scholarly journals N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 769
Author(s):  
Nadishka Jayawardena ◽  
Linde A. Miles ◽  
Laura N. Burga ◽  
Charles Rudin ◽  
Matthias Wolf ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Cancer Therapeutics ◽  
Anthrax Toxin ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Toxin Receptor ◽  
Seneca Valley Virus ◽  
Complex Glycans

Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

scholarly journals Seneca Valley virus attachment and uncoating mediated by its receptor anthrax toxin receptor 1

2018 ◽  
Vol 115 (51) ◽  
pp. 13087-13092 ◽  
Author(s):  
Lin Cao ◽  
Ran Zhang ◽  
Tingting Liu ◽  
Zixian Sun ◽  
Mingxu Hu ◽  
...  
Keyword(s):  
Metal Ion ◽  
Structural Changes ◽  
Attachment Site ◽  
Cell Entry ◽  
Anthrax Toxin ◽  
Virus Propagation ◽  
Virus Attachment ◽  
Toxin Receptor ◽  
Seneca Valley Virus ◽  
Acidic Conditions

Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. SVV mediates cell entry by attachment to the receptor anthrax toxin receptor 1 (ANTXR1). Here we determine atomic structures of mature SVV particles alone and in complex with ANTXR1 in both neutral and acidic conditions, as well as empty “spent” particles in complex with ANTXR1 in acidic conditions by cryoelectron microscopy. SVV engages ANTXR1 mainly by the VP2 DF and VP1 CD loops, leading to structural changes in the VP1 GH loop and VP3 GH loop, which attenuate interprotomer interactions and destabilize the capsid assembly. Despite lying on the edge of the attachment site, VP2 D146 interacts with the metal ion in ANTXR1 and is required for cell entry. Though the individual substitution of most interacting residues abolishes receptor binding and virus propagation, a serine-to-alanine mutation at VP2 S177 significantly increases SVV proliferation. Acidification of the SVV–ANTXR1 complex results in a major reconfiguration of the pentameric capsid assemblies, which rotate ∼20° around the icosahedral fivefold axes to form a previously uncharacterized spent particle resembling a potential uncoating intermediate with remarkable perforations at both two- and threefold axes. These structures provide high-resolution snapshots of SVV entry, highlighting opportunities for anticancer therapeutic optimization.

Pinpointing cysteine oxidation sites by high-resolution proteomics reveals a mechanism of redox-dependent inhibition of human STING

2021 ◽  
Vol 14 (680) ◽  
pp. eaaw4673
Author(s):  
Natalia Zamorano Cuervo ◽  
Audray Fortin ◽  
Elise Caron ◽  
Stéfany Chartier ◽  
Nathalie Grandvaux
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Disulfide Bonds ◽  
Redox Regulation ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Cysteine Oxidation ◽  
Spectrometry Analysis ◽  
Host Interactions ◽  
In Cells

Protein function is regulated by posttranslational modifications (PTMs), among which reversible oxidation of cysteine residues has emerged as a key regulatory mechanism of cellular responses. Given the redox regulation of virus-host interactions, the identification of oxidized cysteine sites in cells is essential to understand the underlying mechanisms involved. Here, we present a proteome-wide identification of reversibly oxidized cysteine sites in oxidant-treated cells using a maleimide-based bioswitch method coupled to mass spectrometry analysis. We identified 2720 unique oxidized cysteine sites within 1473 proteins with distinct abundances, locations, and functions. Oxidized cysteine sites were found in numerous signaling pathways, many relevant to virus-host interactions. We focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway, which is stimulated in response to the detection of cytosolic DNA by cGAS. We demonstrated the reversible oxidation of Cys148 and Cys206 of STING in cells. Molecular analyses led us to establish a model in which Cys148 oxidation is constitutive, whereas Cys206 oxidation is inducible by oxidative stress or by the natural ligand of STING, 2′3′-cGAMP. Our data suggest that the oxidation of Cys206 prevented hyperactivation of STING by causing a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This finding should aid the design of therapies targeting STING that are relevant to autoinflammatory disorders, immunotherapies, and vaccines.

Abstract 416: Sarcomere Disassembly After Unloading is Regulated by Ubiquitination and Acetylation of Capz and α-actinin

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Christopher Solís ◽  
R John Solaro ◽  
Chad M Warren ◽  
Brenda Russell
Keyword(s):  
Ventricular Myocytes ◽  
Mass Spectrometry Analysis ◽  
Actin Binding ◽  
Mechanical Forces ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Time Period ◽  
Omecamtiv Mecarbil ◽  
Myocyte Contractility ◽  
Sarcomere Assembly

Cardiac function mainly depends on the total myocyte mass in the ventricles. Assembly and disassembly of sarcomeres occurs to adjust this mass to altered mechanical demand. In the heart, hypertrophic cardiomyopathy results from myofibrillar assembly controlled by post-translational modification of proteins directed by signaling pathways. More is known about assembly on loading than disassembly on unloading. Here, the hypothesis tested is that unloading of mechanical forces affects acetylation (Ac) and ubiquitination (Ub) of the actin-binding proteins, α-actinin and CapZ. Omecamtiv mecarbil (0.5 μM) and mavacamten (1 μM) were used to increase (load) and decrease (unload) cardiomyocyte tension, respectively, via their action on myosin ATPase. Mavacamten decreased myocyte contractility in rat ventricular myocytes (NRVMs) and caused significant sarcomere disassembly by 6 h and 70% atrophy by 24 h. Assembly was preserved with omecamtiv mecarbil (0.5 μM) over the 24 h time period. Post-translational modification was determined in loaded and unloaded NRVMs at 6 h of drug treatment. Bottom-up mass spectrometry analysis showed single residues in α-actinin and CapZ that were acetylated or ubiquitinated. Acetylation levels appeared to increase in the mavacamten-treated samples while these levels are preserved in untreated and omecamtiv mecarbil-treated samples. Ac and Ub in the Z-discs were quantified on immunofluorescent images. The Z-discs colocalized oligo-Ub (K-48 oligo-Ub linkage) and Ac in untreated samples; this Z-disc localization of Ub and Ac was diminished with unloading. Fluorescence recovery after photobleaching (FRAP) measurements of the dynamics of α-actinin and CapZ after reduced cell tension with mavacamten (1 μM) and omecamtiv mercabil (0.5 μM) are ongoing. Overall, results suggest sarcomere assembly is regulated by mechanical forces through a mechanism involving Ac and Ub of myofibrillar proteins. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis.

scholarly journals Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus

2018 ◽  
Vol 115 (46) ◽  
pp. E10934-E10940 ◽  
Author(s):  
Nadishka Jayawardena ◽  
Laura N. Burga ◽  
Richard A. Easingwood ◽  
Yoshimasa Takizawa ◽  
Matthias Wolf ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protective Antigen ◽  
Therapeutic Option ◽  
Oncolytic Viruses ◽  
Anthrax Toxin ◽  
Bacterial Toxin ◽  
Structural Basis ◽  
Electron Microscopy Analysis ◽  
Toxin Receptor ◽  
Seneca Valley Virus

Recently, the use of oncolytic viruses in cancer therapy has become a realistic therapeutic option. Seneca Valley Virus (SVV) is a newly discovered picornavirus, which has earned a significant reputation as a potent oncolytic agent. Anthrax toxin receptor 1 (ANTXR1), one of the cellular receptors for the protective antigen secreted by Bacillus anthracis, has been identified as the high-affinity cellular receptor for SVV. Here, we report the structure of the SVV-ANTXR1 complex determined by single-particle cryo-electron microscopy analysis at near-atomic resolution. This is an example of a shared receptor structure between a mammalian virus and a bacterial toxin. Our structure shows that ANTXR1 decorates the outer surface of the SVV capsid and interacts with the surface-exposed BC loop and loop II of VP1, “the puff” of VP2 and “the knob” of VP3. Comparison of the receptor-bound capsid structure with the native capsid structure reveals that receptor binding induces minor conformational changes in SVV capsid structure, suggesting the role of ANTXR1 as an attachment receptor. Furthermore, our results demonstrate that the capsid footprint on the receptor is not conserved in anthrax toxin receptor 2 (ANTXR2), thereby providing a molecular mechanism for explaining the exquisite selectivity of SVV for ANTXR1.

rBAT-b0,+AT heterodimer is the main apical reabsorption system for cystine in the kidney

2002 ◽  
Vol 283 (3) ◽  
pp. F540-F548 ◽  
Author(s):  
Esperanza Fernández ◽  
Montserrat Carrascal ◽  
Ferran Rousaud ◽  
Joaquín Abián ◽  
Antonio Zorzano ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Expression Patterns ◽  
Human Kidney ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Spectrometry Analysis ◽  
Sds Page ◽  
Heterologous Cell ◽  
Dibasic Amino Acids

Mutations in the rBAT and b0,+AT genes cause type I and non-type I cystinuria, respectively. The disulfide-linked rBAT-b0,+AT heterodimer mediates high-affinity transport of cystine and dibasic amino acids (b0,+-like activity) in heterologous cell systems. However, the significance of this heterodimer for cystine reabsorption is unknown, as direct evidence for such a complex in vivo is lacking and the expression patterns of rBAT and b0,+AT along the proximal tubule are opposite. We addressed this issue by biochemical means. Western blot analysis of mouse and human kidney brush-border membranes showed that rBAT and b0,+AT were solely expressed as heterodimers of identical size and that both proteins coprecipitated. Moreover, quantitative immunopurification of b0,+AT followed by SDS-PAGE and mass spectrometry analysis established that b0,+AT heterodimerizes exclusively with rBAT. Together with cystine reabsorption data, our results demonstrate that a decreasing expression gradient of heterodimeric rBAT-b0,+AT along the proximal tubule is responsible for virtually all apical cystine reabsorption. As a corollary of the above, there should be an excess of rBAT expression over that of b0,+AT protein in the kidney. Indeed, complete immunodepletion of b0,+AT did not coprecipitate >20–30% of rBAT. Therefore, another rBAT-associated subunit may be present in latter parts of the proximal tubule.

scholarly journals β-N-Acetylglucosamine (O-GlcNAc) Is a Novel Regulator of Mitosis-specific Phosphorylations on Histone H3

2012 ◽  
Vol 287 (15) ◽  
pp. 12195-12203 ◽  
Author(s):  
Jerry J. Fong ◽  
Brenda L. Nguyen ◽  
Robert Bridger ◽  
Estela E. Medrano ◽  
Lance Wells ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Histone H3 ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Cycle Progression ◽  
Spectrometry Analysis ◽  
Biochemical Assays ◽  
M Phase ◽  
Nucleocytoplasmic Proteins

O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.

scholarly journals Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike S protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications

2020 ◽  
Author(s):  
Zeyu Sun ◽  
Keyi Ren ◽  
Xing Zhang ◽  
Jinghua Chen ◽  
Zhengyi Jiang ◽  
...  
Keyword(s):  
Structural Models ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Complex Type ◽  
Post Translational Modifications ◽  
S Protein ◽  
Spectrometry Analysis ◽  
Glycosylation Sites ◽  
Binding Surface ◽  
Structural Details

AbstractThe pneumonia-causing COVID-19 pandemia has prompt worldwide efforts to understand its biological and clinical traits of newly identified HCoV-19 virus. In this study, post-translational modification (PTM) of recombinant HCoV-19 S and hACE2 were characterized by LC-MSMS. We revealed that both proteins were highly decorated with specific proportions of N-glycan subtypes. Out of 21 possible glycosites in HCoV-19 S protein, 20 were confirmed completely occupied by N-glycans, with oligomannose glycans being the most abundant type. All 7 possible glycosylation sites in hACE2 were completely occupied mainly by complex type N-glycans. However, we showed that glycosylation did not directly contribute to the binding affinity between SARS-CoV spike protein and hACE2. Additionally, we also identified multiple sites methylated in both proteins, and multiple prolines in hACE2 were converted to hydroxylproline. Refined structural models were built by adding N-glycan and PTMs to recently published cryo-EM structure of the HCoV-19 S and hACE2 generated with glycosylation sites in the vicinity of binding surface. The PTM and glycan maps of both HCoV-19 S and hACE2 provide additional structural details to study mechanisms underlying host attachment, immune response mediated by S protein and hACE2, as well as knowledge to develop remedies and vaccines desperately needed nowadays.

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