina and Hylton, Norma K and Chindavong, Peter S and Pan, Ling and Tsai, Li-Huei and Haggarty, Stephen J}
}
@article {1636644,
	title = {Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering},
	journal = {mBio},
	volume = {12},
	number = {4},
	year = {2021},
	month = {2021 08 31},
	pages = {e0154821},
	abstract = {During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world{\textquoteright}s population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.},
	keywords = {Animals, Capsid, Cell Nucleus, Chlorocebus aethiops, Herpesvirus 1, Human, Humans, Lipid Metabolism, Nuclear Envelope, Phosphorylation, Static Electricity, Vero Cells, Virus Assembly, Virus Release, Virus Replication},
	issn = {2150-7511},
	doi = {10.1128/mBio.01548-21},
	author = {Thorsen, Michael K and Lai, Alex and Lee, Michelle W and Hoogerheide, David P and Wong, Gerard C L and Freed, Jack H and Heldwein, Ekaterina E}
}
@booklet {https://open.bu.edu/handle/2144/43777,
	title = {Identification of novel apolipoprotein E receptor 2 splice variants and their role in synaptic transmission},
	year = {2021},
	publisher = {OpenBU},
	url = {https://open.bu.edu/handle/2144/43777},
	author = {Omuro,, Kerilyn, C.}
}
@article {1636669,
	title = {Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium},
	journal = {Commun Biol},
	volume = {4},
	number = {1},
	year = {2021},
	month = {2021 03 19},
	pages = {360},
	abstract = {Human (h) carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) function depends upon IgV-mediated homodimerization or heterodimerization with host ligands, including hCEACAM5, hTIM-3, PD-1, and a variety of microbial pathogens. However, there is little structural information available on how hCEACAM1 transitions between monomeric and dimeric states which in the latter case is critical for initiating hCEACAM1 activities. We therefore mutated residues within the hCEACAM1 IgV GFCC{\textquoteright} face including V39, I91, N97, and E99 and examined hCEACAM1 IgV monomer-homodimer exchange using differential scanning fluorimetry, multi-angle light scattering, X-ray crystallography and/or nuclear magnetic resonance. From these studies, we describe hCEACAM1 homodimeric, monomeric and transition states at atomic resolution and its conformational behavior in solution through NMR assignment of the wildtype (WT) hCEACAM1 IgV dimer and N97A mutant monomer. These studies reveal the flexibility of the GFCC{\textquoteright} face and its important role in governing the formation of hCEACAM1 dimers and selective heterodimers.},
	keywords = {Antigens, CD, Cell Adhesion Molecules, Crystallography, X-Ray, Dynamic Light Scattering, Fluorometry, Humans, Magnetic Resonance Spectroscopy, Mutation, Protein Conformation, Protein Multimerization, Structure-Activity Relationship},
	issn = {2399-3642},
	doi = {10.1038/s42003-021-01871-2},
	author = {Gandhi, Amit K and Sun, Zhen-Yu J and Kim, Walter M and Huang, Yu-Hwa and Kondo, Yasuyuki and Bonsor, Daniel A and Sundberg, Eric J and Wagner, Gerhard and Kuchroo, Vijay K and Petsko, Gregory A and Blumberg, Richard S}
}
@article {1636649,
	title = {Structures of the σ receptor enable docking for bioactive ligand discovery},
	journal = {Nature},
	volume = {600},
	number = {7890},
	year = {2021},
	month = {2021 Dec},
	pages = {759-764},
	abstract = {The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes\&nbsp;through structure-based screens of ultra large libraries, enabling study of\&nbsp;underexplored areas of biology.},
	issn = {1476-4687},
	doi = {10.1038/s41586-021-04175-x},
	author = {Alon, Assaf and Lyu, Jiankun and Braz, Joao M and Tummino, Tia A and Craik, Veronica and O{\textquoteright}Meara, Matthew J and Webb, Chase M and Radchenko, Dmytro S and Moroz, Yurii S and Huang, Xi-Ping and Liu, Yongfeng and Roth, Bryan L and Irwin, John J and Basbaum, Allan I and Shoichet, Brian K and Kruse, Andrew C}
}
@article {1636635,
	title = {Conserved Outer Tegument Component UL11 from Herpes Simplex Virus 1 Is an Intrinsically Disordered, RNA-Binding Protein},
	journal = {mBio},
	volume = {11},
	number = {3},
	year = {2020},
	month = {2020 05 05},
	abstract = {A distinguishing morphological feature of all herpesviruses is the multiprotein tegument layer located between the nucleocapsid and lipid envelope of the virion. Tegument proteins play multiple roles in viral replication, including viral assembly, but we do not yet understand their individual functions or how the tegument is assembled and organized. UL11, the smallest tegument protein, is important for several distinct processes in replication, including efficient virion morphogenesis and cell-cell spread. However, the mechanistic understanding of its role in these and other processes is limited in part by the scant knowledge of its biochemical and structural properties. Here, we report that UL11 from herpes simplex virus 1 (HSV-1) is an intrinsically disordered, conformationally dynamic protein that undergoes liquid-liquid phase separation (LLPS) in vitro Intrinsic disorder may underlie the ability of UL11 to exert multiple functions and bind multiple partners. Sequence analysis suggests that not only all UL11 homologs but also all HSV-1 tegument proteins contain intrinsically disordered regions of different lengths. The presence of intrinsic disorder, and potentially, the ability to form LLPS, may thus be a common feature of the tegument proteins. We hypothesize that tegument assembly may involve the formation of a biomolecular condensate, driven by the heterogeneous mixture of intrinsically disordered tegument proteins.IMPORTANCE Herpesvirus virions contain a unique tegument layer sandwiched between the capsid and lipid envelope and composed of multiple copies of about two dozen viral proteins. However, little is known about the structure of the tegument or how it is assembled. Here, we show that a conserved tegument protein UL11 from herpes simplex virus 1, a prototypical alphaherpesvirus, is an intrinsically disordered protein that undergoes liquid-liquid phase separation in vitro Through sequence analysis, we find intrinsically disordered regions of different lengths in all HSV-1 tegument proteins. We hypothesize that intrinsic disorder is a common characteristic of tegument proteins and propose a new model of tegument as a biomolecular condensate.},
	keywords = {Crystallography, Herpesvirus 1, Human, Protein Binding, RNA-Binding Proteins, Viral Structural Proteins},
	issn = {2150-7511},
	doi = {10.1128/mBio.00810-20},
	author = {Metrick, Claire M and Koenigsberg, Andrea L and Heldwein, Ekaterina E}
}
@article {1636629,
	title = {A nanobody targeting the LIN28:let-7 interaction fragment of TUT4 blocks uridylation of let-7},
	journal = {Proc Natl Acad Sci U S A},
	volume = {117},
	number = {9},
	year = {2020},
	month = {2020 03 03},
	pages = {4653-4663},
	abstract = {The LIN28:pre-let-7:TUTase ternary complex regulates pluripotency and oncogenesis by controlling processing of the let-7 family of microRNAs. The complex oligouridylates the 3{\textquoteright} ends of pre-let-7 molecules, leading to their degradation via the DIS3L2 exonuclease. Previous studies suggest that components of this complex are potential therapeutic targets in malignancies that aberrantly express LIN28. In this study we developed a functional epitope selection approach to identify nanobody inhibitors of the LIN28:pre-let-7:TUT4 complex. We demonstrate that one of the identified nanobodies, Nb-S2A4, targets the 106-residue LIN28:let-7 interaction (LLI) fragment of TUT4. Nb-S2A4 can effectively inhibit oligouridylation and monouridylation of pre-let-7g in vitro. Expressing Nb-S2A4 allows maturation of the let-7 species in cells expressing LIN28, highlighting the therapeutic potential of targeting the LLI fragment.},
	keywords = {Animals, Binding Sites, DNA-Binding Proteins, HEK293 Cells, HeLa Cells, Humans, Mice, MicroRNAs, Protein Binding, RNA 3{\textquoteright} End Processing, RNA Stability, RNA-Binding Proteins, Sf9 Cells, Single-Domain Antibodies, Spodoptera},
	issn = {1091-6490},
	doi = {10.1073/pnas.1919409117},
	author = {Yu, Chunxiao and Wang, Longfei and Rowe, R Grant and Han, Areum and Ji, Wanying and McMahon, Conor and Baier, Alexander S and Huang, Yu-Chung and Marion, William and Pearson, Daniel S and Kruse, Andrew C and Daley, George Q and Wu, Hao and Sliz, Piotr}
}
@article {1636633,
	title = {Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication},
	journal = {J Virol},
	volume = {94},
	number = {13},
	year = {2020},
	month = {2020 06 16},
	abstract = {Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation.IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.},
	keywords = {Animals, Cell Line, Chlorocebus aethiops, Humans, Kinetics, Nucleocapsid Proteins, Phosphoproteins, Protein Binding, Ribonucleoproteins, RNA, Messenger, RNA, Viral, RNA-Dependent RNA Polymerase, Transcription, Genetic, Vero Cells, Vesicular Stomatitis, Vesiculovirus, Viral Proteins, Virus Replication},
	issn = {1098-5514},
	doi = {10.1128/JVI.00115-20},
	author = {Bloyet, Louis-Marie and Morin, Benjamin and Vesna Brusic and Gardner, Erica and Ross, Robin A and Vadakkan, Tegy and Kirchhausen, Tomas and Whelan, Sean P J}
}
@article {1636639,
	title = {A Small-Molecule Inhibitor to the Cytokine Interleukin-4},
	journal = {ACS Chem Biol},
	volume = {15},
	number = {10},
	year = {2020},
	month = {2020 10 16},
	pages = {2649-2654},
	abstract = {Interleukin-4 (IL-4) is a multifunctional cytokine and an important regulator of inflammation. When deregulated, IL-4 activity is associated with asthma, allergic inflammation, and multiple types of cancer. While antibody-based inhibitors targeting the soluble cytokine have been evaluated clinically, they failed to achieve their end points in trials. Small-molecule inhibitors are an attractive alternative, but identifying effective chemotypes that inhibit the protein-protein interactions between cytokines and their receptors remains an active area of research. As a result, no small-molecule inhibitors to the soluble IL-4 cytokine have yet been reported. Here, we describe the first IL-4 small-molecule inhibitor identified and characterized through a combination of binding-based approaches and cell-based activity assays. The compound features a nicotinonitrile scaffold with micromolar affinity and potency for the cytokine and disrupts type II IL-4 signaling in cells. Small-molecule inhibitors of these important cell-signaling proteins have implications for numerous immune-related disorders and inform future drug discovery and design efforts for these challenging protein targets.},
	keywords = {Aminopyridines, Humans, Interleukin-4, Ligands, Phosphorylation, Protein Binding, Signal Transduction, Small Molecule Libraries, STAT6 Transcription Factor, THP-1 Cells},
	issn = {1554-8937},
	doi = {10.1021/acschembio.0c00615},
	author = {Quinnell, Sean P and Leifer, Becky S and Nestor, Stephen T and Tan, Kelly and Sheehy, Daniel F and Ceo, Luke and Doyle, Shelby K and Koehler, Angela N and Vegas, Arturo J}
}
@article {1636632,
	title = {Structure-Guided Molecular Grafting of a Complex Broadly Neutralizing Viral Epitope},
	journal = {ACS Infect Dis},
	volume = {6},
	number = {5},
	year = {2020},
	month = {2020 05 08},
	pages = {1182-1191},
	abstract = {Antigenic variation and viral evolution have thwarted traditional influenza vaccination strategies. The broad protection afforded by a "universal" influenza vaccine may come from immunogens that elicit humoral immune responses targeting conserved epitopes on the viral hemagglutinin (HA), such as the receptor-binding site (RBS). Here, we engineered candidate immunogens that use noncirculating, avian influenza HAs as molecular scaffolds to present the broadly neutralizing RBS epitope from historical, circulating H1 influenzas. These "resurfaced" HAs (rsHAs) remove epitopes potentially targeted by strain-specific responses in immune-experienced individuals. Through structure-guided optimization, we improved two antigenically different scaffolds to bind a diverse panel of pan-H1 and H1/H3 cross-reactive bnAbs with high affinity. Subsequent serological and single germinal center B cell analyses from murine prime-boost immunizations show that the rsHAs are both immunogenic and can augment the quality of elicited RBS-directed antibodies. Our structure-guided, RBS grafting approach provides candidate immunogens for selectively presenting a conserved viral epitope.},
	keywords = {Animals, Antibodies, Neutralizing, Antibodies, Viral, Antigen Presentation, Epitopes, Hemagglutinin Glycoproteins, Influenza Virus, Humans, Influenza Vaccines, Mice},
	issn = {2373-8227},
	doi = {10.1021/acsinfecdis.0c00008},
	author = {Bajic, Goran and Maron, Max J and Caradonna, Timothy M and Tian, Ming and Mermelstein, Adam and Fera, Daniela and Kelsoe, Garnett and Kuraoka, Masayuki and Schmidt, Aaron G}
}
@article {1636628,
	title = {A Systematic Approach to HIV-1 Vaccine Immunogen Selection},
	journal = {AIDS Res Hum Retroviruses},
	volume = {36},
	number = {9},
	year = {2020},
	month = {2020 09},
	pages = {762-770},
	abstract = {A tremendous loss of financial and human resources from seven large-scale HIV vaccine efficacy trials suggest a need for a systematic approach to vaccine selection. We conducted a systematic analysis of three important envelope glycoprotein (Env) vaccine candidates: BG505 SOSIP.664, 1086.C gp140, and 1086.C gp120 to determine the most promising by comparing their structure and antigenicity. We found that the BG505 SOSIP trimer and 1086.C gp140 clearly outperformed the 1086.C gp120 monomer. BG505 SOSIP.664 bound the strongest to the most potent and broadest broadly neutralizing antibodies (bnAbs) PG9, PGT145, VRC01, and PGT121. Of interest, although BG505 SOSIP.664 did not bind to the CH58 mAb, 1086.C gp140 bound strongly to this mAb, which belongs to a class of non-neutralizing antibodies that may be protective based on correlates of protection studies of the RV144 HIV vaccine trial. The 1086.C gp120 monomer was the least antigenic of the three vaccine immunogens, binding the weakest to bnAbs and CH58 mAb. Taken together, the evidence provided here combined with previous preclinical immunogenicity and efficacy data strongly argue that the BG505 SOSIP.664 trimer and 1086.C gp140 are likely to be better vaccine immunogens than the monomeric 1086.C gp120, which was just recently tested and shown to be nonefficacious in a phase IIb/III trial. Thus, to best utilize our financial and valuable human resources, we propose a systematic approach by not only comparing structure and antigenicity, but also immunogenicity and efficacy of Env vaccine candidates in the preclinical phase to the selection of only the most promising vaccine candidates for clinical testing.},
	keywords = {AIDS Vaccines, Antibodies, Neutralizing, env Gene Products, Human Immunodeficiency Virus, HIV Antibodies, HIV Envelope Protein gp120, HIV Infections, HIV-1, Humans, Protein Multimerization},
	issn = {1931-8405},
	doi = {10.1089/AID.2019.0239},
	author = {Bontempo, Alexander and Garcia, Maria M and Rivera, Naylene and Cayabyab, Mark J}
}
@article {1636634,
	title = {Topical delivery of siRNA into skin using ionic liquids},
	journal = {J Control Release},
	volume = {323},
	year = {2020},
	month = {2020 07 10},
	pages = {475-482},
	abstract = {Skin diseases such as lupus, cancer, psoriasis, and hyperhidrosis can potentially be treated effectively by suppressing allele-specific genes using small interfering RNA (siRNA). Injections of siRNA into skin, though effective, are painful and cover small surface areas and thus are not suitable as a long-term treatment option. Topical delivery of siRNA is an attractive alternative option to mediate RNA interference (RNAi). However, the barrier function of the epidermis impedes effective permeation of siRNA into the skin. Herein, we describe topical delivery of siRNA using ionic liquids (ILs) capable of complexing with siRNA non-covalently and delivering it effectively. Using complementary and synergistic strategies of ionic liquids, we report delivery of effective doses of siRNA into skin. The first strategy involved the use of hydrophobic cations to robe the siRNA and the second strategy involved the use of choline-geranic acid ionic liquid (CAGE) to enhance its dermal penetration. In vitro studies in porcine skin confirmed the synergistic effect of these strategies in enhancing epidermal and dermal penetration. In vivo application of siRNA formulation to SKH-1E hairless mice significantly suppressed GAPDH expression with no clinical evidence of toxicity. This is a simple, personalized, and scalable platform for effective topical delivery of siRNA for treating genetic skin diseases.},
	keywords = {Administration, Cutaneous, Animals, Ionic Liquids, Mice, RNA Interference, RNA, Small Interfering, Skin, Skin Absorption, Swine},
	issn = {1873-4995},
	doi = {10.1016/j.jconrel.2020.04.038},
	author = {Dharamdasani, Vimisha and Mandal, Abhirup and Qi, Qin M and Suzuki, Isabella and Bentley, Maria Vit{\'o}ria Lopes Badra and Mitragotri, Samir}
}
@article {1636641,
	title = {XLF acts as a flexible connector during non-homologous end joining},
	journal = {Elife},
	volume = {9},
	year = {2020},
	month = {2020 12 08},
	abstract = {Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here, we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of the XRCC4-like factor (XLF), a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku-binding motif (KBM) at the extreme C-terminus are required for end joining. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku, while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.},
	keywords = {Animals, Chromatography, Gel, DNA End-Joining Repair, DNA Repair Enzymes, DNA-Binding Proteins, Immunoblotting, Ovum, Sequence Alignment, Xenopus laevis, Xenopus Proteins},
	issn = {2050-084X},
	doi = {10.7554/eLife.61920},
	author = {Carney, Sean M and Moreno, Andrew T and Piatt, Sadie C and Cisneros-Aguirre, Metztli and Lopezcolorado, Felicia Wednesday and Stark, Jeremy M and Loparo, Joseph J}
}
@article {1572171,
	title = {Identification and Mechanistic Characterization of a Peptide Inhibitor of Glycogen Synthase Kinase (GSK3β) Derived from the Disrupted in Schizophrenia 1 (DISC1) Protein},
	journal = {ACS Chem Neurosci},
	volume = {11},
	number = {24},
	year = {2020},
	month = {2020 12 16},
	pages = {4128-4138},
	abstract = {Glycogen synthase kinase 3-beta (GSK3β) is a critical regulator of several cellular pathways involved in neurodevelopment and neuroplasticity and as such is a potential focus for the discovery of new neurotherapeutics toward the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3β have been focused on developing small molecule inhibitors that compete with adenosine triphosphate (ATP) through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The disrupted in schizophrenia 1 (DISC1) protein has previously been shown to bind and inhibit GSK3β activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3β in a noncompetitive mode distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3β: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3β binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3β.},
	keywords = {Glycogen Synthase Kinase 3 beta, Humans, Nerve Tissue Proteins, Peptides, Phosphorylation},
	issn = {1948-7193},
	doi = {10.1021/acschemneuro.0c00380},
	author = {Saundh, Stephanie L and Patnaik, Debasis and Gagn{\'e}, Steve and Bishop, Joshua A and Lipsit, Sean and Amat, Samat and Pujari, Narsimha and Nambisan, Anand Krishnan and Bigsby, Robert and Murphy, Mary and Tsai, Li-Huei and Haggarty, Stephen J and Leung, Adelaine Kwun-Wai}
}
@article {1720406,
	title = {Non-full-length Water-Soluble CXCR4 and CCR5 Chemokine Receptors: Implication for Overlooked Truncated but Functional Membrane Receptors},
	journal = {iScience},
	volume = {23},
	number = {12},
	year = {2020},
	month = {2020 Dec 18},
	pages = {101670},
	abstract = {It was posited that functionalities of GPCRs require full-length sequences that are negated by residue deletions. Here we report that significantly truncated nfCCR5QTY and nfCXCR4QTY still bind native ligands. Receptor-ligand interactions were discovered from yeast 2-hybrid screening and confirmed by mating selection. Two nfCCR5QTY (SZ218a, SZ190b) and two nfCXCR4QTY (SZ158a, SZ146a) were expressed in E.\&nbsp;coli. Synthesized receptors exhibited α-helical structures and bound respective ligands with reduced affinities. SZ190b and SZ158a were reconverted into non-QTY forms and expressed in HEK293T cells. Reconverted receptors localized on cell membranes and functioned as negative regulators for ligand-induced signaling when co-expressed with full-length receptors. CCR5-SZ190b individually can perform signaling at a reduced level with higher ligand concentration. Our findings provide insight into essential structural components for CCR5 and CXCR4 functionality, while raising the possibility that non-full-length receptors may be resulted from alternative splicing and that pseudo-genes in genomes may be present and functional in living organisms.},
	issn = {2589-0042},
	doi = {10.1016/j.isci.2020.101670},
	author = {Qing, Rui and Tao, Fei and Chatterjee, Pranam and Yang, Gaojie and Qiuyi Han and Chung, Haeyoon and Ni, Jun and Suter, Bernhard P and Kubicek, Jan and Maertens, Barbara and Schubert, Thomas and Blackburn, Camron and Zhang, Shuguang}
}
@article {1572174,
	title = {eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function},
	journal = {Nucleic Acids Res},
	volume = {48},
	number = {11},
	year = {2020},
	month = {2020 06 19},
	pages = {6223-6233},
	abstract = {As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they trigger stress response pathways to protect themselves until transient stresses have passed. Inhibition of translation is a key component of such cellular stress responses and mounting evidence has revealed the importance of a class of tRNA-derived small RNAs called tiRNAs in this process. The most potent of these small RNAs are those with the capability of assembling into tetrameric G-quadruplex (G4) structures. However, the mechanism by which these small RNAs inhibit translation has yet to be elucidated. Here we show that eIF4G, the major scaffolding protein in the translation initiation complex, directly binds G4s and this activity is required for tiRNA-mediated translation repression. Targeting of eIF4G results in an impairment of 40S ribosome scanning on mRNAs leading to the formation of eIF2α-independent stress granules. Our data reveals the mechanism by which tiRNAs inhibit translation and demonstrates novel activity for eIF4G in the regulation of translation.},
	keywords = {Eukaryotic Initiation Factor-2, Eukaryotic Initiation Factor-4F, Eukaryotic Initiation Factor-4G, G-Quadruplexes, Humans, Peptide Chain Initiation, Translational, Phosphoproteins, Protein Biosynthesis, Protein Domains, Ribosome Subunits, Small, Eukaryotic, RNA, Messenger, RNA, Transfer},
	issn = {1362-4962},
	doi = {10.1093/nar/gkaa336},
	author = {Lyons, Shawn M and Kharel, Prakash and Akiyama, Yasutoshi and Ojha, Sandeep and Dave, Dhwani and Tsvetkov, Vladimir and Merrick, William and Ivanov, Pavel and Anderson, Paul}
}
@article {1572173,
	title = {Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus},
	journal = {Mol Cell},
	volume = {78},
	number = {6},
	year = {2020},
	month = {2020 06 18},
	pages = {1207-1223.e8},
	abstract = {Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T\&nbsp;cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T\&nbsp;cell-mediated killing, improving CAR T\&nbsp;cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.},
	keywords = {Aged, Animals, B7-H1 Antigen, Cell Line, Tumor, Female, Gene Expression Regulation, Neoplastic, Humans, Immunotherapy, Immunotherapy, Adoptive, Interferon-gamma, Interferons, Janus Kinase 2, Male, Mice, Mice, Inbred NOD, Middle Aged, Programmed Cell Death 1 Ligand 2 Protein, RNA, Long Noncoding, Signal Transduction, STAT1 Transcription Factor, T-Lymphocytes, Cytotoxic},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2020.05.015},
	author = {Mineo, Marco and Lyons, Shawn M and Zdioruk, Mykola and von Spreckelsen, Niklas and Ferrer-Luna, Ruben and Ito, Hirotaka and Alayo, Quazim A and Kharel, Prakash and Giantini Larsen, Alexandra and Fan, William Y and Auduong, Sophia and Grauwet, Korneel and Passaro, Carmela and Khalsa, Jasneet K and Shah, Khalid and Reardon, David A and Ligon, Keith L and Beroukhim, Rameen and Nakashima, Hiroshi and Ivanov, Pavel and Anderson, Paul J and Lawler, Sean E and Chiocca, E Antonio}
}
@article {1514266,
	title = {CBASS Immunity Uses CARF-Related Effectors to Sense 3{\textquoteright}-5{\textquoteright}- and 2{\textquoteright}-5{\textquoteright}-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection},
	journal = {Cell},
	volume = {182},
	number = {1},
	year = {2020},
	month = {2020 07 09},
	pages = {38-49.e17},
	abstract = {cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of \&gt;2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2{\textquoteright}-5{\textquoteright}- and 3{\textquoteright}-5{\textquoteright}-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.},
	keywords = {Amino Acid Sequence, Bacteria, Bacterial Proteins, Bacteriophages, CRISPR-Cas Systems, Deoxyribonuclease I, Immunity, Ligands, Mutagenesis, Nucleotidyltransferases, Oligonucleotides, Protein Binding, Second Messenger Systems, Signal Transduction},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2020.05.019},
	author = {Lowey, Brianna and Whiteley, Aaron T and Keszei, Alexander F A and Morehouse, Benjamin R and Mathews, Ian T and Antine, Sadie P and Cabrera, Victor J and Kashin, Dmitry and Niemann, Percy and Jain, Mohit and Schwede, Frank and Mekalanos, John J and Shao, Sichen and Lee, Amy S Y and Kranzusch, Philip J}
}
@article {Seo2020.05.12.090472,
	title = {A cyclin-dependent kinase 5-derived peptide inhibits Cdk5/p25 activity and improves neurodegenerative phenotypes},
	journal = {bioRxiv},
	year = {2020},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This effect is mediated by pathological cleavage of the Cdk5 activator p35 to produce the truncated product p25, exhibiting increased stability and altered substrate specificity. The benefit of blocking p25 production has been demonstrated in various rodent and human neurodegenerative models. However, important Cdk5/p35 functions in the developing and adult brain have made it challenging to selectively target the detrimental effects of Cdk5/p25 while sparing the physiological functions of Cdk5/p35. Here, we report a 12-amino acid-long peptide fragment derived from Cdk5 (the Cdk5 inhibitory (Cdk5i) peptide) that shows a high binding affinity toward the Cdk5/p25 complex and can efficiently and selectively inhibit Cdk5/p25 kinase activity. Using cellular assays, mouse neurodegeneration models and human cerebral organoids generated from patient-derived iPSCs, we demonstrate beneficial effects of the Cdk5i peptide on various pathological phenotypes including gliosis, DNA damage, and Tau hyperphosphorylation.Competing Interest StatementS.J.H. is a member of the scientific advisory board of Psy Therapeutics, Frequency Therapeutics, and Vesigen Therapeutics, none of whom were involved in the present study. L.-H. T. and S.J.H. are also co-founders and members of Scientific Advisory Board of Souvien Therapeutics. L.-H. T., S.J.H. and J.S. have licensed intellectual property related to Cdk5 inhibitory peptide. The remaining authors declare no competing interests.},
	doi = {10.1101/2020.05.12.090472},
	url = {https://www.biorxiv.org/content/early/2020/05/14/2020.05.12.090472.1},
	author = {Seo, Jinsoo and Pao, Ping-Chieh and Kritskiy, Oleg and Lee, Audrey and Patnaik, Debasis and Watson, L. Ashely and Bula, Michael and Barker, Scarlett J. and Penney, Jay and Silva, M. Catarina and Stephen J. Haggarty and Tsai, Li-Huei}
}
@article {1501372,
	title = {Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation},
	journal = {Commun Biol},
	volume = {3},
	number = {1},
	year = {2020},
	month = {2020 Mar 17},
	pages = {128},
	abstract = {Programmed cell death-1 (PD-1) inhibits T cell responses. This function relies on interaction with SHP-2. PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM) at Y223 and one immunoreceptor tyrosine-based switch motif (ITSM) at Y248. Only ITSM-Y248 is indispensable for PD-1-mediated inhibitory function but how SHP-2 enzymatic activation is mechanistically regulated by one PD-1 phosphotyrosine remains a puzzle. We found that after PD-1 phosphorylation, SHP-2 can bridge phosphorylated ITSM-Y248 residues on two PD-1 molecules via its amino terminal (N)-SH2 and carboxyterminal (C)-SH2 domains forming a PD-1: PD-1 dimer in live cells. The biophysical ability of SHP-2 to interact with two ITSM-pY248 residues was documented by isothermal titration calorimetry. SHP-2 interaction with two ITSM-pY248 phosphopeptides induced robust enzymatic activation. Our results unravel a mechanism of PD-1: SHP-2 interaction that depends only on ITSM-Y248 and explain how a single docking site within the PD-1 cytoplasmic tail can activate SHP-2 and PD-1-mediated inhibitory function.},
	issn = {2399-3642},
	doi = {10.1038/s42003-020-0845-0},
	author = {Patsoukis, Nikolaos and Duke-Cohan, Jonathan S and Chaudhri, Apoorvi and Aksoylar, Halil-Ibrahim and Wang, Qi and Council, Asia and Berg, Anders and Freeman, Gordon J and Boussiotis, Vassiliki A}
}
@article {1492727,
	title = {An open-source drug discovery platform enables ultra-large virtual screens},
	journal = {Nature},
	volume = {580},
	number = {7805},
	year = {2020},
	month = {2020 04},
	pages = {663-668},
	abstract = {On average, an approved drug currently costs US$2-3~billion and takes more than 10~years to develop1. In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened2. However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4~billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1~billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant (Kd)~=~114~nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins.},
	keywords = {Access to Information, Automation, Cloud Computing, Computer Simulation, Databases, Chemical, Drug Discovery, Drug Evaluation, Preclinical, Kelch-Like ECH-Associated Protein 1, Ligands, Molecular Docking Simulation, Molecular Targeted Therapy, NF-E2-Related Factor 2, Reproducibility of Results, Software, Thermodynamics, User-Computer Interface},
	issn = {1476-4687},
	doi = {10.1038/s41586-020-2117-z},
	author = {Gorgulla, Christoph and Boeszoermenyi, Andras and Wang, Zi-Fu and Fischer, Patrick D and Coote, Paul W and Padmanabha Das, Krishna M and Malets, Yehor S and Radchenko, Dmytro S and Moroz, Yurii S and Scott, David A and Fackeldey, Konstantin and Hoffmann, Moritz and Iavniuk, Iryna and Wagner, Gerhard and Arthanari, Haribabu}
}
@article {Patnaik:2020gs,
	title = {Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration},
	journal = {bioRxiv},
	volume = {27},
	number = {2},
	year = {2020},
	pages = {2020.03.02.973636},
	abstract = {Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative disease. Preventing this loss of genomic integrity that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent, class I histone deacetylase HDAC1 has been identified as a critical protein for protecting neurons from deleterious effects mainly caused by double-strand DNA breaks in Alzheimer{\textquoteright}s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS or FTD will benefit from the identification of small molecules capable of selectively increasing the deacetylase activity of HDAC1 over other structurally similar class I HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive decline associated with AD and Parkinson{\textquoteright}s disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1 and provides protection against genotoxic stress. We show that exifone acts as a mixed, non-essential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Selectivity profiling and estimation of kinetic parameters using biolayer interferometry suggest HDAC1 is a preferential target compared to other class I HDACs and CDK5. Treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in a decrease of histone acetylation, consistent with an intracellular mechanism of deacetylase activation. Moreover, using tauopathy patient-derived iPSC neuronal models subject to oxidative stress through mitochondrial inhibition exifone treatment was neuroprotective. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.},
	author = {Patnaik, Debasis and Pao, Ping-Chieh and Zhao, Wen-Ning and Silva, M Catarina and Hylton, Norma K and Chindavong, Peter S and Pan, Ling and Tsai, Li-Huei and Haggarty, Stephen J}
}
@article {Chang2020.03.03.975086,
	title = {Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis},
	journal = {bioRxiv},
	year = {2020},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {DNA replication is mediated by the coordinated actions of multiple enzymes within replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving replisomes, the remodeling of ssDNA binding protein (SSB) upon replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action.},
	doi = {10.1101/2020.03.03.975086},
	url = {https://www.biorxiv.org/content/early/2020/03/04/2020.03.03.975086},
	author = {Chang, Seungwoo and Thrall, Elizabeth S. and Laureti, Luisa and Pag{\`e}s, Vincent and Loparo, Joseph J.}
}
@article {1646394,
	title = {Angiotensin and biased analogs induce structurally distinct active conformations within a GPCR},
	journal = {Science},
	volume = {367},
	number = {6480},
	year = {2020},
	month = {2020 02 21},
	pages = {888-892},
	abstract = {Biased agonists of G protein-coupled receptors (GPCRs) preferentially activate a subset of downstream signaling pathways. In this work, we present crystal structures of angiotensin II type 1 receptor (AT1R) (2.7 to 2.9 angstroms) bound to three ligands with divergent bias profiles: the balanced endogenous agonist angiotensin II (AngII) and two strongly β-arrestin-biased analogs. Compared with other ligands, AngII promotes more-substantial rearrangements not only at the bottom of the ligand-binding pocket but also in a key polar network in the receptor core, which forms a sodium-binding site in most GPCRs. Divergences from the family consensus in this region, which appears to act as a biased signaling switch, may predispose the AT1R and certain other GPCRs (such as chemokine receptors) to adopt conformations that are capable of activating β-arrestin but not heterotrimeric Gq protein signaling.},
	keywords = {Angiotensin II, beta-Arrestins, Humans, Ligands, Protein Conformation, Receptor, Angiotensin, Type 1, Signal Transduction},
	issn = {1095-9203},
	doi = {10.1126/science.aay9813},
	author = {Wingler, Laura M and Skiba, Meredith A and McMahon, Conor and Staus, Dean P and Kleinhenz, Alissa L W and Suomivuori, Carl-Mikael and Latorraca, Naomi R and Dror, Ron O and Lefkowitz, Robert J and Kruse, Andrew C}
}
@article {1636636,
	title = {Catalytically inactive T7 DNA polymerase imposes a lethal replication roadblock},
	journal = {J Biol Chem},
	volume = {295},
	number = {28},
	year = {2020},
	month = {2020 07 10},
	pages = {9542-9550},
	abstract = {Bacteriophage T7 encodes its own DNA polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA polymerase of low processivity. However, gp5 becomes highly processive upon formation of a complex with Escherichia coli thioredoxin, the product of the trxA gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the polymerase domain were replaced by alanine is highly toxic to E. coli cells. This toxicity depends on the presence of a functional E. coli trxA allele and T7 RNA polymerase-driven expression but is independent of the exonuclease activity of gp5. In vitro, the purified gp5 variant is devoid of any detectable polymerase activity and inhibited DNA synthesis by the replisomes of E. coli and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA polymerase I containing an engineered gp5 thioredoxin-binding domain did not exhibit toxicity. We conclude that gp5 alleles encoding inactive polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.},
	keywords = {Bacteriophage T7, DNA Replication, DNA, Viral, DNA-Directed DNA Polymerase, Escherichia coli, Escherichia coli Proteins, Protein Domains, Thioredoxins},
	issn = {1083-351X},
	doi = {10.1074/jbc.RA120.013738},
	author = {Hernandez, Alfredo J and Lee, Seung-Joo and Chang, Seungwoo and Lee, Jaehun A and Loparo, Joseph J and Richardson, Charles C}
}
@article {1636630,
	title = {Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy},
	journal = {PLoS Biol},
	volume = {18},
	number = {3},
	year = {2020},
	month = {2020 03},
	pages = {e3000618},
	abstract = {Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.},
	keywords = {Animals, Binding Sites, Botulinum Toxins, Type A, Cell Membrane, Cells, Cultured, Crystallography, X-Ray, Female, Gangliosides, Membrane Lipids, Mice, Muscle, Skeletal, Mutation, Neurons, Paralysis, Protein Engineering, Rats, Transgenic, Recombinant Proteins, Synaptotagmins, Tryptophan},
	issn = {1545-7885},
	doi = {10.1371/journal.pbio.3000618},
	author = {Yin, Linxiang and Masuyer, Geoffrey and Zhang, Sicai and Zhang, Jie and Miyashita, Shin-Ichiro and Burgin, David and Lovelock, Laura and Coker, Shu-Fen and Tian-Min Fu and Stenmark, P{\r a}l and Dong, Min}
}
@article {1636631,
	title = {Ddi1 is a ubiquitin-dependent protease},
	journal = {Proc Natl Acad Sci U S A},
	volume = {117},
	number = {14},
	year = {2020},
	month = {2020 04 07},
	pages = {7776-7781},
	abstract = {The Saccharomyces cerevisiae protein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.},
	keywords = {Molecular Chaperones, Polyubiquitin, Proteasome Endopeptidase Complex, Protein Binding, Protein Domains, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Homology, Ubiquitin, Vesicular Transport Proteins},
	issn = {1091-6490},
	doi = {10.1073/pnas.1902298117},
	author = {Yip, Matthew CJ and Bodnar, Nicholas O and Rapoport, Tom A}
}
@article {1636637,
	title = {The protein kinase Akt acts as a coat adaptor in endocytic recycling},
	journal = {Nat Cell Biol},
	volume = {22},
	number = {8},
	year = {2020},
	month = {2020 08},
	pages = {927-933},
	abstract = {Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.},
	keywords = {Adaptor Proteins, Vesicular Transport, Clathrin, GTPase-Activating Proteins, HEK293 Cells, HeLa Cells, Humans, Integrins, Protein Binding, Proto-Oncogene Proteins c-akt, Receptors, Transferrin},
	issn = {1476-4679},
	doi = {10.1038/s41556-020-0530-z},
	author = {Hsu, Jia-Wei and Bai, Ming and Li, Kunhua and Yang, Jia-Shu and Chu, Nam and Cole, Philip A and Eck, Michael J and Li, Jian and Hsu, Victor W}
}
@article {1636651,
	title = {STING cyclic dinucleotide sensing originated in bacteria},
	journal = {Nature},
	volume = {586},
	number = {7829},
	year = {2020},
	month = {2020 10},
	pages = {429-433},
	abstract = {Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP-AMP signalling during viral infection and anti-tumour immunity1-5. STING shares no structural homology with other known signalling proteins6-9, which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD+ cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIR-STING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGAS-STING pathway in prokaryotic defence against bacteriophages.},
	keywords = {Animals, Bacteria, Bacterial Proteins, Bacteriophages, Crystallography, X-Ray, Cyclic GMP, Evolution, Molecular, Membrane Proteins, Models, Molecular, NAD, Nucleotidyltransferases, Second Messenger Systems},
	issn = {1476-4687},
	doi = {10.1038/s41586-020-2719-5},
	author = {Morehouse, Benjamin R and Govande, Apurva A and Millman, Adi and Keszei, Alexander F A and Lowey, Brianna and Ofir, Gal and Shao, Sichen and Sorek, Rotem and Kranzusch, Philip J}
}
@article {1636638,
	title = {Structural basis for capsid recruitment and coat formation during HSV-1 nuclear egress},
	journal = {Elife},
	volume = {9},
	year = {2020},
	month = {2020 06 24},
	abstract = {During herpesvirus infection, egress of nascent viral capsids from the nucleus is mediated by the viral nuclear egress complex (NEC). NEC deforms the inner nuclear membrane (INM) around the capsid by forming a hexagonal array. However, how the NEC coat interacts with the capsid and how curved coats are generated to enable budding is yet unclear. Here, by structure-guided truncations, confocal microscopy, and cryoelectron tomography, we show that binding of the capsid protein UL25 promotes the formation of NEC pentagons rather than hexagons. We hypothesize that during nuclear budding, binding of UL25 situated at the pentagonal capsid vertices to the NEC at the INM promotes formation of NEC pentagons that would anchor the NEC coat to the capsid. Incorporation of NEC pentagons at the points of contact with the vertices would also promote assembly of the curved hexagonal NEC coat around the capsid, leading to productive egress of UL25-decorated capsids.},
	keywords = {Active Transport, Cell Nucleus, Capsid Proteins, Cloning, Molecular, Cryoelectron Microscopy, Escherichia coli, Gene Expression Regulation, Viral, Herpesvirus 1, Human, Microscopy, Confocal, Protein Transport, Virus Replication},
	issn = {2050-084X},
	doi = {10.7554/eLife.56627},
	author = {Draganova, Elizabeth B and Zhang, Jiayan and Zhou, Z Hong and Heldwein, Ekaterina E}
}
@article {1636627,
	title = {QTY code designed thermostable and water-soluble chimeric chemokine receptors with tunable ligand affinity},
	journal = {Proc Natl Acad Sci U S A},
	volume = {116},
	number = {51},
	year = {2019},
	month = {2019 12 17},
	pages = {25668-25676},
	abstract = {Chemokine receptors are of great interest as they play a critical role in many immunological and pathological processes. The ability to study chemokine receptors in aqueous solution without detergent would be significant because natural receptors require detergents to become soluble. We previously reported using the QTY code to design detergent-free chemokine receptors. We here report the design of 2 detergent-free chimeric chemokine receptors that were experimentally unattainable in detergent solution. We designed chimeric receptors by switching the N terminus and 3 extracellular (EC) loops between different receptors. Specifically, we replaced the N terminus and 3 EC loops of CCR5QTY with the N terminus and 3 EC loops of CXCR4. The ligand for CXCR4; namely CXCL12, binds to the chimeric receptor CCR5QTY (7TM)-CXCR4 (N terminus+3 EC loops), but with lower affinity compared to CXCR4; the CCL5 ligand of CCR5 binds the chimeric receptor with \~{}20{\texttimes} lower affinity. The chimeric design helps to elucidate the mechanism of native receptor-ligand interaction. We also show that all detergent-free QTY-designed chemokine receptors, expressed in Escherichia coli, bind to their respective chemokines with affinities in the nanomolar (nM) range, similar to the affinities of native receptors and SF9-produced QTY variants. These QTY-designed receptors exhibit remarkable thermostability in the presence of arginine and retain ligand-binding activity after heat treatment at 60 {\textdegree}C for 4 h and 24 h, and at 100 {\textdegree}C for 10 min. Our design approach enables affordable scale-up production of detergent-free QTY variant chemokine receptors with tunable functionality for various uses.},
	keywords = {Computational Biology, Humans, Ligands, Models, Molecular, Protein Binding, Protein Engineering, Receptors, Chemokine, Recombinant Proteins, Sequence Alignment, Solubility, Water},
	issn = {1091-6490},
	doi = {10.1073/pnas.1909026116},
	author = {Qing, Rui and Qiuyi Han and Skuhersky, Michael and Chung, Haeyoon and Badr, Myriam and Schubert, Thomas and Zhang, Shuguang}
}
@article {1636626,
	title = {The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA},
	journal = {Proc Natl Acad Sci U S A},
	volume = {116},
	number = {4},
	year = {2019},
	month = {2019 01 22},
	pages = {1394-1403},
	abstract = {The factors and mechanisms that govern tRNA stability in bacteria are not well understood. Here, we investigated the influence of posttranscriptional modification of bacterial tRNAs (tRNA modification) on tRNA stability. We focused on ThiI-generated 4-thiouridine (s4U), a modification found in bacterial and archaeal tRNAs. Comprehensive quantification of Vibrio cholerae tRNAs revealed that the abundance of some tRNAs is decreased in a ΔthiI strain in a stationary phase-specific manner. Multiple mechanisms, including rapid degradation of a subset of hypomodified tRNAs, account for the reduced abundance of tRNAs in the absence of thiI Through transposon insertion sequencing, we identified additional tRNA modifications that promote tRNA stability and bacterial viability. Genetic analysis of suppressor mutants as well as biochemical analyses revealed that rapid degradation of hypomodified tRNA is mediated by the RNA degradosome. Elongation factor Tu seems to compete with the RNA degradosome, protecting aminoacyl tRNAs from decay. Together, our observations describe a previously unrecognized bacterial tRNA quality control system in which hypomodification sensitizes tRNAs to decay mediated by the RNA degradosome.},
	keywords = {Archaea, Bacteria, Endoribonucleases, Kinetics, Multienzyme Complexes, Peptide Elongation Factor Tu, Polyribonucleotide Nucleotidyltransferase, Quality Control, RNA Helicases, RNA Processing, Post-Transcriptional, RNA, Transfer, Thiouridine, Vibrio cholerae},
	issn = {1091-6490},
	doi = {10.1073/pnas.1814130116},
	author = {Kimura, Satoshi and Waldor, Matthew K}
}
@article {1654412,
	title = {Affinity maturation in a human humoral response to influenza hemagglutinin},
	journal = {Proc Natl Acad Sci U S A},
	year = {2019},
	month = {2019 Dec 16},
	abstract = {Affinity maturation of the B cell antigen receptor (BCR) is a conserved and crucial component of the adaptive immune response. BCR lineages, inferred from paired heavy- and light-chain sequences of rearranged Ig genes from multiple descendants of the same naive B cell precursor (the lineages{\textquoteright} unmutated common ancestor, "UCA"), make it possible to reconstruct the underlying somatic evolutionary history. We present here an extensive structural and biophysical analysis of a lineage of BCRs directed against the receptor binding site (RBS) of subtype H1 influenza virus hemagglutinin (HA). The lineage includes 8 antibodies detected directly by sequencing, 3 in 1 principal branch and 5 in the other. When bound to HA, the heavy-chain third complementarity determining region (HCDR3) fits with an invariant pose into the RBS, but in each of the 2 branches, the rest of the Fab reorients specifically, from its position in the HA-bound UCA, about a hinge at the base of HCDR3. New contacts generated by the reorientation compensate for contacts lost as the H1 HA mutated during the time between the donor{\textquoteright}s initial exposure and the vaccination that preceded sampling. Our data indicate that a "pluripotent" naive response differentiated, in each branch, into 1 of its possible alternatives. This property of naive BCRs and persistence of multiple branches of their progeny lineages can offer broader protection from evolving pathogens than can a single, linear pathway of somatic mutation.},
	issn = {1091-6490},
	doi = {10.1073/pnas.1915620116},
	author = {McCarthy, Kevin R and Raymond, Donald D and Do, Khoi T and Schmidt, Aaron G and Harrison, Stephen C}
}
@mastersthesis {JarrettSanchezMario2019SaBM,
	title = {Structural and Biochemical Mechanisms of Notch Signal Activation and Inhibition},
	year = {2019},
	type = {PhD Thesis},
	abstract = {Cell signaling encompasses the fundamental processes which enable an organism to perceive and respond to its environment. Productive cell signaling occurs in a series of tightly regulated steps that require stimulus detection, signal transmission, and a downstream response.},
	author = {Jarrett, Sanchez Mario}
}
@article {1468505,
	title = {Structural Basis for Achieving GSK-3β Inhibition with High Potency, Selectivity, and Brain Exposure for Positron Emission Tomography Imaging and Drug Discovery},
	journal = {J Med Chem},
	volume = {62},
	number = {21},
	year = {2019},
	month = {2019 Nov 14},
	pages = {9600-9617},
	abstract = {Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer{\textquoteright}s disease (AD), suggesting application for these compounds in AD diagnosis and identified [C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3β-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC = 0.030 nM) and selective (>10-fold GSK-3β/GSK-3α) GSK-3β inhibitor known to date. Inhibition of CRMP2 and tau phosphorylation, as well as favorable therapeutic window against WNT/β-catenin signaling activation, was observed in cells.},
	issn = {1520-4804},
	doi = {10.1021/acs.jmedchem.9b01030},
	author = {Bernard-Gauthier, Vadim and Mossine, Andrew V and Knight, Ashley and Patnaik, Debasis and Zhao, Wen-Ning and Cheng, Chialin and Krishnan, Hema S and Xuan, Lucius L and Chindavong, Peter S and Reis, Surya A and Chen, Jinshan Michael and Shao, Xia and Stauff, Jenelle and Arteaga, Janna and Sherman, Phillip and Salem, Nicolas and Bonsall, David and Amaral, Brenda and Varlow, Cassis and Wells, Lisa and Martarello, Laurent and Patel, Shil and Liang, Steven H and Kurumbail, Ravi G and Haggarty, Stephen J and Scott, Peter J H and Vasdev, Neil}
}
@article {1453550,
	title = {E46K-like α-synuclein mutants increase lipid interactions and disrupt membrane selectivity},
	journal = {J Biol Chem},
	volume = {294},
	number = {25},
	year = {2019},
	month = {2019 Jun 21},
	pages = {9799-9812},
	abstract = {Parkinson{\textquoteright}s disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn{\textquoteright}s interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn{\textquoteright}s N-terminal region ( E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA-responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other "synucleinopathies." In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants{\textquoteright} avidity for synaptic vesicle-like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn{\textquoteright}s curvature selectivity. Indiscriminate binding can shift αSyn{\textquoteright}s subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.},
	issn = {1083-351X},
	doi = {10.1074/jbc.RA118.006551},
	author = {Rovere, Matteo and Powers, Alex E and Jiang, Haiyang and Pitino, Julia C and Fonseca-Ornelas, Luis and Patel, Dushyant S and Achille, Alessandro and Langen, Ralf and Varkey, Jobin and Bartels, Tim}
}
@article {1442673,
	title = {Antibodies to a Conserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism},
	journal = {Cell},
	volume = {177},
	number = {5},
	year = {2019},
	month = {2019 May 16},
	pages = {1124-1135.e16},
	abstract = {Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in\&nbsp;vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2019.03.048},
	author = {Watanabe, Akiko and McCarthy, Kevin R and Kuraoka, Masayuki and Schmidt, Aaron G and Adachi, Yu and Onodera, Taishi and Tonouchi, Keisuke and Caradonna, Timothy M and Bajic, Goran and Song, Shengli and McGee, Charles E and Sempowski, Gregory D and Feng, Feng and Urick, Patricia and Kepler, Thomas B and Takahashi, Yoshimasa and Harrison, Stephen C and Kelsoe, Garnett}
}
@article {1455936,
	title = {Bispecific Forkhead Transcription Factor FoxN3 Recognizes Two Distinct Motifs with Different DNA Shapes},
	journal = {Mol Cell},
	volume = {74},
	number = {2},
	year = {2019},
	month = {2019 Apr 18},
	pages = {245-253.e6},
	abstract = {Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition.},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2019.01.019},
	author = {Rogers, Julia M and Waters, Colin T and Seegar, Tom C M and Jarrett, Sanchez M and Hallworth, Amelia N and Blacklow, Stephen C and Bulyk, Martha L}
}
@article {1468730,
	title = {Identification of small molecule inhibitors targeting the Zika virus envelope protein},
	journal = {Antiviral Res},
	volume = {164},
	year = {2019},
	month = {2019 Apr},
	pages = {147-153},
	abstract = {The recent emergence of Zika virus, a mosquito-borne flavivirus, in the Americas has shed light on the severe neurological diseases associated with infection, notably congenital microcephaly in newborns and Guillain-Barr{\'e} syndrome in adults. Despite the recent focus on Zika virus, there are currently no approved vaccines or antiviral therapies available to treat or prevent infection. In this study we established a competitive amplified luminescent proximity homogeneous assay (ALPHAscreen) to identify small molecule inhibitors targeting the envelope protein of Zika virus (Zika E). We utilized this assay to screen two libraries of nearly 27,000 compounds and identified seven novel inhibitors of Zika E. Characterization of these primary screening leads demonstrated that inhibition of Zika virus occurs at non-cytotoxic concentrations for all seven lead compounds. In addition, we found that all seven lead compounds have potent activity against the closely related dengue virus 2 but not vesicular stomatitis virus, an unrelated enveloped virus. Biochemical experiments indicate that these compounds act by preventing E-mediated membrane fusion. This work highlights a new method for the discovery and optimization of direct-acting antivirals targeting the E protein of Zika and other flaviviruses.},
	issn = {1872-9096},
	doi = {10.1016/j.antiviral.2019.02.008},
	author = {Pitts, Jared and Hsia, Chih-Yun and Lian, Wenlong and Wang, Jinhua and Pfeil, Marc-Philipp and Kwiatkowski, Nicholas and Li, Zhengnian and Jang, Jaebong and Gray, Nathanael S and Yang, Priscilla L}
}
@article {1429554,
	title = {Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models},
	journal = {Elife},
	volume = {8},
	year = {2019},
	month = {2019 Mar 25},
	abstract = {Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01-175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4, to trigger tau ubiquitination and proteasomal degradation. QC-01-175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01-175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated -knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.},
	issn = {2050-084X},
	doi = {10.7554/eLife.45457},
	author = {Silva, M Catarina and Ferguson, Fleur M and Cai, Quan and Donovan, Katherine A and Nandi, Ghata and Patnaik, Debasis and Zhang, Tinghu and Huang, Hai-Tsang and Lucente, Diane E and Dickerson, Bradford C and Mitchison, Timothy J. and Fischer, Eric S and Gray, Nathanael S and Haggarty, Stephen J}
}
@article {1468729,
	title = {Small Molecules Targeting the Flavivirus E Protein with Broad-Spectrum Activity and Antiviral Efficacy in Vivo},
	journal = {ACS Infect Dis},
	volume = {5},
	number = {3},
	year = {2019},
	month = {2019 Mar 08},
	pages = {460-472},
	abstract = {Vaccines and antivirals to combat dengue, Zika, and other flavivirus pathogens present a major, unmet medical need. Vaccine development has been severely challenged by the antigenic diversity of these viruses and the propensity of non-neutralizing, cross-reactive antibodies to facilitate cellular infection and increase disease severity. As an alternative, direct-acting antivirals targeting the flavivirus envelope protein, E, have the potential to act via an analogous mode of action without the risk of antibody-dependent enhancement of infection and disease. We previously discovered that structurally diverse small molecule inhibitors of the dengue virus E protein exhibit varying levels of antiviral activity against other flaviviruses in cell culture. Here, we demonstrate that the broad-spectrum activity of several cyanohydrazones against dengue, Zika, and Japanese encephalitis viruses is due to specific inhibition of E-mediated membrane fusion during viral entry and provide proof of concept for pharmacological inhibition of E as an antiviral strategy in vivo.},
	issn = {2373-8227},
	doi = {10.1021/acsinfecdis.8b00322},
	author = {Li, Pi-Chun and Jang, Jaebong and Hsia, Chih-Yun and Groomes, Patrice V and Lian, Wenlong and de Wispelaere, Melissanne and Pitts, Jared D and Wang, Jinhua and Kwiatkowski, Nicholas and Gray, Nathanael S and Yang, Priscilla L}
}
@article {1468731,
	title = {TRAIP is a master regulator of DNA interstrand crosslink repair},
	journal = {Nature},
	volume = {567},
	number = {7747},
	year = {2019},
	month = {2019 03},
	pages = {267-272},
	abstract = {Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.},
	keywords = {Animals, DNA, DNA Helicases, DNA Repair, DNA Replication, Female, Humans, Minichromosome Maintenance Complex Component 7, N-Glycosyl Hydrolases, Protein Binding, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination, Xenopus},
	issn = {1476-4687},
	doi = {10.1038/s41586-019-1002-0},
	author = {Wu, R Alex and Semlow, Daniel R and Kamimae-Lanning, Ashley N and Kochenova, Olga V and Chistol, Gheorghe and Hodskinson, Michael R and Amunugama, Ravindra and Sparks, Justin L and Wang, Meng and Deng, Lin and Mimoso, Claudia A and Low, Emily and Patel, Ketan J and Walter, Johannes C}
}
@article {1455947,
	title = {Structural basis for KCTD-mediated rapid desensitization of GABA signalling},
	journal = {Nature},
	volume = {567},
	number = {7746},
	year = {2019},
	month = {2019 03},
	pages = {127-131},
	abstract = {The GABAB (γ-aminobutyric acid type B) receptor is one of the principal inhibitory neurotransmitter receptors in the brain, and it signals through heterotrimeric G proteins to activate a variety of effectors, including G-protein-coupled inwardly~rectifying potassium channels (GIRKs)1,2. GABAB-receptor signalling is tightly regulated by auxiliary subunits called KCTDs, which control the kinetics of GIRK activation and desensitization3-5. However, the mechanistic basis for KCTD modulation of GABAB signalling remains incompletely understood. Here, using a combination of X-ray crystallography, electron microscopy, and functional and biochemical experiments, we reveal the molecular details of KCTD binding to both GABAB receptors and G-protein βγ subunits. KCTDs associate with the receptor by forming an asymmetric pentameric ring around a region of the receptor carboxy-terminal tail, while a second KCTD domain, H1, engages in a symmetric interaction with five copies of Gβγ in which the G-protein subunits also interact directly with one another. We further show that KCTD binding to Gβγ is highly cooperative, defining a model in which KCTD proteins cooperatively strip G proteins from GIRK channels to induce rapid desensitization following receptor activation. These results provide a framework for understanding the molecular basis for the precise temporal control of GABAB signalling by KCTD proteins.},
	keywords = {Crystallography, X-Ray, G Protein-Coupled Inwardly-Rectifying Potassium Channels, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Humans, Intracellular Signaling Peptides and Proteins, Microscopy, Electron, Models, Biological, Models, Molecular, Nerve Tissue Proteins, Protein Binding, Protein Domains, Proteins, Receptors, GABA-B, Signal Transduction},
	issn = {1476-4687},
	doi = {10.1038/s41586-019-0990-0},
	author = {Zheng, Sanduo and Abreu, Nohely and Levitz, Joshua and Kruse, Andrew C}
}
@article {1453551,
	title = {Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes},
	journal = {Methods Mol Biol},
	volume = {1948},
	year = {2019},
	month = {2019},
	pages = {123-143},
	abstract = {α-Synuclein{\textquoteright}s physiology and pathology have been linked by numerous reports to its ability to bind and remodel membranes, especially at synaptic terminals. It is therefore critical for researchers investigating the determinants of these interactions to rely on methods capable of providing an accurate and complete physicochemical snapshot of the binding events. Circular dichroism (CD) and isothermal titration calorimetry (ITC) are established techniques for the study of binding equilibria in biological systems and, especially when used in combination, allow a thorough characterization of the protein-lipid interplay.Here we provide general guidelines and describe some common pitfalls of these experiments. This protocol describes the preparation of small unilamellar vesicles (SUVs), mimicking the curved bilayers α-synuclein normally interacts with, the CD-monitored titration of α-synuclein with SUVs, the ITC (lipid-into-protein) experiment, and the subsequent data analysis using an n independent binding site model.},
	keywords = {alpha-Synuclein, Calorimetry, Cell Membrane, Circular Dichroism, Data Analysis, Lipid Bilayers, Liposomes, Protein Binding, Temperature},
	issn = {1940-6029},
	doi = {10.1007/978-1-4939-9124-2_11},
	author = {Rovere, Matteo}
}
@mastersthesis {RichesonKatherine2019PEoM,
	title = {Paradoxical Effects on Mitotic Exit Induced by a Small Molecule Inhibitor of APC/CCdc20},
	year = {2019},
	type = {PhD Thesis},
	abstract = {The Anaphase Promoting Complex/Cyclosome (APC/C) is a mega-dalton ubiquitin ligase that regulates chromosome segregation and mitotic exit by targeting destruction box (D-box)-containing substrates for degradation by the proteasome. Because accurate chromosome segregation requires precise timing of APC/C activation, APC/C is subject to highly complex regulation through binding of essential activators and inhibitory protein complexes. The APC/C is activated by Cdc20, which acts as a substrate receptor and is inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated in response to improper kinetochore-microtubule attachments. We previously identified apcin, a small molecule ligand of the D-box receptor of Cdc20, as an inhibitor of APC/C that competitively inhibits substrate binding in vitro. We show that although apcin inhibits substrate degradation and delays mitotic exit in human cells, high endogenous levels of Cdc20 limit apcin{\textquoteright}s effectiveness. Surprisingly, we found that apcin paradoxically accelerates substrate degradation and promotes mitotic exit in cells with high SAC activity. Biochemical studies indicate that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin{\textquoteright}s behavior as an antagonist of Cdc20 can thus result in either net inhibition of APC/C, delaying mitotic exit when SAC activity is low, or net activation of APC/C, promoting mitotic exit when SAC activity is high. Genetic experiments suggest that the dual behaviors of apcin arise from targeting a common binding site in Cdc20, the D-box receptor, which is required for both substrate ubiquitination as well as efficient APC/C inhibition by MCC. Therefore, we establish a new mechanism through which a small molecule, by targeting a single site on a dynamic protein interface, can lead to opposing biological effects depending on the regulatory context.},
	author = {Richeson, Katherine}
}
@mastersthesis {SchmidtHaydenRiley2019SaBI,
	title = {Structural and Biochemical Investigations of Sigma Receptors},
	year = {2019},
	school = {ProQuest Dissertations Publishing},
	type = {PhD Thesis},
	abstract = {The sigma-1 and sigma-2 receptors are a pair of enigmatic membrane proteins that are promising drug targets for the treatment of several conditions. Ligands targeting the sigma-1 receptor may be useful for the treatment of neuropathic pain, ischemic stroke, Alzheimer{\textquoteright}s disease, and other neurodegenerative diseases. Ligands targeting the sigma-2 receptor hold promise for the treatment and imaging of cancer, and for the treatment of the negative symptoms of schizophrenia. Since the discovery of these receptors, myriad small molecules have been identified that bind to these receptors with high affinity. However, despite their pharmacological tractability and therapeutic potential, the basic molecular functions of the sigma receptors remain ambiguous. The work presented in this dissertation makes key advances towards understanding the molecular biology of both sigma-1 and sigma-2 receptors. In this dissertation, I describe the first crystal structures of the human sigma-1 receptor bound to five different small molecule ligands, including classical antagonist and an agonist. These structures provide a model of the sigma-1 receptor{\textquoteright}s structure, reveal how it binds a multitude of small ligands with high (\&lt;100 nM) affinity, and suggests a structural mechanism by which agonists may differ from antagonists. Additionally, a detailed analysis of sigma-1 receptor ligand-binding kinetics provides key insights into receptor-ligand interactions. Notably, this work reveals that the association between the sigma-1 receptor and its ligands is a multi-step process that is rate limited by a conformational change in the receptor prior to ligand binding. Finally, we identified the gene that codes for the sigma-2 receptor as Tmem97, resolving a nearly 40-year old pharmacological mystery. The identification of the sigma-2 receptor as TMEM97 unities two previously independent fields of study, simultaneously providing the sigma-2 receptor field with access to modern molecular biological techniques and the TMEM97 field with a diverse collection of pharmacological tools. Collectively, the work described here represents progress towards a molecular understanding of sigma receptor function, which will be crucial to realizing the full therapeutic potential of these enigmatic receptors.},
	keywords = {Biochemistry, Biophysics, Molecular Biology},
	isbn = {9798684634802},
	author = {Schmidt, Hayden Riley}
}
@article {1429787,
	title = {Structures and gating mechanism of human TRPM2},
	journal = {Science},
	volume = {362},
	number = {6421},
	year = {2018},
	month = {2018 12 21},
	abstract = {Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain responsible for binding adenosine diphosphate (ADP)-ribose (ADPR), and both ADPR and calcium (Ca) are required for TRPM2 activation. Here we report cryo-electron microscopy structures of human TRPM2 alone, with ADPR, and with ADPR and Ca NUDT9H forms both intra- and intersubunit interactions with the N-terminal TRPM homology region (MHR1/2/3) in the apo state but undergoes conformational changes upon ADPR binding, resulting in rotation of MHR1/2 and disruption of the intersubunit interaction. The binding of Ca further engages transmembrane helices and the conserved TRP helix to cause conformational changes at the MHR arm and the lower gating pore to potentiate channel opening. These findings explain the molecular mechanism of concerted TRPM2 gating by ADPR and Ca and provide insights into the gating mechanism of other TRP channels.},
	keywords = {Adenosine Diphosphate Ribose, Calcium, Cryoelectron Microscopy, Humans, Ion Channel Gating, Protein Domains, Protein Multimerization, Pyrophosphatases, TRPM Cation Channels},
	issn = {1095-9203},
	doi = {10.1126/science.aav4809},
	author = {Wang, Longfei and Tian-Min Fu and Zhou, Yiming and Xia, Shiyu and Greka, Anna and Wu, Hao}
}
@article {1455946,
	title = {High resolution X-ray and NMR structural study of human T-cell immunoglobulin and mucin domain containing protein-3},
	journal = {Sci Rep},
	volume = {8},
	number = {1},
	year = {2018},
	month = {2018 Nov 30},
	pages = {17512},
	abstract = {T-cell immunoglobulin and mucin domain containing protein-3 (TIM-3) is an important immune regulator. Here, we describe a novel high resolution (1.7 {\r A}) crystal structure of the human (h)TIM-3 N-terminal variable immunoglobulin (IgV) domain with bound calcium (Ca) that was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Significant conformational differences were observed in the B-C, C{\textquoteright}-C" and C{\textquoteright}-D loops of hTIM-3 compared to mouse (m)TIM-3, hTIM-1 and hTIM-4. Further, the conformation of the C-C{\textquoteright} loop of hTIM-3 was notably different from hTIM-4. Consistent with the known metal ion-dependent binding of phosphatidylserine (PtdSer) to mTIM-3 and mTIM-4, the NMR spectral analysis and crystal structure of Ca-bound hTIM-3 revealed that residues in the hTIM-3 F-G loop coordinate binding to Ca. In addition, we established a novel biochemical assay to define hTIM-3 functionality as determined by binding to human carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1). These studies provide new insights useful for understanding and targeting hTIM-3.},
	issn = {2045-2322},
	doi = {10.1038/s41598-018-35754-0},
	author = {Gandhi, Amit K and Kim, Walter M and Sun, Zhen-Yu J and Huang, Yu-Hwa and Bonsor, Daniel A and Sundberg, Eric J and Kondo, Yasuyuki and Wagner, Gerhard and Kuchroo, Vijay K and Petsko, Gregory and Blumberg, Richard S}
}
@article {1469808,
	title = {Structure-Based Evolution of Low Nanomolar O-GlcNAc Transferase Inhibitors},
	journal = {J Am Chem Soc},
	volume = {140},
	number = {42},
	year = {2018},
	month = {2018 10 24},
	pages = {13542-13545},
	abstract = {Reversible glycosylation of nuclear and cytoplasmic proteins is an important regulatory mechanism across metazoans. One enzyme, O-linked N-acetylglucosamine transferase (OGT), is responsible for all nucleocytoplasmic glycosylation and there is a well-known need for potent, cell-permeable inhibitors to interrogate OGT function. Here we report the structure-based evolution of OGT inhibitors culminating in compounds with low nanomolar inhibitory potency and on-target cellular activity. In addition to disclosing useful OGT inhibitors, the structures we report provide insight into how to inhibit glycosyltransferases, a family of enzymes that has been notoriously refractory to inhibitor development.},
	keywords = {Drug Design, Enzyme Inhibitors, HCT116 Cells, HEK293 Cells, Humans, Molecular Docking Simulation, N-Acetylglucosaminyltransferases, Small Molecule Libraries},
	issn = {1520-5126},
	doi = {10.1021/jacs.8b07328},
	author = {Martin, Sara E S and Tan, Zhi-Wei and Itkonen, Harri M and Duveau, Damien Y and Paulo, Joao A and Janetzko, John and Boutz, Paul L and T{\"o}rk, Lisa and Moss, Frederick A and Thomas, Craig J and Gygi, Steven P and Lazarus, Michael B and Walker, Suzanne}
}
@article {1351038,
	title = {Fusion surface structure, function, and dynamics of gamete fusogen HAP2},
	journal = {Elife},
	volume = {7},
	year = {2018},
	month = {2018 10 03},
	abstract = {HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loops of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the two fusion loops, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.},
	issn = {2050-084X},
	doi = {10.7554/eLife.39772},
	author = {Feng, Juan and Dong, Xianchi and Pinello, Jennifer and Zhang, Jun and Lu, Chafen and Iacob, Roxana E and Engen, John R and Snell, William J and Springer, Timothy A.}
}
@mastersthesis {LoGerfoPhilipJ2019DoaS,
	title = {Development of a Small Molecule Inhibitor of Interleukin-18 for the Prevention of Vein Graft Failure or Other Inflammatory Diseases},
	year = {2018},
	type = {Master{\textquoteright}s Thesis},
	abstract = {Overexpression of Interleukin-18 (IL18), a pleiotropic pro-inflammatory cytokine has been implicated in the early pathogenesis of intimal hyperplasia (IH), the leading cause of delayed graft failure. IL18 Binding Protein (IL18BP) is the natural inhibitor of IL18. Although recombinant IL-18BP is being clinically tested as a potential therapy to treat IL-18 associated conditions, a small molecule inhibitor of IL-18 may be clinically more amenable and feasible as a potential therapy to prevent IH development or other chronic inflammatory diseases.},
	author = {LoGerfo, Philip J}
}
@article {1331355,
	title = {A single XLF dimer bridges DNA ends during nonhomologous end joining},
	journal = {Nat Struct Mol Biol},
	volume = {25},
	number = {9},
	year = {2018},
	month = {2018 Sep},
	pages = {877-884},
	abstract = {Nonhomologous end joining (NHEJ) is the primary pathway of DNA double-strand-break repair in vertebrate cells, yet how NHEJ factors assemble a synaptic complex that bridges DNA ends remains unclear. To address the role of XRCC4-like factor (XLF) in synaptic-complex assembly, we used single-molecule fluorescence imaging in Xenopus laevis egg extract, a system that efficiently joins DNA ends. We found that a single XLF dimer binds DNA substrates just before the formation of a ligation-competent synaptic complex between DNA ends. The interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient formation of this synaptic complex. Our results indicate that, in contrast to a model in which filaments of XLF and XRCC4 bridge DNA ends, binding of a single XLF dimer facilitates the assembly of a stoichiometrically well-defined synaptic complex.},
	issn = {1545-9985},
	doi = {10.1038/s41594-018-0120-y},
	author = {Graham, Thomas G W and Carney, Sean M and Walter, Johannes C and Loparo, Joseph J}
}
@article {1329365,
	title = {The dynamic nature of the conserved tegument protein UL37 of herpesviruses},
	journal = {J Biol Chem},
	year = {2018},
	month = {2018 Aug 30},
	abstract = {In all herpesviruses, the space between the capsid shell and the lipid envelope is occupied by the unique tegument layer composed of proteins that in addition to structural, play many other roles in the viral replication. UL37 is a highly conserved tegument protein that has activities ranging from virion morphogenesis to directional capsid trafficking to manipulation of the host innate immune response and binds multiple partners. The N-terminal half of UL37 (UL37N) has a compact bean-shaped alpha-helical structure that contains a surface region essential for neuroinvasion. However, no biochemical or structural information is currently available for the C-terminal half of UL37 (UL37C) that mediates most of its interactions with multiple binding partners. Here, we show that PRV UL37C is a conformationally flexible monomer composed of an elongated folded core and an unstructured C-tail. This elongated structure, along with that of its binding partner UL36, explains the nature of filamentous tegument structures bridging the capsid and the envelope. We propose that the dynamic nature of UL37 underlies its ability to perform diverse roles during viral replication.},
	issn = {1083-351X},
	doi = {10.1074/jbc.RA118.004481},
	author = {Koenigsberg, Andrea L and Heldwein, Ekaterina}
}
@article {1327860,
	title = {TMC1 Forms the Pore of Mechanosensory Transduction Channels in Vertebrate Inner Ear Hair Cells},
	journal = {Neuron},
	volume = {99},
	number = {4},
	year = {2018},
	month = {2018 Aug 22},
	pages = {736-753.e6},
	abstract = {The proteins that form the permeation pathway of mechanosensory transduction channels in inner-ear\&nbsp;hair cells have not been definitively identified. Genetic, anatomical, and physiological evidence support a role for transmembrane channel-like protein (TMC) 1 in hair cell sensory transduction, yet the molecular function of TMC proteins remains unclear. Here, we provide biochemical evidence suggesting TMC1 assembles as a dimer, along with structural and sequence analyses suggesting similarity to dimeric TMEM16 channels. To identify the pore region of TMC1, we used cysteine mutagenesis and expressed mutant TMC1 in hair cells of Tmc1/2-null mice. Cysteine-modification reagents rapidly and irreversibly altered permeation properties of mechanosensory transduction. We propose that TMC1 is structurally similar to TMEM16 channels and includes ten transmembrane domains with four domains, S4-S7, that line the channel pore. The data provide compelling evidence that TMC1 is a pore-forming component of sensory transduction channels in auditory and vestibular hair cells.},
	issn = {1097-4199},
	doi = {10.1016/j.neuron.2018.07.033},
	author = {Pan, Bifeng and Akyuz, Nurunisa and Liu, Xiao-Ping and Asai, Yukako and Nist-Lund, Carl and Kurima, Kiyoto and Derfler, Bruce H and Gy{\"o}rgy, Bence and Limapichat, Walrati and Walujkar, Sanket and Wimalasena, Lahiru N and Sotomayor, Marcos and Corey, David P. and Holt, Jeffrey R}
}
@article {1327865,
	title = {Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein},
	journal = {Cell Chem Biol},
	volume = {25},
	number = {8},
	year = {2018},
	month = {2018 Aug 16},
	pages = {1006-1016.e8},
	abstract = {Viral envelope proteins are required for productive viral entry and initiation of infection. Although the humoral immune system provides ample evidence for targeting envelope proteins as an antiviral strategy, there are few pharmacological interventions that have this mode of action. In contrast to classical antiviral targets such as viral proteases and polymerases, viral envelope proteins as a class do not have a well-conserved active site that can be rationally targeted with small molecules. We previously identified compounds that inhibit dengue virus by binding to its envelope protein, E. Here, we show that these small molecules inhibit dengue virus fusion and map the binding site of these compounds to a specific pocket on E. We further demonstrate inhibition of Zika, West Nile, and Japanese encephalitis viruses by these compounds, providing pharmacological evidence for the pocket as a target for developing broad-spectrum antivirals against multiple, mosquito-borne flavivirus pathogens.},
	issn = {2451-9448},
	doi = {10.1016/j.chembiol.2018.05.011},
	author = {de Wispelaere, Melissanne and Lian, Wenlong and Potisopon, Supanee and Li, Pi-Chun and Jang, Jaebong and Ficarro, Scott B and Clark, Margaret J and Zhu, Xuling and Kaplan, Jenifer B and Pitts, Jared D and Wales, Thomas E and Wang, Jinhua and Engen, John R and Marto, Jarrod A and Gray, Nathanael S and Yang, Priscilla L}
}
@article {1455945,
	title = {Hydrophobic Residue in Escherichia coli Thioredoxin Critical for the Processivity of T7 DNA Polymerase},
	journal = {Biochemistry},
	volume = {57},
	number = {40},
	year = {2018},
	month = {2018 10 09},
	pages = {5807-5817},
	abstract = {Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to enhance the processivity of its DNA polymerase, a requirement for the growth of phage T7. The evolutionarily conserved structure and high degree of homology of amino acid sequence of the thioredoxin family imply that homologues from other organisms might also interact with T7 DNA polymerase to support the phage growth. Despite the structural resemblance, human thioredoxin, whose X-ray crystallographic structure overlaps with that of the E. coli protein, cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by surface plasmon resonance and thus does not increase the processivity. Homologous scanning analysis using this nonfunctional homologue reveals that the 60 N-terminal and the 12 C-terminal amino acid residues of E. coli thioredoxin can be substituted for its human counterpart without significantly affecting phage growth. Comparison of chimeric thioredoxins, followed by site-directed mutagenesis, identifies leucine 95 as a critical element. This residue may contribute to hydrophobic interactions with the thioredoxin-binding loop of the polymerase; levels of DNA binding and thus nucleotide polymerization are significantly decreased in the absence of this residue. The results suggest that the specific interactions at the interface of thioredoxin and DNA polymerase, rather than the overall structure, are important in the interactions that promote high processivity.},
	keywords = {Bacteriophage T7, DNA-Directed DNA Polymerase, Escherichia coli, Escherichia coli Proteins, Humans, Hydrophobic and Hydrophilic Interactions, Protein Structure, Secondary, Surface Plasmon Resonance, Thioredoxins},
	issn = {1520-4995},
	doi = {10.1021/acs.biochem.8b00341},
	author = {Lee, Seung-Joo and Tran, Ngoc Q and Lee, Joseph and Richardson, Charles C}
}
@article {1327866,
	title = {Discovery of Immunologically Inspired Small Molecules that Target the Viral Envelope Protein},
	journal = {ACS Infect Dis},
	year = {2018},
	month = {2018 Jul 20},
	abstract = {Dengue virus is a major human pathogen that infects over 350 million people annually leading to approximately 500,000 hospitali-zations due to severe dengue. Since the only marketed vaccine, Dengvaxia, has recently been shown to increase disease severity in those lacking natural immunity, antivirals to prevent or treat DENV infection represent a large, unmet medical need. Small mole-cules that target the dengue virus envelope protein, E, on the surface of the virion could act analogously to antibodies by engaging E extracellularly to block infection, but a shortage of target-based assays suitable for screening and medicinal chemistry studies has limited efforts in this area. Here we demonstrate that the dengue envelope (E) protein offers a tractable drug target for preventing dengue infection by developing a target-based assay using a recombinantly expressed dengue serotype 2 E protein. We performed a high-throughput screen of ~20,000 compounds followed by secondary assays to confirm target-binding and antiviral activity and counter-screens to exclude compounds with non-specific activities. These efforts yielded 8 distinct chemical leads that inhibit dengue infection by binding to E and preventing E-mediated membrane fusion with potencies equal to or greater than previously described small molecule inhibitors of E. We show that a subset of these compounds inhibit viruses representative of the other three dengue serotypes and Zika virus. This work provides tools for discovery and optimization of direct-acting antivirals against dengue E and shows that this approach may be useful in developing antivirals with broad-spectrum activity against other flavivirus pathogens.},
	issn = {2373-8227},
	doi = {10.1021/acsinfecdis.8b00127},
	author = {Lian, Wenlong and Jang, Jaebong and Potisopon, Supanee and Li, Pi-Chun and Rahmeh, Amal and Wang, Jinhua and Kwiatkowski, Nicholas P and Gray, Nathanael S and Yang, Priscilla L}
}
@article {1318125,
	title = {NEDD9 targets to promote endothelial fibrosis and pulmonary arterial hypertension},
	journal = {Sci Transl Med},
	volume = {10},
	number = {445},
	year = {2018},
	month = {2018 Jun 13},
	abstract = {Germline mutations involving small mothers against decapentaplegic-transforming growth factor-β (SMAD-TGF-β) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-β ligand trap and, thus, was not contingent on TGF-β signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-β-independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.},
	issn = {1946-6242},
	doi = {10.1126/scitranslmed.aap7294},
	author = {Samokhin, Andriy O and Stephens, Thomas and Wertheim, Bradley M and Wang, Rui-Sheng and Vargas, Sara O and Yung, Lai-Ming and Cao, Minwei and Brown, Marcel and Arons, Elena and Dieffenbach, Paul B and Fewell, Jason G and Matar, Majed and Bowman, Frederick P and Haley, Kathleen J and Alba, George A and Marino, Stefano M and Kumar, Rahul and Rosas, Ivan O and Waxman, Aaron B and Oldham, William M and Khanna, Dinesh and Graham, Brian B and Seo, Sachiko and Gladyshev, Vadim N. and Yu, Paul B and Fredenburgh, Laura E and Loscalzo, Joseph and Leopold, Jane A and Maron, Bradley A}
}
@article {1327863,
	title = {Small-Molecule Inhibitors Disrupt let-7 Oligouridylation and Release the Selective Blockade of let-7 Processing by LIN28},
	journal = {Cell Rep},
	volume = {23},
	number = {10},
	year = {2018},
	month = {2018 Jun 05},
	pages = {3091-3101},
	abstract = {LIN28 is an RNA-binding protein that regulates the maturation of the let-7 family of microRNAs by bipartite interactions with let-7 precursors through its two distinct cold shock and zinc-knuckle domains. Through inhibition of let-7 biogenesis, LIN28 functions as a pluripotency factor, as well as a driver of tumorigenesis. Here, we report a fluorescence polarization assay to identify small-molecule inhibitors for\&nbsp;both domains of LIN28 involved in let-7 interactions. Of 101,017 compounds screened, six inhibit LIN28:let-7 binding and impair LIN28-mediated let-7 oligouridylation. Upon further characterization, we demonstrate that the LIN28 inhibitor TPEN destabilizes the zinc-knuckle domain of LIN28, while LI71 binds the cold shock domain to suppress LIN28{\textquoteright}s activity against let-7 in leukemia cells and embryonic stem cells. Our results demonstrate selective pharmacologic inhibition of individual domains of LIN28 and provide a foundation for therapeutic inhibition of the let-7 biogenesis pathway in LIN28-driven diseases.},
	issn = {2211-1247},
	doi = {10.1016/j.celrep.2018.04.116},
	author = {Wang, Longfei and Rowe, R Grant and Jaimes, Adriana and Yu, Chunxiao and Nam, Yunsun and Pearson, Daniel S and Jin Zhang and Xie, Xiangyu and Marion, William and Heffron, Gregory J and Daley, George Q and Sliz, Piotr}
}
@mastersthesis {DebnathAnik2018ESMf,
	title = {Engineering Symbiotic Microbiota for in Situ Delivery of Therapeutic Proteins},
	year = {2018},
	type = {PhD Thesis},
	abstract = {As symbiotic members of the human microbiome, Lactobacilli are an emerging class of living biotherapeutic agents capable of sustained and targeted drug delivery of protein biologics to the many organs they naturally inhabit. However, progress to this end remains limited by the paucity of genetic tools for manipulating these bacteria. We used synthetic biology techniques to build a tunable, broad host range expression system, and applied in silico techniques for de novo design of robust, strain-optimized secretion signals that direct protein export. Together, these components provide an approach to rapidly engineer niche-adapted Lactobacilli for in situ delivery of therapeutic proteins. We leveraged this platform to engineer model symbionts from the gastrointestinal and female genital tracts to secrete single domain antibody fragments, as candidate vectors for IBD immunotherapy and HIV immunoprophylaxis. This work advances Lactobacillus as a vehicle for drug delivery, and serves as a platform to accelerate development of diverse, microbiome-based medicines capable of addressing myriad disease.},
	author = {Debnath, Anik}
}
@article {1327864,
	title = {Identifying the Protein Interactions of the Cytosolic Iron-Sulfur Cluster Targeting Complex Essential for Its Assembly and Recognition of Apo-Targets},
	journal = {Biochemistry},
	volume = {57},
	number = {16},
	year = {2018},
	month = {2018 04 24},
	pages = {2349-2358},
	abstract = {The cytosolic iron-sulfur cluster assembly (CIA) system assembles iron-sulfur (FeS) cluster cofactors and inserts them into \&gt;20 apoprotein targets residing in the cytosol and nucleus. Three CIA proteins, called Cia1, Cia2, and Met18 in yeast, form the targeting complex responsible for apo-target recognition. There is little information about the structure of this complex or its mechanism of CIA substrate recognition. Herein, we exploit affinity co-purification and size exclusion chromatography to determine the subunit connectivity and stoichiometry of the CIA targeting complex. We conclude that Cia2 is the organizing center of the targeting complex, which contains one Met18, two Cia1, and four Cia2 polypeptides. To probe target recognition specificity, we utilize the CIA substrates Leu1 and Rad3 as well as the Escherichia coli FeS-binding transcription factor FNR (fumerate nitrate reductase). We demonstrate that both of the yeast CIA substrates are recognized, whereas the bacterial protein is not. Thus, while the targeting complex exhibits flexible target recognition in vitro, it cannot promiscuously recognize any FeS protein. Additionally, we demonstrate that the full CIA targeting complex is required to stably bind Leu1 in vitro, whereas the Met18-Cia2 subcomplex is sufficient to recognize Rad3. Together, these results allow us to propose a unifying model for the architecture of this highly conserved complex and demonstrate what component or subcomplexes are vital for target identification.},
	keywords = {Cell Cycle Proteins, Cell Nucleus, Cytosol, DNA Helicases, Hydro-Lyases, Iron-Sulfur Proteins, Protein Binding, Protein Interaction Maps, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors},
	issn = {1520-4995},
	doi = {10.1021/acs.biochem.7b00072},
	author = {Vo, Amanda and Fleischman, Nicholas M and Froehlich, Mary J and Lee, Claudia Y and Cosman, Jessica A and Glynn, Calina A and Hassan, Zanub O and Perlstein, Deborah L}
}
@article {1327862,
	title = {Structural basis for LeishIF4E-1 modulation by an interacting protein in the human parasite Leishmania major},
	journal = {Nucleic Acids Res},
	volume = {46},
	number = {7},
	year = {2018},
	month = {2018 Apr 20},
	pages = {3791-3801},
	abstract = {Leishmania parasites are unicellular pathogens that are transmitted to humans through the bite of infected sandflies. Most of the regulation of their gene expression occurs post-transcriptionally, and the different patterns of gene expression required throughout the parasites{\textquoteright} life cycle are regulated at the level of translation. Here, we report the X-ray crystal structure of the Leishmania cap-binding isoform 1, LeishIF4E-1, bound to a protein fragment of previously unknown function, Leish4E-IP1, that binds tightly to LeishIF4E-1. The molecular structure, coupled to NMR spectroscopy experiments and in vitro cap-binding assays, reveal that Leish4E-IP1 allosterically destabilizes the binding of LeishIF4E-1 to the 5{\textquoteright} mRNA cap. We propose mechanisms through which Leish4E-IP1-mediated LeishIF4E-1 inhibition could regulate translation initiation in the human parasite.},
	issn = {1362-4962},
	doi = {10.1093/nar/gky194},
	author = {Meleppattu, Shimi and Arthanari, Haribabu and Zinoviev, Alexandra and Boeszoermenyi, Andras and Wagner, Gerhard and Shapira, Michal and L{\'e}ger-Abraham, M{\'e}lissa}
}
@article {1322671,
	title = {Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis},
	journal = {Nature},
	volume = {556},
	number = {7699},
	year = {2018},
	month = {2018 04 05},
	pages = {118-121},
	abstract = {The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 {\r A}, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.},
	keywords = {Bacillus subtilis, Biocatalysis, Cell Wall, Crystallography, X-Ray, Escherichia coli, Models, Molecular, Nucleotidyltransferases, Peptidoglycan, Protein Domains, Protein Folding, Structure-Activity Relationship, Thermus thermophilus},
	issn = {1476-4687},
	doi = {10.1038/nature25985},
	author = {Sjodt, Megan and Brock, Kelly and Dobihal, Genevieve and Rohs, Patricia D A and Green, Anna G and Hopf, Thomas A and Meeske, Alexander J and Srisuknimit, Veerasak and Kahne, Daniel and Walker, Suzanne and Marks, Debora S and Bernhardt, Thomas G and Rudner, David Z and Kruse, Andrew C}
}
@article {1328331,
	title = {Refolding of helical soluble α-synuclein through transient interaction with lipid interfaces},
	journal = {FEBS Lett},
	volume = {592},
	number = {9},
	year = {2018},
	month = {2018 May},
	pages = {1464-1472},
	abstract = {α-Synuclein (αSyn) is a key player in the pathogenesis of Parkinson{\textquoteright}s disease and other synucleinopathies. Here, we report the existence of a novel soluble α-helical conformer of αSyn, obtained through transient interaction with lipid interfaces, and propose dynamic oligomerization as the mechanism underlying its stability. The conformational space of αSyn appears to be highly context-dependent, and lipid bilayers might thus play crucial roles as molecular chaperones in a cellular environment.},
	issn = {1873-3468},
	doi = {10.1002/1873-3468.13047},
	author = {Rovere, Matteo and Sanderson, John B and Fonseca-Ornelas, Luis and Patel, Dushyant S and Bartels, Tim}
}
@article {1318963,
	title = {O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix},
	journal = {J Am Chem Soc},
	volume = {140},
	number = {10},
	year = {2018},
	month = {2018 03 14},
	pages = {3510-3513},
	abstract = {The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously assess OGT activity across \&gt;6000 human proteins. With this assay we examined the contribution to substrate selection of a conserved asparagine ladder within the lumen of OGT{\textquoteright}s superhelical tetratricopeptide repeat (TPR) domain. When five asparagines were mutated, OGT retained significant activity against short peptides, but showed limited limited glycosylation of protein substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes the majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain.},
	keywords = {Asparagine, Humans, N-Acetylglucosaminyltransferases, Protein Array Analysis, Proteins, Substrate Specificity, Tetratricopeptide Repeat},
	issn = {1520-5126},
	doi = {10.1021/jacs.7b13546},
	author = {Levine, Zebulon G and Fan, Chenguang and Melicher, Michael S and Orman, Marina and Benjamin, Tania and Walker, Suzanne}
}
@article {1328332,
	title = {Substrate binding to BamD triggers a conformational change in BamA to control membrane insertion},
	journal = {Proc Natl Acad Sci U S A},
	volume = {115},
	number = {10},
	year = {2018},
	month = {2018 03 06},
	pages = {2359-2364},
	abstract = {The β-barrel assembly machine (Bam) complex folds and inserts integral membrane proteins into the outer membrane of Gram-negative bacteria. The two essential components of the complex, BamA and BamD, both interact with substrates, but how the two coordinate with each other during assembly is not clear. To elucidate aspects of this process we slowed the assembly of an essential β-barrel substrate of the Bam complex, LptD, by changing a conserved residue near the C terminus. This defective substrate is recruited to the Bam complex via BamD but is unable to integrate into the membrane efficiently. Changes in the extracellular loops of BamA partially restore assembly kinetics, implying that BamA fails to engage this defective substrate. We conclude that substrate binding to BamD activates BamA by regulating extracellular loop interactions for folding and membrane integration.},
	keywords = {Bacterial Outer Membrane Proteins, Escherichia coli Proteins, Kinetics, Models, Molecular, Periplasm, Protein Binding, Protein Conformation, Protein Folding},
	issn = {1091-6490},
	doi = {10.1073/pnas.1711727115},
	author = {Lee, James and Sutterlin, Holly A and Wzorek, Joseph S and Mandler, Michael D and Hagan, Christine L and Grabowicz, Marcin and Tomasek, David and May, Mary D and Hart, Elizabeth M and Silhavy, Thomas J and Kahne, Daniel}
}
@article {1318962,
	title = {Evolution of sequence-defined highly functionalized nucleic acid polymers},
	journal = {Nat Chem},
	volume = {10},
	number = {4},
	year = {2018},
	month = {2018 Apr},
	pages = {420-427},
	abstract = {The evolution of sequence-defined synthetic polymers made of building blocks beyond those compatible with polymerase enzymes or the ribosome has the potential to generate new classes of receptors, catalysts and materials. Here we describe a ligase-mediated DNA-templated polymerization and in vitro selection\&nbsp;system to evolve highly functionalized nucleic acid polymers (HFNAPs) made from 32 building blocks that contain eight chemically diverse side chains on a DNA backbone. Through iterated cycles of polymer translation, selection and reverse translation, we discovered HFNAPs that bind proprotein convertase subtilisin/kexin type 9 (PCSK9) and interleukin-6, two protein targets implicated in human diseases. Mutation and reselection of an active PCSK9-binding polymer yielded evolved polymers with high affinity (K = 3 nM). This evolved polymer potently inhibited the binding between PCSK9 and the low-density lipoprotein receptor. Structure-activity relationship studies revealed that specific side chains at defined positions in the polymers are required for binding to their respective targets. Our findings expand the chemical space of evolvable polymers to include densely functionalized nucleic acids with diverse, researcher-defined chemical repertoires.},
	issn = {1755-4349},
	doi = {10.1038/s41557-018-0008-9},
	author = {Zhen Chen and Lichtor, Phillip A and Berliner, Adrian P and Chen, Jonathan C and Liu, David R}
}
@article {1318964,
	title = {Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires},
	journal = {Immunity},
	volume = {48},
	number = {1},
	year = {2018},
	month = {2018 Jan 16},
	pages = {174-184.e9},
	abstract = {Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.},
	issn = {1097-4180},
	doi = {10.1016/j.immuni.2017.12.009},
	author = {McCarthy, Kevin R and Watanabe, Akiko and Kuraoka, Masayuki and Do, Khoi T and McGee, Charles E and Sempowski, Gregory D and Kepler, Thomas B and Schmidt, Aaron G and Kelsoe, Garnett and Harrison, Stephen C}
}
@article {1327859,
	title = {Conserved epitope on influenza-virus hemagglutinin head defined by a vaccine-induced antibody},
	journal = {Proc Natl Acad Sci U S A},
	volume = {115},
	number = {1},
	year = {2018},
	month = {2018 01 02},
	pages = {168-173},
	abstract = {Circulating influenza viruses evade neutralization in their human hosts by acquiring escape mutations at epitopes of prevalent antibodies. A goal for next-generation influenza vaccines is to reduce escape likelihood by selectively eliciting antibodies recognizing conserved surfaces on the viral hemagglutinin (HA). The receptor-binding site (RBS) on the HA "head" and a region near the fusion peptide on the HA "stem" are two such sites. We describe here a human antibody clonal lineage, designated CL6649, members of which bind a third conserved site ("lateral patch") on the side of the H1-subtype, HA head. A crystal structure of HA with bound Fab6649 shows the conserved antibody footprint. The site was invariant in isolates from 1977 (seasonal) to 2012 (pdm2009); antibodies in CL6649 recognize HAs from the entire period. In 2013, human H1 viruses acquired mutations in this epitope that were retained in subsequent seasons, prompting modification of the H1 vaccine component in 2017. The mutations inhibit Fab6649 binding. We infer from the rapid spread of these mutations in circulating H1 influenza viruses that the previously subdominant, conserved lateral patch had become immunodominant for individuals with B-cell memory imprinted by earlier H1 exposure. We suggest that introduction of the pdm2009 H1 virus, to which most of the broadly prevalent, neutralizing antibodies did not bind, conferred a selective advantage in the immune systems of infected hosts to recall of memory B cells that recognized the lateral patch, the principal exposed epitope that did not change when pdm2009 displaced previous seasonal H1 viruses.},
	keywords = {Antibodies, Monoclonal, Antibodies, Viral, Epitopes, Hemagglutinin Glycoproteins, Influenza Virus, Humans, Immunologic Memory, Influenza Vaccines},
	issn = {1091-6490},
	doi = {10.1073/pnas.1715471115},
	author = {Raymond, Donald D and Bajic, Goran and Ferdman, Jack and Suphaphiphat, Pirada and Settembre, Ethan C and Moody, M Anthony and Schmidt, Aaron G and Harrison, Stephen C}
}
@article {1636625,
	title = {Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen},
	journal = {Proc Natl Acad Sci U S A},
	volume = {115},
	number = {40},
	year = {2018},
	month = {2018 10 02},
	pages = {10124-10129},
	abstract = {The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified Amycolatopsis sp. AA4 as the producing strain and Streptomyces coelicolor M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Staphylococcus aureus Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces S. aureus infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.},
	keywords = {Anthraquinones, Anti-Bacterial Agents, DNA, Bacterial, DNA, Ribosomal, Microbial Sensitivity Tests, RNA, Ribosomal, 16S, Streptomyces coelicolor},
	issn = {1091-6490},
	doi = {10.1073/pnas.1807613115},
	author = {Pishchany, Gleb and Mevers, Emily and Ndousse-Fetter, Sula and Horvath, Dennis J and Paludo, Camila R and Silva-Junior, Eduardo A and Koren, Sergey and Skaar, Eric P and Clardy, Jon and Kolter, Roberto}
}
@article {1636650,
	title = {Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch},
	journal = {Cell},
	volume = {173},
	number = {2},
	year = {2018},
	month = {2018 04 05},
	pages = {430-442.e17},
	abstract = {Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2β2) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT\&amp;RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our\&nbsp;findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.},
	keywords = {Base Sequence, beta-Globins, beta-Thalassemia, Binding Sites, Carrier Proteins, Cell Line, Chromatin, Clustered Regularly Interspaced Short Palindromic Repeats, Erythroid Cells, Fetal Hemoglobin, gamma-Globins, Gene Editing, Humans, Nuclear Proteins, Promoter Regions, Genetic, Protein Isoforms, Repressor Proteins, Zinc Fingers},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2018.03.016},
	author = {Liu, Nan and Hargreaves, Victoria V and Zhu, Qian and Kurland, Jesse V and Hong, Jiyoung and Kim, Woo Jin and Sher, Falak and Macias-Trevino, Claudio and Rogers, Julia M and Kurita, Ryo and Nakamura, Yukio and Yuan, Guo-Cheng and Bauer, Daniel E and Xu, Jian and Bulyk, Martha L and Orkin, Stuart H}
}
@mastersthesis {ChenZhen2018EoDF,
	title = {Evolution of Diversely Functionalized Nucleic Acid Polymers},
	year = {2018},
	type = {PhD Thesis},
	abstract = {Darwinian evolution gave rise to biopolymers with a myriad of folded structures and functions, as well as all the diverse life forms on earth that are supported by those molecules. Amazingly, the essence of molecular evolution can be recapitulated in a test tube with purely biochemical manipulations outside of living cells. For nearly three decades, researchers have been evolving biopolymers {\textendash} and even biomimetic polymers that are not produced in any living system {\textendash} by subjecting diverse libraries of polymers to iterated cycles of templated synthesis, functional selection, and amplification. In principle, any class of sequence-defined polymer whose synthesis can be templated by an amplifiable genetic material (such as DNA) is evolvable in this manner. To date, however, the evolution of polymers made of building blocks beyond those compatible with polymerase enzymes or the ribosome has not been demonstrated. To explore new kinds of evolvable sequence-defined polymers and discover new classes of receptors, catalysts, and materials, we used a ligase-mediated DNA-templated polymerization system and in vitro selection to evolve highly functionalized nucleic acid polymers (HFNAPs) made from 32 building blocks containing eight chemically diverse side-chains on a DNA backbone. Through iterated cycles of polymer translation, selection, and reverse translation, we discovered HFNAPs that bind PCSK9 and IL-6, two protein targets implicated in human diseases. Mutation and reselection of an active PCSK9-binding polymer yielded evolved polymers with high affinity (KD = 3 nM). This evolved polymer potently inhibited binding between PCSK9 and the LDL receptor. Structure-activity relationship studies revealed that specific side-chains at defined positions in the polymers are required for binding to their respective targets. Our findings expand the chemical space of evolvable polymers to include densely functionalized nucleic acids with diverse, researcher-defined chemical repertoires. SELEX; Sequence-defined synthetic polymer},
	keywords = {Chemistry, General},
	author = {Zhen Chen}
}
@article {1636621,
	title = {Characterization of TauC3 antibody and demonstration of its potential to block tau propagation},
	journal = {PLoS One},
	volume = {12},
	number = {5},
	year = {2017},
	month = {2017},
	pages = {e0177914},
	abstract = {The spread of neurofibrillary tangle (NFT) pathology through the human brain is a hallmark of Alzheimer{\textquoteright}s disease (AD), which is thought to be caused by the propagation of "seeding" competent soluble misfolded tau. "TauC3", a C-terminally truncated form of tau that is generated by caspase-3 cleavage at D421, has previously been observed in NFTs and has been implicated in tau toxicity. Here we show that TauC3 is found in the seeding competent high molecular weight (HMW) protein fraction of human AD brain. Using a specific TauC3 antibody, we were able to substantially block the HMW tau seeding activity of human AD brain extracts in an in vitro tau seeding FRET assay. We propose that TauC3 could contribute to the templated tau misfolding that leads to NFT spread in AD brains.},
	keywords = {Aged, 80 and over, Alzheimer Disease, Antibodies, Antibody Specificity, Brain, Caspase 3, Cell Line, Female, Humans, Male, Middle Aged, Neurofibrillary Tangles, Protein Folding, tau Proteins},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0177914},
	author = {Nicholls, Samantha B and DeVos, Sarah L and Commins, Caitlin and Nobuhara, Chloe and Bennett, Rachel E and Corjuc, Diana L and Maury, Eduardo and Eftekharzadeh, Bahareh and Akingbade, Ololade and Fan, Zhanyun and Roe, Allyson D and Takeda, Shuko and Wegmann, Susanne and Hyman, Bradley T}
}
@article {1636622,
	title = {Identification of the gene that codes for the σ receptor},
	journal = {Proc Natl Acad Sci U S A},
	volume = {114},
	number = {27},
	year = {2017},
	month = {2017 07 03},
	pages = {7160-7165},
	abstract = {The σ2 receptor is an enigmatic protein that has attracted significant attention because of its involvement in diseases as diverse as cancer and neurological disorders. Unlike virtually all other receptors of medical interest, it has eluded molecular cloning since its discovery, and the gene that codes for the receptor remains unknown, precluding the use of modern biological methods to study its function. Using a chemical biology approach, we purified the σ2 receptor from tissue, revealing its identity as TMEM97, an endoplasmic reticulum-resident transmembrane protein that regulates the sterol transporter NPC1. We show that TMEM97 possesses the full suite of molecular properties that define the σ2 receptor, and we identify Asp29 and Asp56 as essential for ligand recognition. Cloning the σ2 receptor resolves a longstanding mystery and will enable therapeutic targeting of this potential drug target.},
	keywords = {Alzheimer Disease, Animals, Aspartic Acid, Carrier Proteins, Cattle, Cholesterol, Endoplasmic Reticulum, Gene Expression Regulation, Humans, Insecta, Intracellular Signaling Peptides and Proteins, Ligands, Liver, MCF-7 Cells, Membrane Glycoproteins, Membrane Proteins, Niemann-Pick C1 Protein, PC12 Cells, Protein Binding, Rats, Receptors, sigma, Recombinant Proteins, RNA, Small Interfering, Schizophrenia},
	issn = {1091-6490},
	doi = {10.1073/pnas.1705154114},
	author = {Alon, Assaf and Schmidt, Hayden R and Wood, Michael D and Sahn, James J and Martin, Stephen F and Kruse, Andrew C}
}
@mastersthesis {EscuderoSilvia2017DRoM,
	title = {Direct Regulation of Mitochondrial Fatty Acid Oxidation by Anti-Apoptotic MCL-1},
	year = {2017},
	type = {PhD Thesis},
	abstract = {Apoptosis is a form of programmed cell death essential to normal development and tissue homeostasis. BCL-2 family proteins are key regulators of the apoptotic pathway and control the life-death decision rendered by cells in response to stress. Localized mainly at the outer mitochondrial membrane, BCL-2 family proteins engage in a variety of heterodimeric and homooligomeric protein interactions, mediated by their conserved BCL-2 Homology (BH) domains, to determine cell fate. MCL-1 is a member of the BCL-2 family and regulates cell death by exerting potent anti-apoptotic activity. At the mitochondria, MCL-1 neutralizes pro-apoptotic BCL-2 family members, including NOXA, PUMA, BIM, and BAK, such that its pathologic overexpression contributes to the development and chemoresistance of many human cancers. Emerging data underscore that MCL-1 has unique attributes among BCL-2 anti-apoptotic proteins with respect to both amino acid composition and function. For example, unlike its anti-apoptotic homologues, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in organism homeostasis. Thus, to explore novel mechanistic role(s) of MCL-1, we undertook an exploratory proteomic study to uncover potential targets of the MCL-1 BH3 domain. Here, we report that MCL-1, and specifically its mitochondrial matrix isoform, directly engages very long chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid β-oxidation pathway. The interaction is mediated by the α-helical MCL-1 BH3 domain, which binds VLCAD at a surface groove adjacent to the enzyme active site. Deletion of endogenous Mcl-1 impairs fatty acid β-oxidation, resulting in selective accumulation of long-chain fatty acylcarnitines, as seen in VLCAD-deficient patients. Our identification of a BH3-mediated interaction between MCL-1 and VLCAD reveals a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.},
	author = {Escudero, Silvia}
}
@article {1170231,
	title = {Targeted Nanotherapeutics Encapsulating Liver X Receptor Agonist GW3965 Enhance Antiatherogenic Effects without Adverse Effects on Hepatic Lipid Metabolism in Ldlr Mice},
	journal = {Adv Healthc Mater},
	volume = {6},
	number = {20},
	year = {2017},
	month = {2017 Oct},
	abstract = {The pharmacological manipulation of liver X receptors (LXRs) has been an attractive therapeutic strategy for atherosclerosis treatment as they control reverse cholesterol transport and inflammatory response. This study presents the development and efficacy of nanoparticles (NPs) incorporating the synthetic LXR agonist GW3965 (GW) in targeting atherosclerotic lesions. Collagen IV (Col IV) targeting ligands are employed to functionalize the NPs to improve targeting to the atherosclerotic plaque, and formulation parameters such as the length of the polyethylene glycol (PEG) coating molecules are systematically optimized. In vitro studies indicate that the GW-encapsulated NPs upregulate the LXR target genes and downregulate proinflammatory mediator in macrophages. The Col IV-targeted NPs encapsulating GW (Col IV-GW-NPs) successfully reaches atherosclerotic lesions when administered for 5 weeks to mice with preexisting lesions, substantially reducing macrophage content (≈30\%) compared to the PBS group, which is with greater efficacy versus nontargeting NPs encapsulating GW (GW-NPs) (≈18\%). In addition, mice administered the Col IV-GW-NPs do not demonstrate increased hepatic lipid biosynthesis or hyperlipidemia during the treatment period, unlike mice injected with the free GW. These findings suggest a new form of LXR-based therapeutics capable of enhanced delivery of the LXR agonist to atherosclerotic lesions without altering hepatic lipid metabolism.},
	keywords = {Animals, Atherosclerosis, Benzoates, Benzylamines, Cells, Cultured, Cholesterol, Collagen Type IV, Disease Models, Animal, Drug Carriers, Lipid Metabolism, Liver, Liver X Receptors, Macrophages, Mice, Mice, Inbred C57BL, Mice, Knockout, Nanomedicine, Nanoparticles, Polyethylene Glycols, Receptors, LDL, Triglycerides},
	issn = {2192-2659},
	doi = {10.1002/adhm.201700313},
	author = {Yu, Mikyung and Amengual, Jaume and Menon, Arjun and Kamaly, Nazila and Zhou, Felix and Xu, Xiaoding and Saw, Phei Er and Lee, Seung-Joo and Si, Kevin and Ortega, Carleena Angelica and Choi, Won Il and Lee, In-Hyun and Bdour, Yazan and Shi, Jinjun and Mahmoudi, Morteza and Jon, Sangyong and Fisher, Edward A and Farokhzad, Omid C}
}
@article {1245906,
	title = {The Kinetochore Receptor for the Cohesin Loading Complex},
	journal = {Cell},
	volume = {171},
	number = {1},
	year = {2017},
	month = {2017 Sep 21},
	pages = {72-84.e13},
	abstract = {The ring-shaped cohesin complex brings together distant DNA domains to maintain, express, and segregate the genome. Establishing specific chromosomal linkages depends on cohesin recruitment to defined loci. One such locus is the budding yeast centromere, which is a paradigm for targeted cohesin loading. The kinetochore, a multiprotein complex that connects centromeres to microtubules, drives the recruitment of high levels of cohesin to link sister chromatids together. We have exploited this system to determine the mechanism of specific cohesin recruitment. We show that phosphorylation of the Ctf19 kinetochore protein by a conserved kinase, DDK, provides a binding site for the Scc2/4 cohesin loading complex, thereby directing cohesin loading to centromeres. A similar mechanism targets cohesin to chromosomes in vertebrates. These findings represent a complete molecular description of targeted cohesin loading, a phenomenon with wide-ranging importance in chromosome segregation and, in multicellular organisms, transcription regulation.},
	keywords = {Cell Cycle Proteins, Centromere, Chromosomal Proteins, Non-Histone, Cytoskeletal Proteins, Kinetochores, Multiprotein Complexes, Phosphorylation, Phylogeny, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, X-Ray Diffraction},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2017.08.017},
	author = {Hinshaw, Stephen M and Makrantoni, Vasso and Harrison, Stephen C and Marston, Ad{\`e}le L}
}
@article {1322667,
	title = {Binding of NAD-Glycohydrolase to Streptolysin O Stabilizes Both Toxins and Promotes Virulence of Group A },
	journal = {mBio},
	volume = {8},
	number = {5},
	year = {2017},
	month = {2017 09 12},
	abstract = {The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD+-glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations in nga, the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence.IMPORTANCE The global increase in invasive GAS infections in the 1980s was associated with the emergence of an M1T1 clone that harbors a 36-kb pathogenicity island, which codes for increased expression of toxins SLO and NADase. Polymorphisms in NADase that render it catalytically inactive can be detected in clinical isolates, including invasive strains. However, such isolates continue to produce full-length NADase. The rationale for this observation is not completely understood. This study characterizes the binding interaction between NADase and SLO and reports that the expression of each toxin is crucial for maximal expression and stability of the other. By this mechanism, the presence of both toxins increases toxicity to keratinocytes and is predicted to enhance GAS survival in the human host. These observations provide an explanation for conservation of full-length NADase expression even when it lacks enzymatic activity and suggest a critical role for binding of NADase to SLO in GAS pathogenesis.},
	keywords = {Bacterial Proteins, Cells, Cultured, Culture Media, Cytotoxins, Humans, Keratinocytes, NAD+ Nucleosidase, Operon, Point Mutation, Protein Binding, Protein Stability, Streptococcal Infections, Streptococcus pyogenes, Streptolysins, Virulence, Virulence Factors},
	issn = {2150-7511},
	doi = {10.1128/mBio.01382-17},
	author = {Velarde, Jorge J and O{\textquoteright}Seaghdha, Maghnus and Baddal, Buket and Bastiat-Sempe, Benedicte and Wessels, Michael R}
}
@article {1170241,
	title = {A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase Activity},
	journal = {Dev Cell},
	volume = {42},
	number = {1},
	year = {2017},
	month = {2017 07 10},
	pages = {37-51.e8},
	abstract = {Kinesin-8 motors regulate the size of microtubule structures, using length-dependent accumulation at the plus end to preferentially disassemble long microtubules. Despite extensive study, the kinesin-8 depolymerase mechanism remains under debate. Here, we provide evidence for an alternative, tubulin curvature-sensing model of microtubule depolymerization by the budding yeast kinesin-8, Kip3. Kinesin-8/Kip3 uses ATP hydrolysis, like other kinesins, for stepping on the microtubule lattice, but at the plus end Kip3 undergoes a switch: its ATPase activity is~suppressed when it binds tightly to the curved conformation of tubulin. This prolongs plus-end binding, stabilizes protofilament curvature, and ultimately promotes microtubule disassembly. The tubulin curvature-sensing model is supported by our identification of Kip3 structural elements necessary and sufficient for plus-end binding and depolymerase activity, as well as by the identification of an α-tubulin residue specifically required for the Kip3-curved tubulin interaction. Together, these findings elucidate a major regulatory mechanism controlling the size of cellular microtubule structures.},
	keywords = {Adenosine Triphosphate, Animals, Biocatalysis, Hydrolysis, Kinesin, Microtubules, Models, Biological, Models, Molecular, Mutant Proteins, Polymerization, Protein Binding, Protein Structure, Secondary, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sus scrofa, Tubulin},
	issn = {1878-1551},
	doi = {10.1016/j.devcel.2017.06.011},
	author = {Arellano-Santoyo, Hugo and Geyer, Elisabeth A and Stokasimov, Ema and Chen, Geng-Yuan and Su, Xiaolei and Hancock, William and Rice, Luke M and Pellman, David}
}
@article {1178826,
	title = {Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins},
	journal = {J Biol Chem},
	volume = {292},
	number = {19},
	year = {2017},
	month = {2017 05 12},
	pages = {8007-8018},
	abstract = {The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.},
	keywords = {Adenosine Triphosphatases, Ascomycota, Crystallography, X-Ray, Cytosol, Fungal Proteins, HSP70 Heat-Shock Proteins, Mutation, Peptides, Protein Binding, Protein Domains, Protein Processing, Post-Translational, Protein Transport, Ribosomes, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins},
	issn = {1083-351X},
	doi = {10.1074/jbc.M116.761122},
	author = {Tripathi, Arati and Mandon, Elisabet C and Gilmore, Reid and Rapoport, Tom A}
}
@article {1103081,
	title = {A network of cis and trans interactions is required for ParB spreading},
	journal = {Nucleic Acids Res},
	volume = {45},
	number = {12},
	year = {2017},
	month = {2017 Jul 07},
	pages = {7106-7117},
	abstract = {Most bacteria utilize the highly conserved parABS partitioning system in plasmid and chromosome segregation. This system depends on a DNA-binding protein ParB, which binds specifically to the centromere DNA sequence parS and to adjacent non-specific DNA over multiple kilobases in a phenomenon called spreading. Previous single-molecule experiments in combination with genetic, biochemical and computational studies have argued that ParB spreading requires cooperative interactions between ParB dimers including DNA bridging and possible nearest-neighbor interactions. A recent structure of a ParB homolog co-crystallized with parS revealed that ParB dimers tetramerize to form a higher order nucleoprotein complex. Using this structure as a guide, we systematically ablated a series of proposed intermolecular interactions in the Bacillus subtilis ParB (BsSpo0J) and characterized their effect on spreading using both in vivo and in vitro assays. In particular, we measured DNA compaction mediated by BsSpo0J using a recently developed single-molecule method to simultaneously visualize protein binding on single DNA molecules and changes in DNA conformation without protein labeling. Our results indicate that residues acting as hubs for multiple interactions frequently led to the most severe spreading defects when mutated, and that a network of both cis and trans interactions between ParB dimers is necessary for spreading.},
	keywords = {Amino Acid Motifs, Bacillus subtilis, Bacterial Proteins, Binding Sites, Centromere, Chromosome Segregation, Chromosomes, Bacterial, Cloning, Molecular, Crystallography, X-Ray, DNA, Bacterial, Escherichia coli, Gene Expression, Kinetics, Models, Molecular, Plasmids, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Recombinant Proteins, Substrate Specificity},
	issn = {1362-4962},
	doi = {10.1093/nar/gkx271},
	author = {Song, Dan and Rodrigues, Kristen and Graham, Thomas G W and Loparo, Joseph J}
}
@article {1066321,
	title = {Genome-wide identification and characterization of Notch transcription complex-binding sequence-paired sites in leukemia cells},
	journal = {Sci Signal},
	volume = {10},
	number = {477},
	year = {2017},
	month = {2017 May 02},
	abstract = {Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer-binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and have been linked to the Notch responsiveness of a few genes. To assess the overall contribution of SPSs to Notch-dependent gene regulation, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay and applied insights from these in vitro studies to Notch-"addicted" T cell acute lymphoblastic leukemia (T-ALL) cells. We found that SPSs contributed to the regulation of about a third of direct Notch target genes. Although originally described in promoters, SPSs are present mainly in long-range enhancers, including an enhancer containing a newly described SPS that regulates  expression. Our work provides a general method for identifying SPSs in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells.},
	keywords = {Animals, Enhancer Elements, Genetic, Gene Expression Profiling, Gene Expression Regulation, Leukemic, Genome, Human, Humans, Mice, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma, Promoter Regions, Genetic, Protein Binding, Receptors, Notch, Signal Transduction, Transcription Factors, Tumor Cells, Cultured},
	issn = {1937-9145},
	doi = {10.1126/scisignal.aag1598},
	author = {Severson, Eric and Arnett, Kelly L and Wang, Hongfang and Zang, Chongzhi and Taing, Len and Liu, Hudan and Pear, Warren S and Shirley Liu, X and Blacklow, Stephen C and Aster, Jon C}
}
@article {1170471,
	title = {NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function},
	journal = {J Biol Chem},
	volume = {292},
	number = {17},
	year = {2017},
	month = {2017 04 28},
	pages = {7052-7065},
	abstract = {Nitrovasodilators relax vascular smooth-muscle cells in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or the leucine zipper (LZ) domain of the myosin light-chain phosphatase component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G (PKG)-Iα. Despite the importance of vasodilation in cardiovascular homeostasis and therapy, our structural understanding of the MBS CC interaction with LZ PKG-1α has remained limited. Here, we report the 3D NMR solution structure of homodimeric CC MBS in which amino acids 932-967 form a coiled-coil of two monomeric α-helices in parallel orientation. We found that the structure is stabilized by non-covalent interactions, with dominant contributions from hydrophobic residues at  and  heptad positions. Using NMR chemical-shift perturbation (CSP) analysis, we identified a subset of hydrophobic and charged residues of CC MBS (localized within and adjacent to the C-terminal region) contributing to the dimer-dimer interaction interface between homodimeric CC MBS and homodimeric LZ PKG-Iα. N backbone relaxation NMR revealed the dynamic features of the CC MBS interface residues identified by NMR CSP. Paramagnetic relaxation enhancement- and CSP-NMR-guided HADDOCK modeling of the dimer-dimer interface of the heterotetrameric complex exhibits the involvement of non-covalent intermolecular interactions that are localized within and adjacent to the C-terminal regions of each homodimer. These results deepen our understanding of the binding restraints of this CC MBS{\textperiodcentered}LZ PKG-Iα low-affinity heterotetrameric complex and allow reevaluation of the role(s) of myosin light-chain phosphatase partner polypeptides in regulation of vascular smooth-muscle cell contractility.},
	keywords = {Animals, Circular Dichroism, Cyclic GMP-Dependent Protein Kinase Type I, Hydrophobic and Hydrophilic Interactions, Leucine Zippers, Magnetic Resonance Spectroscopy, Mice, Molecular Dynamics Simulation, Muscle, Smooth, Vascular, Myosins, Protein Binding, Protein Domains, Protein Multimerization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Spectrophotometry, Ultraviolet, Static Electricity},
	issn = {1083-351X},
	doi = {10.1074/jbc.M117.781260},
	author = {Sharma, Alok K and Birrane, Gabriel and Anklin, Clemens and Rigby, Alan C and Alper, Seth L}
}
@article {1322670,
	title = {Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling},
	journal = {Cell},
	volume = {169},
	number = {2},
	year = {2017},
	month = {2017 Apr 06},
	pages = {338-349.e11},
	abstract = {G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the β2 adrenoceptor. These receptors co-localize with a variety of\&nbsp;G proteins even before receptor activation, and activated receptors are largely sequestered from G\&nbsp;proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as\&nbsp;a\&nbsp;potential regulator of β2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.},
	keywords = {Ascorbate Peroxidases, beta-Arrestins, Biotin, GTP-Binding Proteins, HEK293 Cells, Humans, Oligopeptides, Protein Engineering, Receptor, Angiotensin, Type 1, Signal Transduction, Staining and Labeling},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2017.03.028},
	author = {Paek, Jaeho and Kalocsay, Marian and Staus, Dean P and Wingler, Laura and Pascolutti, Roberta and Paulo, Joao A and Gygi, Steven P and Kruse, Andrew C}
}
@article {1180371,
	title = {Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter},
	journal = {Proc Natl Acad Sci U S A},
	volume = {114},
	number = {14},
	year = {2017},
	month = {2017 04 04},
	pages = {E2846-E2851},
	abstract = {The calcium (Ca2+) uniporter of mitochondria is a holocomplex consisting of the Ca2+-conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn2+), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn2+ specifically, whereas in the WT the two rings bind Mn2+ cooperatively, resulting in \~{}1,000-fold higher apparent affinity. Ca2+ can specifically displace the bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.},
	keywords = {Amino Acid Motifs, Binding Sites, Caenorhabditis elegans Proteins, Calcium, Magnetic Resonance Spectroscopy, Manganese, Mitochondrial Membrane Transport Proteins, Molecular Dynamics Simulation, Mutation, Ruthenium Compounds},
	issn = {1091-6490},
	doi = {10.1073/pnas.1620316114},
	author = {Cao, Chan and Wang, Shuqing and Cui, Tanxing and Su, Xun-Cheng and Chou, James J}
}
@article {991191,
	title = {Conformational equilibria and intrinsic affinities define integrin activation},
	journal = {EMBO J},
	volume = {36},
	number = {5},
	year = {2017},
	month = {2017 03 01},
	pages = {629-645},
	abstract = {We show that the three conformational states of integrin αβ have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact αβ On the surface of K562 cells, αβ is 99.8\% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.},
	keywords = {Cell Line, Humans, Integrin alpha5beta1, Models, Molecular, Protein Binding, Protein Conformation, Thermodynamics},
	issn = {1460-2075},
	doi = {10.15252/embj.201695803},
	author = {Li, Jing and Su, Yang and Xia, Wei and Qin, Yan and Humphries, Martin J and Vestweber, Dietmar and Caba{\~n}as, Carlos and Lu, Chafen and Springer, Timothy A.}
}
@article {953736,
	title = {Covalently circularized nanodiscs for studying membrane proteins and viral entry},
	journal = {Nat Methods},
	volume = {14},
	number = {1},
	year = {2017},
	month = {2017 01},
	pages = {49-52},
	abstract = {We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a β-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.},
	keywords = {Humans, Lipid Bilayers, Models, Molecular, Nanostructures, Nuclear Magnetic Resonance, Biomolecular, Poliomyelitis, Poliovirus, Receptors, Neurotensin, Virus Internalization, Voltage-Dependent Anion Channel 1},
	issn = {1548-7105},
	doi = {10.1038/nmeth.4079},
	author = {Nasr, Mahmoud L and Baptista, Diego and Strauss, Mike and Sun, Zhen-Yu J and Grigoriu, Simina and Huser, Sonja and Pl{\"u}ckthun, Andreas and Hagn, Franz and Walz, Thomas and Hogle, James M and Wagner, Gerhard}
}
@article {1636623,
	title = {AID Recognizes Structured DNA for Class Switch Recombination},
	journal = {Mol Cell},
	volume = {67},
	number = {3},
	year = {2017},
	month = {2017 Aug 03},
	pages = {361-373.e4},
	abstract = {Activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. Mechanisms of AID targeting and catalysis remain elusive despite its critical immunological roles and\&nbsp;off-target effects in tumorigenesis. Here, we produced active human AID and revealed its preferred recognition and deamination of structured substrates. G-quadruplex (G4)-containing substrates mimicking the mammalian immunoglobulin switch regions are particularly good AID substrates in\&nbsp;vitro. By solving crystal structures of maltose binding protein (MBP)-fused AID alone and in complex with deoxycytidine monophosphate, we surprisingly identify a bifurcated substrate-binding surface that explains structured substrate recognition by capturing two adjacent single-stranded overhangs simultaneously. Moreover, G4 substrates induce cooperative AID oligomerization. Structure-based mutations that disrupt bifurcated substrate recognition or oligomerization both compromise CSR in splenic B cells. Collectively, our data implicate intrinsic preference of AID for structured substrates and uncover the importance of G4 recognition and oligomerization of AID in CSR.},
	keywords = {Animals, Antibody Diversity, APOBEC Deaminases, B-Lymphocytes, Cytidine Deaminase, DNA, Humans, Immunoglobulin Class Switching, Immunoglobulin Switch Region, Mice, Models, Molecular, Mutation, Nucleic Acid Conformation, Protein Binding, Protein Conformation, Recombination, Genetic, Spleen, Structure-Activity Relationship, Substrate Specificity},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2017.06.034},
	author = {Qiao, Qi and Wang, Li and Meng, Fei-Long and Hwang, Joyce K and Alt, Frederick W and Wu, Hao}
}
@article {1636624,
	title = {Chemical Interaction among Termite-Associated Microbes},
	journal = {J Chem Ecol},
	volume = {43},
	number = {11-12},
	year = {2017},
	month = {2017 Dec},
	pages = {1078-1085},
	abstract = {Bacteria and fungi in shared environments compete with one another for common substrates, and this competition typically involves microbially-produced small molecules. An investigation of one shared environmental niche, the carton material of the Formosan subterranean termite Coptotermes formosanus, identified the participants on one of these molecular exchanges. Molecular characterization of several termite-associated actinobacteria strains identified eleven known antimicrobial metabolites that may aid in protecting the C. formosanus colony from pathogenic fungal infections. One particular actinobacterial-derived small molecule, bafilomycin C1, elicited a strong chemical response from Trichoderma harzianum, a common soil saprophyte. Upon purification and structure elucidation, three major fungal metabolites were identified, t22-azaphilone, cryptenol, and homodimericin A. Both t22-azaphilone and homodimericin A are strongly upregulated, 123- and 38-fold, respectively, when exposed to bafilomycin C1, suggesting each play a role in defending T. harzianum from the toxic effect of bafilomycin C1.},
	keywords = {Actinobacteria, Animals, Anti-Infective Agents, Benzopyrans, Isoptera, Macrolides, Magnetic Resonance Spectroscopy, Microbial Sensitivity Tests, Pigments, Biological, Polyketides, Streptomyces, Trichoderma},
	issn = {1573-1561},
	doi = {10.1007/s10886-017-0900-6},
	author = {Mevers, Emily and Chouvenc, Thomas and Su, Nan-Yao and Clardy, Jon}
}
@mastersthesis {Arellano-SantoyoHugo2017RoMP,
	title = {Regulation of Microtubule Plus-End Dynamics by Molecular Motors},
	year = {2017},
	type = {PhD Thesis},
	abstract = {The microtubule cytoskeleton is the structural base of key cellular processes such as axonal growth, intracellular transport and cell division. These carefully orchestrated processes rely on the precise control of microtubule function and architecture. Microtubules, however, are dynamic polymers that stochastically transition between periods of growth and shrinkage. Therefore, the cell must regulate microtubule dynamics to achieve control over the length and stability of the microtubule cytoskeleton to accurately perform its cellular functions. Kinesins are best known for their ability to move processively on microtubules and carry cargo. Nevertheless, some kinesin families can also regulate microtubule dynamics and organize microtubule structures. Kinesin-8s play a central role in regulating the microtubule cytoskeleton by controlling microtubule length. Although extensive studies have been conducted, the mechanism by which kinesin-8 disassemble microtubules has remained unclear. My thesis work identified the central features of this mechanism for the yeast kinesin-8/Kip3. Like other kinesins, kinesin-8/Kip3, uses ATP hydrolysis for stepping on the microtubule lattice. Upon binding to curved tubulin at the plus-end, Kip3 undergoes a switch in activity resulting in strong tubulin binding, thereby promoting microtubule depolymerization. Our model is supported by the identification of the structural elements in the motor domain and in tubulin required for plus-end recognition, long-range processivity and microtubule depolymerization by Kip3. To obtain a comprehensive understanding of the Kip3-tubulin interaction we obtained high-resolution structures using cryo-electron microscopy of Kip3 bound to stabilized microtubules. This approach identified intra and intermolecular interactions that underlie Kip3{\textquoteright}s ability to switch from a motile kinesin to a depolymerase at the microtubule end. The coexistence of the two activities in kinesin-8/Kip3, gives rise to its emergent property of length-dependent microtubule disassembly. Another challenge in the regulation of microtubule structures is in the control of the nucleation and architecture of the microtubule polymer. To overcome these challenges we developed a programmable DNA-origami microtubule seed, which can template defined microtubule structures. This tool will enable future studies on the regulation of microtubule architecture, such as the microtubule doublet structures found in cilia. Together, these findings elucidate regulatory mechanisms whereby nanometer-size molecules can measure and regulate micron-sized cellular microtubule structures.},
	keywords = {Biology, Biophysics, General, Molecular},
	author = {Arellano-Santoyo, Hugo}
}
@mastersthesis {SongDan2017SSoB,
	title = {Single-molecule Studies of Bacterial Chromosome Organization and Segregation},
	year = {2017},
	school = {ProQuest Dissertations Publishing},
	abstract = {The bacterial chromosome is dramatically compacted into a subcellular structure called nucleoid. Simultaneously, it must be organized to allow the protein machinery for essential cellular processes to gain access to the DNA. Bacteria lack the histones that play a critical role in chromosome compaction in other kingdoms of life; instead they have evolved other mechanisms to compact, organize, and segregate their chromosomes. Particularly, a group of chromosomal architectural proteins called nucleoid-associated proteins (NAPs) and the highly conserved structural maintenance of chromosomes (SMC) condensin complex are two of the major players in bacterial chromosome condensation and organization. Emerging single-molecule techniques, which can detect transient changes in DNA structure with high spatial and temporal resolutions, have contributed significantly to our understanding of how NAPs and SMC family members condense DNA. However, most nanomanipulation methods only measure the change in extension of DNA as a proxy to investigate protein-DNA interactions; they are unable to directly measure protein binding on DNA without fluorescently labeling the protein. In this dissertation, I describe a new approach to simultaneously monitor the association of proteins with DNA and changes in DNA conformation, while bypassing the difficult procedure of labeling proteins. Our method is demonstrated to be a relatively simple yet highly quantitative assay to study any DNA-binding protein in general. The second part of this dissertation focuses on the study of ParB, which is part of the parABS partitioning system required in chromosome segregation for most bacteria. ParB binds specifically to the centromere DNA sequence parS and to adjacent non-specific DNA in a phenomenon called spreading. Previous studies have argued that ParB spreading requires cooperative interactions between ParB dimers including DNA bridging and possible nearest-neighbor interactions. A recent structure of a ParB homolog co-crystalized with parS revealed that ParB dimers form a higher order tetrameric complex. Using this structure as a guide, a series of proposed intermolecular interactions in the Bacillus subtilis ParB was ablated to investigate their effect on spreading. Our results, based on in vivo and in vitro characterizations, demonstrated that a network of both cis and trans interactions between ParB dimers is necessary for spreading.},
	keywords = {Biophysics, Genetics, microbiology},
	isbn = {9798678154866},
	author = {Song, Dan}
}
@article {1019291,
	title = {A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions},
	journal = {ACS Infect Dis},
	volume = {3},
	number = {2},
	year = {2017},
	month = {2017 02 10},
	pages = {112-118},
	abstract = {Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.},
	keywords = {Allosteric Regulation, Allosteric Site, Antiviral Agents, Crystallography, X-Ray, Cytomegalovirus, DNA-Binding Proteins, DNA-Directed DNA Polymerase, High-Throughput Screening Assays, Humans, Lysine, Models, Molecular, Protein Binding, Protein Conformation, Small Molecule Libraries, Viral Proteins},
	issn = {2373-8227},
	doi = {10.1021/acsinfecdis.6b00079},
	author = {Chen, Han and Coseno, Molly and Ficarro, Scott B and Mansueto, My Sam and Komazin-Meredith, Gloria and Boissel, Sandrine and Filman, David J and Marto, Jarrod A and Hogle, James M and Coen, Donald M}
}
@article {1170226,
	title = {Structural characterization of the C-terminal coiled-coil domains of wild-type and kidney disease-associated mutants of apolipoprotein L1},
	journal = {FEBS J},
	volume = {283},
	number = {10},
	year = {2016},
	month = {2016 05},
	pages = {1846-62},
	abstract = {Trypanosomes that cause sleeping sickness endocytose apolipoprotein L1 (APOL1)-containing trypanolytic factors from human serum, leading to trypanolytic death through generation of APOL1-associated lytic pores in trypanosomal membranes. The trypanosome Trypanosoma brucei rhodesiense counteracts trypanolysis by expressing the surface protein serum response-associated (SRA), which can bind APOL1 common variant G0 to block its trypanolytic activity. However, two missense variants in the C terminal predicted coiled-coil (CC) domains of human APOL1 G1 (S342G/I384M) and G2 (ΔN388Y389) decrease or abrogate APOL1 binding to T. brucei rhodesiense SRA, thus preserving APOL1 trypanolytic activity. These evolutionarily selected APOL1 missense variants, found at a high frequency in some populations of African descent, also confer elevated risk of kidney disease. Understanding the SRA-APOL1 interaction and the role of APOL1 G1 and G2 variants in kidney disease demands structural characterization of the APOL1 CC domain. Using CD, heteronuclear NMR, and molecular dynamics (MD) simulation on structural homology models, we report here unique and dynamic solution conformations of nephropathy variants G1 and G2 as compared with the common variant G0. Conformational plasticity in G1 and G2 CC domains led to interhelical α1-α2 approximation coupled with secondary structural changes and delimited motional properties absent in the G0 CC domain. The G1 substitutions conferred local structural changes principally along helix α1, whereas the G2 deletion altered the structure of both helix α2 and helix α1. These dynamic features of APOL1 CC variants likely reflect their intrinsic structural properties, and should help interpret future APOL1 structural studies and define the contribution of APOL1 risk variants to kidney disease.},
	keywords = {Amino Acid Sequence, Animals, Apolipoprotein L1, Apolipoproteins, Circular Dichroism, Cloning, Molecular, Humans, Kidney Diseases, Lipoproteins, HDL, Molecular Dynamics Simulation, Mutation, Missense, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Sequence Homology, Amino Acid, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Trypanosoma brucei rhodesiense},
	issn = {1742-4658},
	doi = {10.1111/febs.13706},
	author = {Sharma, Alok K and Friedman, David J and Pollak, Martin R. and Alper, Seth L}
}
@article {953741,
	title = {Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes},
	journal = {Elife},
	volume = {5},
	year = {2016},
	month = {2016 11 11},
	abstract = {Heterochromatin is a conserved feature of eukaryotic chromosomes with central roles in regulation of gene expression and maintenance of genome stability. Heterochromatin formation involves spreading of chromatin-modifying factors away from initiation points over large DNA domains by poorly understood mechanisms. In , heterochromatin formation requires the SIR complex, which contains subunits with histone-modifying, histone-binding, and self-association activities. Here, we analyze binding of the Sir proteins to reconstituted mono-, di-, tri-, and tetra-nucleosomal chromatin templates and show that key Sir-Sir interactions bridge only sites on different nucleosomes but not sites on the same nucleosome, and are therefore {\textquoteright}interrupted{\textquoteright} with respect to sites on the same nucleosome. We observe maximal binding affinity and cooperativity to unmodified di-nucleosomes and propose that nucleosome pairs bearing unmodified histone H4-lysine16 and H3-lysine79 form the fundamental units of Sir chromatin binding and that cooperative binding requiring two appropriately modified nucleosomes mediates selective Sir recruitment and spreading.},
	keywords = {Heterochromatin, Nucleosomes, Saccharomyces cerevisiae, Silent Information Regulator Proteins, Saccharomyces cerevisiae},
	issn = {2050-084X},
	doi = {10.7554/eLife.17556},
	author = {Behrouzi, Reza and Lu, Chenning and Currie, Mark A and Jih, Gloria and Iglesias, Nahid and Moazed, Danesh}
}
@article {1170236,
	title = {Selvamicin, an atypical antifungal polyene from two alternative genomic contexts},
	journal = {Proc Natl Acad Sci U S A},
	volume = {113},
	number = {46},
	year = {2016},
	month = {2016 11 15},
	pages = {12940-12945},
	abstract = {The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin{\textquoteright}s shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin{\textquoteright}s biosynthesis including a subcluster of genes consistent with selvamicin{\textquoteright}s 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.},
	keywords = {Actinobacteria, Animals, Antifungal Agents, Ants, Candida albicans, Gene Transfer, Horizontal, Genome, Bacterial, Genomics, Glycosylation, Macrolides, Multigene Family, Plasmids, Polyenes},
	issn = {1091-6490},
	doi = {10.1073/pnas.1613285113},
	author = {Van Arnam, Ethan B and Ruzzini, Antonio C and Sit, Clarissa S and Horn, Heidi and Pinto-Tom{\'a}s, Adri{\'a}n A and Currie, Cameron R and Clardy, Jon}
}
@article {1178831,
	title = {The Aggregation Paths and Products of Aβ42 Dimers Are Distinct from Those of the Aβ42 Monomer},
	journal = {Biochemistry},
	volume = {55},
	number = {44},
	year = {2016},
	month = {2016 Nov 08},
	pages = {6150-6161},
	abstract = {Extracts of Alzheimer{\textquoteright}s disease (AD) brain that contain what appear to be sodium dodecyl sulfate-stable amyloid β-protein (Aβ) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated the Aβ monomer, consist primarily of Aβ42, and resist denaturation by chaotropic agents. In nature, covalently cross-linked Aβ dimers could be generated in two ways: by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aβ42 monomer to produce DiY and Q-K dimers and then used a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by the Aβ monomer. The results suggest that the cross-links disfavor fibril formation from Aβ dimers, thereby enhancing the concentration of soluble aggregates akin to those in aqueous extracts of AD brain. Thus, it seems that Aβ dimers may play an important role in determining the formation of soluble rather than insoluble aggregates.},
	keywords = {Amyloid beta-Peptides, Chromatography, High Pressure Liquid, Circular Dichroism, Dimerization, Fluorescence, Mass Spectrometry, Molecular Weight},
	issn = {1520-4995},
	doi = {10.1021/acs.biochem.6b00453},
	author = {O{\textquoteright}Malley, Tiernan T and Witbold, William M and Linse, Sara and Walsh, Dominic M}
}
@article {953746,
	title = {Mitochondrial Sirtuin Network Reveals Dynamic SIRT3-Dependent Deacetylation in Response to Membrane Depolarization},
	journal = {Cell},
	volume = {167},
	number = {4},
	year = {2016},
	month = {2016 11 03},
	pages = {985-1000.e21},
	abstract = {Mitochondrial sirtuins, SIRT3-5, are NAD-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss~of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In~vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.},
	keywords = {Acetylation, Adenosine Triphosphatases, Animals, Carrier Proteins, HeLa Cells, Humans, Immunoprecipitation, Membrane Proteins, Mice, Mitochondria, Mitochondrial Proteins, Mitochondrial Proton-Translocating ATPases, Protein Interaction Maps, Sirtuin 3, Sirtuins},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2016.10.016},
	author = {Yang, Wen and Nagasawa, Koji and M{\"u}nch, Christian and Xu, Yingjie and Satterstrom, Kyle and Jeong, Seungmin and Hayes, Sebastian D and Jedrychowski, Mark P and Vyas, F Sejal and Zaganjor, Elma and Guarani, Virginia and Ringel, Alison E and Gygi, Steven P and Harper, J Wade and Haigis, Marcia C}
}
@article {953751,
	title = {Structure of the MIND Complex Defines a Regulatory Focus for Yeast Kinetochore Assembly},
	journal = {Cell},
	volume = {167},
	number = {4},
	year = {2016},
	month = {2016 11 03},
	pages = {1014-1027.e12},
	abstract = {Kinetochores connect centromeric nucleosomes with~mitotic-spindle microtubules through conserved, cross-interacting protein subassemblies. In budding yeast, the heterotetrameric MIND complex (Mtw1, Nnf1, Nsl1, Dsn1), ortholog of the metazoan Mis12 complex, joins the centromere-proximal components, Mif2 and COMA, with the principal microtubule-binding component, the Ndc80 complex (Ndc80C). We report the crystal structure of Kluyveromyces lactis MIND and examine its partner interactions, to understand the connection from a centromeric nucleosome to a much larger microtubule. MIND resembles an elongated, asymmetric Y; two globular heads project from a coiled-coil shaft. An N-terminal extension of Dsn1 from one head regulates interactions of the other head, blocking binding of Mif2 and COMA. Dsn1 phosphorylation by Ipl1/Aurora B relieves this autoinhibition, enabling MIND~to join an assembling kinetochore. A C-terminal extension of Dsn1 recruits Ndc80C to the opposite end of the shaft. The structure and properties of MIND show how it integrates phospho-regulatory inputs for kinetochore assembly and disassembly.},
	keywords = {Chromosomal Proteins, Non-Histone, Crystallography, X-Ray, Fungal Proteins, Kinetochores, Kluyveromyces, Multiprotein Complexes},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2016.10.011},
	author = {Dimitrova, Yoana N and Jenni, Simon and Valverde, Roberto and Khin, Yadana and Harrison, Stephen C}
}
@article {889726,
	title = {Structure and Dynamics of PD-L1 and an Ultra-High-Affinity PD-1 Receptor Mutant},
	journal = {Structure},
	volume = {24},
	number = {10},
	year = {2016},
	month = {2016 Oct 04},
	pages = {1719-1728},
	abstract = {The immune checkpoint receptor PD-1 and its ligand, PD-L1, have emerged as key regulators of anti-tumor~immunity in humans. Recently, we reported an ultra-high-affinity PD-1 mutant, termed high-affinity consensus (HAC) PD-1, which shows superior therapeutic efficacy in mice compared with antibodies. However, the molecular details underlying the action of this agent remain incompletely understood, and a molecular view of PD-1/PD-L1 interactions in general is only beginning to emerge. Here, we report the structure of HAC PD-1 in complex with PD-L1, showing that it binds PD-L1 using a unique set of polar interactions. Biophysical studies and long-timescale molecular dynamics experiments reveal the mechanisms by which ten point mutations confer a 35,000-fold enhancement in binding affinity, and offer atomic-scale views of the role of conformational dynamics in PD-1/PD-L1 interactions. Finally, we show that the HAC PD-1 exhibits pH-dependent affinity, with pseudo-irreversible binding in a low pH setting akin to the tumor microenvironment.},
	keywords = {B7-H1 Antigen, Binding Sites, Crystallography, X-Ray, Humans, Hydrogen-Ion Concentration, Models, Molecular, Molecular Dynamics Simulation, Point Mutation, Programmed Cell Death 1 Receptor, Protein Binding, Protein Structure, Secondary},
	issn = {1878-4186},
	doi = {10.1016/j.str.2016.06.026},
	author = {Pascolutti, Roberta and Sun, Xianqiang and Kao, Joseph and Maute, Roy L and Ring, Aaron M and Bowman, Gregory R and Kruse, Andrew C}
}
@article {927866,
	title = {Homodimericin A: A Complex Hexacyclic Fungal Metabolite},
	journal = {J Am Chem Soc},
	volume = {138},
	number = {38},
	year = {2016},
	month = {2016 09 28},
	pages = {12324-7},
	abstract = {Microbes sense and respond to their environment with small molecules, and discovering these molecules and identifying their functions informs chemistry, biology, and medicine. As part of a study of molecular exchanges between termite-associated actinobacteria and pathogenic fungi, we uncovered a remarkable fungal metabolite, homodimericin A, which is strongly upregulated by the bacterial metabolite bafilomycin C1. Homodimericin A is a hexacyclic polyketide with a carbon backbone containing eight contiguous stereogenic carbons in a C20 hexacyclic core. Only half of its carbon atoms have an attached hydrogen, which presented a significant challenge for NMR-based structural analysis. In spite of its microbial production and rich stereochemistry, homodimericin A occurs naturally as a racemic mixture. A plausible nonenzymatic reaction cascade leading from two identical achiral monomers to homodimericin A is presented, and homodimericin A{\textquoteright}s formation by this path, a six-electron oxidation, could be a response to oxidative stress triggered by bafilomycin C1.},
	keywords = {Antifungal Agents, Deoxyribonuclease (Pyrimidine Dimer), Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Structure, Polyketides, Trichoderma, Up-Regulation, Viral Proteins},
	issn = {1520-5126},
	doi = {10.1021/jacs.6b07588},
	author = {Mevers, Emily and Saur{\'\i}, Josep and Liu, Yizhou and Moser, Arvin and Ramadhar, Timothy R and Varlan, Maria and Williamson, R Thomas and Martin, Gary E and Clardy, Jon}
}
@article {871021,
	title = {Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling},
	journal = {Cell},
	volume = {166},
	number = {5},
	year = {2016},
	month = {2016 Aug 25},
	pages = {1176-1187.e14},
	abstract = {In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and~cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the~binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol. },
	keywords = {Animals, Cholesterol, Crystallography, X-Ray, Hedgehog Proteins, Mice, NIH 3T3 Cells, Oxysterols, Protein Binding, Protein Conformation, Signal Transduction, Smoothened Receptor, Xenopus laevis},
	issn = {1097-4172},
	doi = {10.1016/j.cell.2016.08.003},
	author = {Huang, Pengxiang and Nedelcu, Daniel and Watanabe, Miyako and Jao, Cindy and Kim, Youngchang and Liu, Jing and Salic, Adrian}
}
@article {871026,
	title = {Electrostatic Interactions between Elongated Monomers Drive Filamentation of Drosophila Shrub, a Metazoan ESCRT-III Protein},
	journal = {Cell Rep},
	volume = {16},
	number = {5},
	year = {2016},
	month = {2016 08 02},
	pages = {1211-1217},
	abstract = {The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.},
	keywords = {Amino Acid Sequence, Animals, Cytokinesis, Drosophila, Drosophila Proteins, Endosomal Sorting Complexes Required for Transport, Humans, Membrane Fusion, Multivesicular Bodies, Nerve Tissue Proteins, Polymers, Protein Multimerization, Protein Transport, Static Electricity, Yeasts},
	issn = {2211-1247},
	doi = {10.1016/j.celrep.2016.06.093},
	author = {McMillan, Brian J and Tibbe, Christine and Jeon, Hyesung and Drabek, Andrew A and Klein, Thomas and Blacklow, Stephen C}
}
@article {871016,
	title = {Antiparallel protocadherin homodimers use distinct affinity- and specificity-mediating regions in cadherin repeats 1-4},
	journal = {Elife},
	volume = {5},
	year = {2016},
	month = {2016 07 29},
	abstract = {Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3, a member of the γ subfamily of clustered Pcdhs. Structure and sequence comparisons of α, β, and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families.},
	keywords = {Cadherins, Crystallography, X-Ray, Humans, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Protein Binding, Protein Conformation, Protein Multimerization, Protocadherins},
	issn = {2050-084X},
	doi = {10.7554/eLife.18449},
	author = {Nicoludis, John M and Vogt, Bennett E and Green, Anna G and Sch{\"a}rfe, Charlotta Pi and Marks, Debora S and Gaudet, Rachelle}
}
@article {871011,
	title = {Direct Targeting of β-Catenin by a Small Molecule Stimulates Proteasomal Degradation and Suppresses Oncogenic Wnt/β-Catenin Signaling},
	journal = {Cell Rep},
	volume = {16},
	number = {1},
	year = {2016},
	month = {2016 06 28},
	pages = {28-36},
	abstract = {The Wnt/β-catenin signaling pathway plays a major role in tissue homeostasis, and its dysregulation can lead to various human diseases. Aberrant activation of β-catenin is oncogenic and is a critical driver in the development and progression of human cancers. Despite the significant potential of targeting the oncogenic β-catenin pathway for cancer therapy, the development of specific inhibitors remains insufficient. Using a T~cell factor (TCF)-dependent luciferase-reporter system, we screened for small-molecule compounds that act against Wnt/β-catenin signaling and identified MSAB (methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate) as a selective inhibitor of Wnt/β-catenin signaling. MSAB shows potent anti-tumor effects selectively on Wnt-dependent cancer cells in~vitro and in mouse cancer models. MSAB binds to β-catenin, promoting its degradation, and specifically downregulates Wnt/β-catenin target genes. Our findings might represent an effective therapeutic strategy for cancers addicted to the Wnt/β-catenin signaling pathway.},
	keywords = {Animals, Benzoates, beta Catenin, Cell Line, Tumor, meta-Aminobenzoates, Mice, Oncogenes, Proteasome Endopeptidase Complex, Proteolysis, Small Molecule Libraries, Sulfonamides, Wnt Signaling Pathway, Xenograft Model Antitumor Assays},
	issn = {2211-1247},
	doi = {10.1016/j.celrep.2016.05.071},
	author = {Hwang, So-Young and Deng, Xianming and Byun, Sanguine and Lee, Chan and Lee, Seung-Joo and Suh, Hyunsuk and Zhang, Jianming and Kang, Qiaofeng and Zhang, Ting and Westover, Kenneth D and Mandinova, Anna and Lee, Sam W}
}
@article {1180376,
	title = {Architecture of the mitochondrial calcium uniporter},
	journal = {Nature},
	volume = {533},
	number = {7602},
	year = {2016},
	month = {2016 05 12},
	pages = {269-73},
	abstract = {Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca(2+)) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity. Ca(2+) entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca(2+)-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture, and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.},
	keywords = {Amino Acid Motifs, Animals, Caenorhabditis elegans, Calcium Channels, Microscopy, Electron, Mitochondria, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Tertiary, Structure-Activity Relationship},
	issn = {1476-4687},
	doi = {10.1038/nature17656},
	author = {Oxenoid, Kirill and Dong, Ying and Cao, Chan and Cui, Tanxing and Sancak, Yasemin and Markhard, Andrew L and Grabarek, Zenon and Kong, Liangliang and Liu, Zhijun and Ouyang, Bo and Cong, Yao and Mootha, Vamsi K and Chou, James J}
}
@article {927861,
	title = {Structural Basis for Substrate Selectivity of the E3 Ligase COP1},
	journal = {Structure},
	volume = {24},
	number = {5},
	year = {2016},
	month = {2016 05 03},
	pages = {687-696},
	abstract = {COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. Here we report structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed β propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the β propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for~motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation.},
	keywords = {Arabidopsis, Arabidopsis Proteins, Binding Sites, Humans, Intracellular Signaling Peptides and Proteins, Molecular Docking Simulation, Peptide Fragments, Protein Binding, Protein-Serine-Threonine Kinases, Substrate Specificity, Ubiquitin-Protein Ligases, WD40 Repeats},
	issn = {1878-4186},
	doi = {10.1016/j.str.2016.03.002},
	author = {Uljon, Sacha and Xu, Xiang and Durzynska, Izabela and Stein, Sarah and Adelmant, Guillaume and Marto, Jarrod A and Pear, Warren S and Blacklow, Stephen C}
}
@article {871006,
	title = {Crystal structure of the human σ1 receptor},
	journal = {Nature},
	volume = {532},
	number = {7600},
	year = {2016},
	month = {2016 Apr 28},
	pages = {527-30},
	abstract = {The human σ1 receptor is an enigmatic endoplasmic-reticulum-resident transmembrane protein implicated in a variety of disorders including depression, drug addiction, and neuropathic pain. Recently, an additional connection to amyotrophic lateral sclerosis has emerged from studies of human genetics and mouse models. Unlike many transmembrane receptors that belong to large, extensively studied families such as G-protein-coupled receptors or ligand-gated ion channels, the σ1 receptor is an evolutionary isolate with no discernible similarity to any other human protein. Despite its increasingly clear importance in human physiology and disease, the molecular architecture of the σ1 receptor and its regulation by drug-like compounds remain poorly defined. Here we report crystal structures of the human σ1 receptor in complex with two chemically divergent ligands, PD144418 and 4-IBP. The structures reveal a trimeric architecture with a single transmembrane domain in each protomer. The carboxy-terminal domain of the receptor shows an extensive flat, hydrophobic membrane-proximal surface, suggesting an intimate association with the cytosolic surface of the endoplasmic reticulum membrane in cells. This domain includes a cupin-like β-barrel with the ligand-binding site buried at its centre. This large, hydrophobic ligand-binding cavity shows remarkable plasticity in ligand recognition, binding the two ligands in similar positions despite dissimilar chemical structures. Taken together, these results reveal the overall architecture, oligomerization state, and molecular basis for ligand recognition by this important but poorly understood protein. },
	keywords = {Benzamides, Binding Sites, Crystallography, X-Ray, Endoplasmic Reticulum, Humans, Hydrophobic and Hydrophilic Interactions, Intracellular Membranes, Isoxazoles, Ligands, Models, Molecular, Piperidines, Protein Structure, Tertiary, Pyridines, Receptors, sigma, Substrate Specificity},
	issn = {1476-4687},
	doi = {10.1038/nature17391},
	author = {Schmidt, Hayden R and Zheng, Sanduo and Gurpinar, Esin and Koehl, Antoine and Manglik, Aashish and Kruse, Andrew C}
}
@article {871001,
	title = {GNF-2 Inhibits Dengue Virus by Targeting Abl Kinases and the Viral E Protein},
	journal = {Cell Chem Biol},
	volume = {23},
	number = {4},
	year = {2016},
	month = {2016 04 21},
	pages = {443-52},
	abstract = {Dengue virus infects more than 300 million people annually, yet there is no widely protective vaccine or drugs against the virus. Efforts to develop antivirals against classical targets such as the viral protease and polymerase have not yielded drugs that have advanced to the clinic. Here, we show that the allosteric Abl kinase inhibitor GNF-2 interferes with dengue virus replication via activity mediated by cellular Abl kinases but additionally blocks viral entry via an Abl-independent mechanism. To characterize this newly discovered antiviral activity, we developed disubstituted pyrimidines that block dengue virus entry with structure-activity relationships distinct from those driving kinase inhibition. We demonstrate that biotin- and fluorophore-conjugated derivatives of GNF-2 interact with the dengue glycoprotein, E, in the pre-fusion conformation that exists on the virion surface, and that this interaction inhibits viral entry. This study establishes GNF-2 as an antiviral compound with polypharmacological activity and provides "lead" compounds for further optimization efforts.},
	keywords = {Animals, Antiviral Agents, Dengue Virus, Dose-Response Relationship, Drug, Humans, Mice, Microbial Sensitivity Tests, Molecular Structure, NIH 3T3 Cells, Protein Kinase Inhibitors, Proto-Oncogene Proteins c-abl, Pyrimidines, Structure-Activity Relationship, Viral Envelope Proteins},
	issn = {2451-9448},
	doi = {10.1016/j.chembiol.2016.03.010},
	author = {Clark, Margaret J and Miduturu, Chandra and Schmidt, Aaron G and Zhu, Xuling and Pitts, Jared D and Wang, Jinhua and Potisopon, Supanee and Zhang, Jianming and Wojciechowski, Amy and Hann Chu, Justin Jang and Gray, Nathanael S and Yang, Priscilla L}
}
@article {870996,
	title = {A Tunable Protein Piston That Breaks Membranes to Release Encapsulated Cargo},
	journal = {ACS Synth Biol},
	volume = {5},
	number = {4},
	year = {2016},
	month = {2016 Apr 15},
	pages = {303-11},
	abstract = {Movement of molecules across membranes in response to a stimulus is a key component of cellular programming. Here, we characterize and manipulate the response of a protein-based piston capable of puncturing membranes in a pH-dependent manner. Our protein actuator consists of modified R bodies found in a bacterial endosymbiont of paramecium. We express and purify R bodies from in E. coli; these pistons undergo multiple rounds of rapid extension and retraction. We developed a high throughput screen for mutants with altered pH sensitivity for tuning of the extension process. We show that the R bodies are capable of acting as synthetic pH-dependent pistons that can puncture E. coli membranes to release the trapped content. As such, these protein machines present a novel way to selectively rupture membrane compartments and will be important for programming cellular compartmentalization. },
	keywords = {Amino Acid Sequence, Bacterial Proteins, Cell Wall, Circular Dichroism, Escherichia coli, Genetic Vectors, Hydrogen-Ion Concentration, Inclusion Bodies, Microscopy, Electron, Molecular Sequence Data, Mutagenesis, Site-Directed, Osmolar Concentration, Recombinant Proteins, Sequence Alignment, Spectrophotometry},
	issn = {2161-5063},
	doi = {10.1021/acssynbio.5b00237},
	author = {Polka, Jessica K and Silver, Pamela A}
}
@article {870986,
	title = {A general approach to visualize protein binding and DNA conformation without protein labelling},
	journal = {Nat Commun},
	volume = {7},
	year = {2016},
	month = {2016 Mar 08},
	pages = {10976},
	abstract = {Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein-DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein-DNA interactions. },
	keywords = {Bacillus subtilis, Bacterial Proteins, Biochemistry, DNA, Bacterial, DNA-Binding Proteins, Fluorescence, Kinetics, Microscopy, Fluorescence, Nucleic Acid Conformation, Protein Binding},
	issn = {2041-1723},
	doi = {10.1038/ncomms10976},
	author = {Song, Dan and Graham, Thomas G W and Loparo, Joseph J}
}
@article {870991,
	title = {NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus},
	journal = {PLoS Pathog},
	volume = {12},
	number = {3},
	year = {2016},
	month = {2016 Mar},
	pages = {e1005468},
	abstract = {A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. },
	keywords = {Antigens, Bacterial, Bacterial Proteins, Bacterial Toxins, Cell Survival, Epithelial Cells, Exotoxins, Humans, Intracellular Space, Keratinocytes, Lysosomes, NAD+ Nucleosidase, Protein Transport, Recombinant Proteins, Streptococcal Infections, Streptococcus pyogenes, Streptolysins, Virulence},
	issn = {1553-7374},
	doi = {10.1371/journal.ppat.1005468},
	author = {Sharma, Onkar and O{\textquoteright}Seaghdha, Maghnus and Velarde, Jorge J and Wessels, Michael R}
}
@article {870981,
	title = {Nicastrin functions to sterically hinder γ-secretase-substrate interactions driven by substrate transmembrane domain},
	journal = {Proc Natl Acad Sci U S A},
	volume = {113},
	number = {5},
	year = {2016},
	month = {2016 Feb 02},
	pages = {E509-18},
	abstract = {γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate{\textquoteright}s ectodomain by α- or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid--another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.},
	keywords = {Amyloid Precursor Protein Secretases, Lipid Bilayers, Membrane Glycoproteins, Substrate Specificity},
	issn = {1091-6490},
	doi = {10.1073/pnas.1512952113},
	author = {Bolduc, David M and Montagna, Daniel R and Gu, Yongli and Selkoe, Dennis J and Wolfe, Michael S}
}
@article {970151,
	title = {Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex},
	journal = {EMBO J},
	volume = {34},
	number = {23},
	year = {2015},
	month = {2015 Dec 02},
	pages = {2937-52},
	abstract = {Herpesvirus nucleocapsids escape from the nucleus in a process orchestrated by a highly conserved, viral nuclear egress complex. In human cytomegalovirus, the complex consists of two proteins, UL50 and UL53. We solved structures of versions of UL53 and the complex by X-ray crystallography. The UL53 structures, determined at 1.93 and 3.0 {\r A} resolution, contained unexpected features including a Bergerat fold resembling that found in certain nucleotide-binding proteins, and a Cys3His zinc finger. Substitutions of zinc-coordinating residues decreased UL50-UL53 co-localization in transfected cells, and, when incorporated into the HCMV genome, ablated viral replication. The structure of the complex, determined at 2.47 {\r A} resolution, revealed a mechanism of heterodimerization in which UL50 clamps onto helices of UL53 like a vise. Substitutions of particular residues on the interaction interface disrupted UL50-UL53 co-localization in transfected cells and abolished virus production. The structures and the identification of contacts can be harnessed toward the rational design of novel and highly specific antiviral drugs and will aid in the detailed understanding of nuclear egress. },
	keywords = {Crystallography, X-Ray, Genome, Viral, Herpesviridae, Protein Structure, Secondary, Viral Proteins, Virus Replication},
	issn = {1460-2075},
	doi = {10.15252/embj.201592651},
	author = {Lye, Ming F and Sharma, Mayuri and El Omari, Kamel and Filman, David J and Schuermann, Jonathan P and Hogle, James M and Coen, Donald M}
}
@article {1180381,
	title = {Structure and multistate function of the transmembrane electron transporter CcdA},
	journal = {Nat Struct Mol Biol},
	volume = {22},
	number = {10},
	year = {2015},
	month = {2015 Oct},
	pages = {809-14},
	abstract = {The mechanism by which transmembrane reductases use a single pair of cysteine residues to relay electrons between protein substrates across biological membranes is a long-standing mystery in thiol-redox biochemistry. Here we show the NMR structure of a reduced-state mimic of archaeal CcdA, a protein that transfers electrons across the inner membrane, by using a redox-active NMR sample. The two cysteine positions in CcdA are separated by 20 {\r A}. Whereas one is accessible to the cytoplasm, the other resides in the protein core, thus implying that conformational exchange is required for periplasmic accessibility. In vivo mixed disulfide-trapping experiments validated the functional positioning of the cysteines, and in vitro accessibility results confirmed conformational exchange. Our NMR and functional data together show the existence of multiple conformational states and suggest a four-state model for relaying electrons from cytosolic to periplasmic redox substrates.},
	keywords = {Amino Acid Sequence, Archaeal Proteins, Carrier Proteins, Computational Biology, Cysteine, Electrons, Membrane Proteins, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Oxidoreductases, Plasmids, Protein Conformation},
	issn = {1545-9985},
	doi = {10.1038/nsmb.3099},
	author = {Williamson, Jessica A and Cho, Seung-Hyun and Ye, Jiqing and Collet, Jean-Francois and Beckwith, Jonathan R and Chou, James J}
}
@article {1180386,
	title = {Substrate-modulated ADP/ATP-transporter dynamics revealed by NMR relaxation dispersion},
	journal = {Nat Struct Mol Biol},
	volume = {22},
	number = {8},
	year = {2015},
	month = {2015 Aug},
	pages = {636-41},
	abstract = {The ADP/ATP carrier (AAC) transports ADP and ATP across the inner mitochondrial membrane. Unlike most transporters, which have two-fold direct or inverted quasisymmetry, AAC has apparent three-fold rotational symmetry. Further, its transport rate is relatively fast for transporters that carry large solutes. Here, we study the yeast AAC carrier 3 by obtaining comprehensive NMR relaxation dispersion measurements, which provide residue-specific information on the protein{\textquoteright}s conformational exchange. Our data indicate that AAC is predominantly in the cytosol-facing open state and converts to a sparsely populated state in an asymmetric manner despite its three-fold structural symmetry. Binding of the substrate ADP substantially increases the rate of conformational exchange, whereas the inhibitor CATR slows the exchange. These results suggest that although the transporter catalyzes the translocation of substrate the substrate also facilitates interconversion between alternating states, and this interconversion may be relevant to the transport function.},
	keywords = {Adenosine Diphosphate, Algorithms, Binding Sites, Kinetics, Magnetic Resonance Spectroscopy, Mitochondrial ADP, ATP Translocases, Models, Molecular, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Saccharomyces cerevisiae Proteins, Substrate Specificity, Thermodynamics},
	issn = {1545-9985},
	doi = {10.1038/nsmb.3059},
	author = {Br{\"u}schweiler, Sven and Yang, Qin and Run, Changqing and Chou, James J}
}
@article {1178836,
	title = {The Familial British Dementia Mutation Promotes Formation of Neurotoxic Cystine Cross-linked Amyloid Bri (ABri) Oligomers},
	journal = {J Biol Chem},
	volume = {290},
	number = {27},
	year = {2015},
	month = {2015 Jul 03},
	pages = {16502-16},
	abstract = {Familial British dementia (FBD) is an inherited neurodegenerative disease believed to result from a mutation in the BRI2 gene. Post-translational processing of wild type BRI2 and FBD-BRI2 result in the production of a 23-residue long Bri peptide and a 34-amino acid long ABri peptide, respectively, and ABri is found deposited in the brains of individuals with FBD. Similarities in the neuropathology and clinical presentation shared by FBD and Alzheimer disease (AD) have led some to suggest that ABri and the AD-associated amyloid β-protein (Aβ) are molecular equivalents that trigger analogous pathogenic cascades. But the sequences and innate properties of ABri and Aβ are quite different, notably ABri contains two cysteine residues that can form disulfide bonds. Thus we sought to determine whether ABri was neurotoxic and if this activity was regulated by oxidation and/or aggregation. Crucially, the type of oxidative cross-linking dramatically influenced both ABri aggregation and toxicity. Cyclization of Bri and ABri resulted in production of biologically inert monomers that showed no propensity to assemble, whereas reduced ABri and reduced Bri aggregated forming thioflavin T-positive amyloid fibrils that lacked significant toxic activity. ABri was more prone to form inter-molecular disulfide bonds than Bri and the formation of covalently stabilized ABri oligomers was associated with toxicity. These results suggest that extension of the C-terminal of Bri causes a shift in the type of disulfide bonds formed and that structures built from covalently cross-linked oligomers can interact with neurons and compromise their function and viability. },
	keywords = {Amyloid, Animals, Cerebral Amyloid Angiopathy, Familial, Cystine, Humans, Long-Term Potentiation, Male, Membrane Glycoproteins, Mice, Mice, Inbred C57BL, Mutation, Neurotoxins, Oxidation-Reduction, Rats, Rats, Sprague-Dawley},
	issn = {1083-351X},
	doi = {10.1074/jbc.M115.652263},
	author = {Cantlon, Adam and Frigerio, Carlo Sala and Freir, Darragh B and Boland, Barry and Jin, Ming and Walsh, Dominic M}
}
@article {927856,
	title = {A tail of two sites: a bipartite mechanism for recognition of notch ligands by mind bomb E3 ligases},
	journal = {Mol Cell},
	volume = {57},
	number = {5},
	year = {2015},
	month = {2015 Mar 05},
	pages = {912-924},
	abstract = {Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.},
	keywords = {Amino Acid Sequence, Animals, Blotting, Western, Calcium-Binding Proteins, Cell Line, Tumor, Crystallography, X-Ray, Drosophila melanogaster, Drosophila Proteins, Epitopes, HEK293 Cells, Humans, Intercellular Signaling Peptides and Proteins, Jagged-1 Protein, Ligands, Membrane Proteins, Models, Molecular, Molecular Sequence Data, Mutation, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Receptors, Notch, Sequence Homology, Amino Acid, Serrate-Jagged Proteins, Ubiquitin-Protein Ligases, Wnt1 Protein},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2015.01.019},
	author = {McMillan, Brian J and Schnute, Bj{\"o}rn and Ohlenhard, Nadja and Zimmerman, Brandon and Miles, Laura and Beglova, Natalia and Klein, Thomas and Blacklow, Stephen C}
}
@article {870976,
	title = {Purification of α-synuclein from human brain reveals an instability of endogenous multimers as the protein approaches purity},
	journal = {Biochemistry},
	volume = {54},
	number = {2},
	year = {2015},
	month = {2015 Jan 20},
	pages = {279-92},
	abstract = {Despite two decades of research, the structure-function relationships of endogenous, physiological forms of α-synuclein (αSyn) are not well understood. Most in vitro studies of this Parkinson{\textquoteright}s disease-related protein have focused on recombinant αSyn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that αSyn exists in considerable part in neurons, erythrocytes, and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant αSyn, physiological tetramers purified from human erythrocytes have substantial α-helical content and resist pathological aggregation into β-sheet rich fibers. Here, we report the first method to fully purify soluble αSyn from the most relevant source, human brain. We describe protocols that purify αSyn to homogeneity from nondiseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction, and affinity chromatographies. Cross-linking of the starting material and the partially purified chromatographic fractions revealed abundant αSyn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue β-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived αSyn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying αSyn to homogeneity destabilizes native, α-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests existence of a stabilizing cofactor (e.g., a small lipid) present inside neurons that is lost during final purification. },
	keywords = {alpha-Synuclein, beta-Synuclein, Brain Chemistry, Chemical Precipitation, Chromatography, Gel, Chromatography, Ion Exchange, Circular Dichroism, Humans, Mass Spectrometry, Protein Multimerization, Protein Stability, Protein Structure, Secondary},
	issn = {1520-4995},
	doi = {10.1021/bi501188a},
	author = {Luth, Eric S and Bartels, Tim and Dettmer, Ulf and Kim, Nora C and Selkoe, Dennis J}
}
@article {1322668,
	title = {CEACAM1 regulates TIM-3-mediated tolerance and exhaustion},
	journal = {Nature},
	volume = {517},
	number = {7534},
	year = {2015},
	month = {2015 01 15},
	pages = {386-90},
	abstract = {T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.},
	keywords = {Animals, Antigens, CD, Autoimmunity, Cell Adhesion Molecules, Cell Line, Colorectal Neoplasms, Disease Models, Animal, Female, Hepatitis A Virus Cellular Receptor 2, Humans, Immune Tolerance, Inflammation, Ligands, Male, Membrane Proteins, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Models, Molecular, Mucous Membrane, Protein Conformation, Protein Multimerization, Receptors, Virus, T-Lymphocytes},
	issn = {1476-4687},
	doi = {10.1038/nature13848},
	author = {Huang, Yu-Hwa and Zhu, Chen and Kondo, Yasuyuki and Anderson, Ana C and Gandhi, Amit and Russell, Andrew and Dougan, Stephanie K and Petersen, Britt-Sabina and Melum, Espen and Pertel, Thomas and Clayton, Kiera L and Raab, Monika and Chen, Qiang and Beauchemin, Nicole and Yazaki, Paul J and Pyzik, Michal and Ostrowski, Mario A and Glickman, Jonathan N and Rudd, Christopher E and Ploegh, Hidde L and Franke, Andre and Petsko, Gregory A and Kuchroo, Vijay K and Blumberg, Richard S}
}
@article {870971,
	title = {Soluble Aβ oligomers are rapidly sequestered from brain ISF in vivo and bind GM1 ganglioside on cellular membranes},
	journal = {Neuron},
	volume = {82},
	number = {2},
	year = {2014},
	month = {2014 Apr 16},
	pages = {308-19},
	abstract = {Soluble Aβ oligomers contribute importantly to synaptotoxicity in Alzheimer{\textquoteright}s disease, but their dynamics in vivo remain unclear. Here, we found that soluble Aβ oligomers were sequestered from brain interstitial fluid onto brain membranes much more rapidly than nontoxic monomers and were recovered in part as bound to GM1 ganglioside on membranes. Aβ oligomers bound strongly to GM1 ganglioside, and blocking the sialic acid residue on GM1 decreased oligomer-mediated LTP impairment in mouse hippocampal slices. In a hAPP transgenic mouse model, substantial levels of GM1-bound Aβ$_{4}$$_{2}$ were recovered from brain membrane fractions. We also detected GM1-bound Aβ in human CSF, and its levels correlated with Aβ$_{4}$$_{2}$, suggesting its potential as a biomarker of Aβ-related membrane dysfunction. Together, these findings highlight a mechanism whereby hydrophobic Aβ oligomers become sequestered onto GM1 ganglioside and presumably other lipids on neuronal membranes, where they may induce progressive functional and structural changes.},
	keywords = {Age Factors, Alzheimer Disease, Amyloid beta-Peptides, Amyloid beta-Protein Precursor, Animals, Brain, Cell Membrane, Dose-Response Relationship, Drug, Embryo, Mammalian, Extracellular Fluid, G(M1) Ganglioside, Gangliosides, hippocampus, Humans, In Vitro Techniques, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Peptide Fragments},
	issn = {1097-4199},
	doi = {10.1016/j.neuron.2014.02.027},
	author = {Soyon Hong and Ostaszewski, Beth L and Yang, Ting and O{\textquoteright}Malley, Tiernan T and Jin, Ming and Yanagisawa, Katsuhiko and Shaomin Li and Bartels, Tim and Selkoe, Dennis J}
}
@article {1178841,
	title = {Aβ dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies},
	journal = {Biochem J},
	volume = {461},
	number = {3},
	year = {2014},
	month = {2014 Aug 01},
	pages = {413-26},
	abstract = {Dimers of Aβ (amyloid β-protein) are believed to play an important role in Alzheimer{\textquoteright}s disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [Aβ](DiY) (dityrosine cross-linked Aβ). For comparison, we used the Aβ monomer and a design dimer cross-linked by replacement of Ser{\texttwosuperior}$^{6}$ with cystine [AβS26C]$_{2}$. We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [Aβ](DiY) and [AβS26C]$_{2}$ have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than Aβ monomers. Our results indicate that the link between Aβ dimers and Alzheimer{\textquoteright}s disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.},
	keywords = {Alzheimer Disease, Amino Acid Substitution, Amyloid beta-Peptides, Animals, Cerebellum, Dimerization, Evoked Potentials, Humans, Injections, Intraventricular, Kinetics, Long-Term Potentiation, Male, Nerve Tissue Proteins, Neurons, Nuclear Magnetic Resonance, Biomolecular, Peptide Fragments, Protein Conformation, Protein Multimerization, Rats, Rats, Wistar, Recombinant Proteins, Solubility, Synapses},
	issn = {1470-8728},
	doi = {10.1042/BJ20140219},
	author = {O{\textquoteright}Malley, Tiernan T and Oktaviani, Nur Alia and Zhang, Dainan and Aleksey Lomakin and O{\textquoteright}Nuallain, Brian and Linse, Sara and Benedek, George B and Rowan, Michael J and Mulder, Frans A A and Walsh, Dominic M}
}
@article {870966,
	title = {N-alpha-acetylation of α-synuclein increases its helical folding propensity, GM1 binding specificity and resistance to aggregation},
	journal = {PLoS One},
	volume = {9},
	number = {7},
	year = {2014},
	month = {2014},
	pages = {e103727},
	abstract = {A switch in the conformational properties of α-synuclein (αS) is hypothesized to be a key step in the pathogenic mechanism of Parkinson{\textquoteright}s disease (PD). Whereas the beta-sheet-rich state of αS has long been associated with its pathological aggregation in PD, a partially alpha-helical state was found to be related to physiological lipid binding; this suggests a potential role of the alpha-helical state in controlling synaptic vesicle cycling and resistance to β-sheet rich aggregation. N-terminal acetylation is the predominant post-translational modification of mammalian αS. Using circular dichroism, isothermal titration calorimetry, and fluorescence spectroscopy, we have analyzed the effects of N-terminal acetylation on the propensity of recombinant human αS to form the two conformational states in interaction with lipid membranes. Small unilamellar vesicles of negatively charged lipids served as model membranes. Consistent with previous NMR studies using phosphatidylserine, we found that membrane-induced α-helical folding was enhanced by N-terminal acetylation and that greater exothermic heat could be measured upon vesicle binding of the modified protein. Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes. The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation. Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1. },
	keywords = {Acetylation, alpha-Synuclein, Amyloid, G(M1) Ganglioside, Humans, Protein Aggregates, Protein Binding, Protein Folding, Protein Processing, Post-Translational, Protein Structure, Secondary, Thermodynamics},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0103727},
	author = {Bartels, Tim and Kim, Nora C and Luth, Eric S and Selkoe, Dennis J}
}
