2023
Plummer-Medeiros AM, Culbertson AT, Morales-Perez CL, Liao M.
Activity and Structural Dynamics of Human ABCA1 in a Lipid Membrane. J Mol Biol 2023;435(8):168038.
AbstractThe 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'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-Å 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.
Morimoto Y, Yamashita N, Daimon T, Hirose H, Yamano S, Haratake N, Ishikawa S, Bhattacharya A, Fushimi A, Ahmad R, Takahashi H, Dashevsky O, Mitsiades C, Kufe D.
MUC1-C is a master regulator of MICA/B NKG2D ligand and exosome secretion in human cancer cells. J Immunother Cancer 2023;11(2)
AbstractBACKGROUND: 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→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.
Curreri AM, Kim J, Dunne M, Angsantikul P, Goetz M, Gao Y, Mitragotri S.
Deep Eutectic Solvents for Subcutaneous Delivery of Protein Therapeutics. Adv Sci (Weinh) 2023;:e2205389.
AbstractProteins 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.
Zuo L, Kuo W-T, Cao F, Chanez-Paredes SD, Zeve D, Mannam P, Jean-François Léa, Day A, Vallen Graham W, Sweat YY, Shashikanth N, Breault DT, Turner JR.
Tacrolimus-binding protein FKBP8 directs myosin light chain kinase-dependent barrier regulation and is a potential therapeutic target in Crohn's disease. Gut 2023;72(5):870-881.
AbstractOBJECTIVE: Intestinal barrier loss is a Crohn'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.
2022
Thorsen MK.
Biochemical, Biophysical and Structural Characterization of the Conserved Herpesviral Nuclear Egress Complex. Tufts University-Graduate School of Biomedical Sciences 2022;
AbstractTo 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.
Leavitt A, Yirmiya E, Amitai G, Lu A, Garb J, Herbst E, Morehouse BR, Hobbs SJ, Antine SP, Sun Z-YJ, Kranzusch PJ, Sorek R.
Viruses inhibit TIR gcADPR signalling to overcome bacterial defence. Nature 2022;611(7935):326-331.
AbstractThe 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 'sponges' 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 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' 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.
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 [Internet]. bioRxiv 2022;
Publisher's VersionAbstractThe 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.
Chanez-Paredes SD, Abtahi S, Zha J, Zuo L, He W, Turner JR.
Selective perijunctional MLCK1 recruitment in Crohn’s disease: Identification of essential structural domains [Internet]. bioRxiv 2022;
Publisher's VersionAbstractIntestinal 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’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’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’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.
Chang S, Thrall ES, Laureti L, Piatt SC, Pagès V, Loparo JJ.
Compartmentalization of the replication fork by single-stranded DNA-binding protein regulates translesion synthesis. Nat Struct Mol Biol 2022;29(9):932-941.
AbstractProcessivity 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.
Park EY, Rawson S, Schmoker A, Kim B-W, Oh S, Song KK, Jeon H, Eck M.
Cryo-EM structure of a RAS/RAF recruitment complex [Internet]. bioRxiv 2022;
Publisher's VersionAbstractCryo-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.
Feng J, Dong X, Su Y, Lu C, Springer TA.
Monomeric prefusion structure of an extremophile gamete fusogen and stepwise formation of the postfusion trimeric state. Nat Commun 2022;13(1):4064.
AbstractHere, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 °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.
Bonazza K, Iacob RE, Hudson NE, Li J, Lu C, Engen JR, Springer TA.
Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its -glycosylated N- and C-linker. Elife 2022;11
AbstractHemostasis 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.
Jiang H, Chiang CY, Chen Z, Nathan S, D'Agostino G, Paulo JA, Song G, Zhu H, Gabelli SB, Cole PA.
Enzymatic analysis of WWP2 E3 ubiquitin ligase using protein microarrays identifies autophagy-related substrates. J Biol Chem 2022;298(5):101854.
AbstractWWP2 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 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.
Erlandson SC, Wang J, Jiang H, Rockman HA, Kruse AC.
Engineering and characterization of a long half-life relaxin receptor RXFP1 agonist [Internet]. bioRxiv 2022;
Publisher's VersionAbstractRelaxin-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.
Duncan-Lowey B.
Effectors of Cell Death in Bacterial Antiphage Defense. Harvard University Graduate School of Arts and Sciences 2022;
AbstractBacteria 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.
Velarde JJ, Piai A, Lichtenstein IJ, Lynskey NN, Chou JJ, Wessels MR.
Structure of the Streptococcus pyogenes NAD Glycohydrolase Translocation Domain and Its Essential Role in Toxin Binding to Oropharyngeal Keratinocytes. J Bacteriol 2022;204(1):e0036621.
AbstractThe 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.
Huang P, Wierbowski BM, Lian T, Chan C, García-Linares S, Jiang J, Salic A.
Structural basis for catalyzed assembly of the Sonic hedgehog-Patched1 signaling complex. Dev Cell 2022;57(5):670-685.e8.
AbstractThe 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.
Jiang H.
Mechanistic Studies of NEDD4 Family HECT E3 Ubiquitin Ligases. Johns Hopkins University 2022;
Abstract
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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
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Harvey EP, Shin J-E, Skiba MA, Nemeth GR, Hurley JD, Wellner A, Shaw AY, Miranda VG, Min JK, Liu CC, Marks DS, Kruse AC.
An in silico method to assess antibody fragment polyreactivity [Internet]. bioRxiv 2022;
Publisher's VersionAbstractAntibodies 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{\"ıve synthetic camelid antibody fragment (‘nanobody’) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our model’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.
