User Publications

Recent Publications using CMI resources.  Thanks to all our users who share their publications.
Wang Z, Zhang D, Qiu X, Inuzuka H, Xiong Y, Liu J, Chen L, Chen H, Xie L, Kaniskan ÜH, Chen X, Jin J, Wei W. Structurally Specific Z-DNA Proteolysis Targeting Chimera Enables Targeted Degradation of Adenosine Deaminase Acting on RNA 1. J Am Chem Soc 2024;146(11):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.
Sharma P, Zhang X, Ly K, Zhang Y, Hu Y, Ye AY, Hu J, Kim JH, Lou M, Wang C, Celuzza Q, Kondo Y, Furukawa K, Bundle DR, Furukawa K, Alt FW, Winau F. The lipid globotriaosylceramide promotes germinal center B cell responses and antiviral immunity. Science 2024;383(6684):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.
Stinson BM, Carney SM, Walter JC, Loparo JJ. Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining. Nat Commun 2024;15(1):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.
Hauser BM, Sangesland M, Lam EC, St Denis KJ, Sheehan ML, Vu ML, Cheng AH, Sordilla S, Lamson DT, Almawi AW, Balazs AB, Lingwood D, Schmidt AG. Heterologous Sarbecovirus Receptor Binding Domains as Scaffolds for SARS-CoV-2 Receptor Binding Motif Presentation. ACS Infect Dis 2024;10(2):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.
Chanez-Paredes SD, Abtahi S, Zha J, Li E, Marsischky G, Zuo L, Grey MJ, He W, Turner JR. Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants. J Biol Chem 2024;300(2):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'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 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.
Lim S, Reilly CB, Barghouti Z, Marelli B, Way JC, Silver PA. Tardigrade secretory proteins protect biological structures from desiccation [Internet]. bioRxiv 2023; Publisher's VersionAbstract
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 (“SAHS”) 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.
Ames A, Seman M, Larkin A, Raiymbek G, Chen Z, Levashkevich A, Biteen JS, Ragunathan K. Identification of a novel, tunable interface in the HP1 protein, Swi6, that underpins epigenetic inheritance. bioRxiv 2023;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.
Sharma P, Zhang X, Ly K, Zhang Y, Hu Y, Ye AY, Hu J, Kim JH, Lou M, Wang C, Celuzza Q, Kondo Y, Furukawa K, Bundle DR, Furukawa K, Alt FW, Winau F. The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity [Internet]. bioRxiv 2023; Publisher's VersionAbstract
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.
Swift ML, Zhou R, Syed A, Moreau LA, Tomasik B, Tainer JA, Konstantinopoulos PA, D'Andrea AD, He YJ, Chowdhury D. Dynamics of the DYNLL1-MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs. Nat Struct Mol Biol 2023;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.
Lim Y, Tamayo-Orrego L, Schmid E, Tarnauskaite Z, Kochenova OV, Gruar R, Muramatsu S, Lynch L, Schlie AV, Carroll PL, Chistol G, Reijns MAM, Kanemaki MT, Jackson AP, Walter JC. In silico protein interaction screening uncovers DONSON's role in replication initiation. Science 2023;381(6664):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.
Xie L, Yin Y, Jayakar S, Kawaguchi R, Wang Q, Peterson S, Shi C, Turnes BL, Zhang Z, Oses-Prieto J, Li J, Burlingame A, Woolf CJ, Geschwind D, Rasband M, Benowitz LI. The oncomodulin receptor ArmC10 enables axon regeneration in mice after nerve injury and neurite outgrowth in human iPSC-derived sensory neurons. Sci Transl Med 2023;15(708):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.
Tummino TA. Applications and Confounds in Drug Discovery and Repurposing [Internet]. University of California, San Francisco, Pharmaceutical Sciences and Pharmacogenomics 2023;(30570041) Publisher's VersionAbstract

The process of discovering a new drug 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 drug candidates from artifacts and suboptimal leads. In fact, it has been said that a Drug Hunter’s 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 application of computational drug discovery and repurposing to identify new treatments for diseases. Secondly, it is the mechanistic understanding of two artifacts common in early-stage drug discovery and repurposing that if used appropriately, should remove potential false-positive screening hits from being pursued as lead candidates.

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

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

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

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

Chapter 5 applies the large-scale docking technique on the lipid-binding G-protein coupled cannabinoid-1 (CB1) receptor. Here, we demonstrate the concept of “new chemistry for new biology” 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.

Simpson MS, De Luca H, Cauthorn S, Luong P, Udeshi ND, Svinkina T, Schmeider SS, Carr SA, Grey MJ, Lencer WI. Structural basis for recognition of unfolded proteins by the ER stress sensor ERN1/IRE1α [Internet]. bioRxiv 2023; Publisher's VersionAbstract
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.
Shlosman I, Fivenson EM, Gilman MSA, Sisley TA, Walker S, Bernhardt TG, Kruse AC, Loparo JJ. Allosteric activation of cell wall synthesis during bacterial growth. Nat Commun 2023;14(1):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.
A M, Wales TE, Zhou H, Draga-Coletă S-V, Gorgulla C, Blackmore KA, Mittenbühler MJ, Kim CR, Bogoslavski D, Zhang Q, Wang Z-F, Jedrychowski MP, Seo H-S, Song K, Xu AZ, Sebastian L, Gygi SP, Arthanari H, Dhe-Paganon S, Griffin PR, Engen JR, Spiegelman BM. Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α. Mol Cell 2023;83(11):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 Å 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.
Fram B, Truebridge I, Su Y, Riesselman AJ, Ingraham JB, Passera A, Napier E, Thadani NN, Lim S, Roberts K, Kaur G, Stiffler M, Marks DS, Bahl CD, Khan AR, Sander C, Gauthier NP. Simultaneous enhancement of multiple functional properties using evolution-informed protein design. bioRxiv 2023;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.
Piatt SC. Disordered Proteins Regulate Stress- and Damage-Dependent DNA Binding [Internet]. Harvard University - Biophysics 2023; Publisher's VersionAbstract
Three-dimensional structure has been used as a starting point to characterize protein function since the advent of X-ray crystallography in the 1950s. However, not all proteins bear stable tertiary or even secondary structures. Referred to as intrinsically disordered protein 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 E. coli single-stranded binding protein (SSB), the C-terminus of X. laevis 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 DNA during times of cellular stress and DNA 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 DNA ends. Finally, preliminary NMR and DNA binding data show that phosphorylations to the ESR1 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 DNA 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, DNA repair, and the transcription of genes in response to DNA damage.
Park Y, Taguchi A, Baidin V, Kahne D, Walker S. A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW. Angew Chem Int Ed Engl 2023;62(25):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 Förster 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.
Pao P-C, Seo J, Lee A, Kritskiy O, Patnaik D, Penney J, Raju RM, Geigenmuller U, Silva CM, Lucente DE, Gusella JF, Dickerson BC, Loon A, Yu MX, Bula M, Yu M, Haggarty SJ, Tsai L-H. A Cdk5-derived peptide inhibits Cdk5/p25 activity and improves neurodegenerative phenotypes. Proc Natl Acad Sci U S A 2023;120(16):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's therapeutic potential.
Chanez-Paredes SD, Abtahi S, Zha J, Zuo L, He W, Turner JR. Distinct steady-state properties and TNF responses of epithelial long myosin light chain kinase (MLCK) splice variants [Internet]. bioRxiv 2023; Publisher's VersionAbstract
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’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’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.
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