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.

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.

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 Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

2009 ◽  
Vol 9 (1) ◽  
pp. 98 ◽  
Author(s):  
Tao Wu ◽  
Tiezheng Yuan ◽  
Sau-Na Tsai ◽  
Chunmei Wang ◽  
Sai-Ming Sun ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Histone H3 ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modifications ◽  
Spectrometry Analysis

scholarly journals Proteomic Identification of an Endogenous Synaptic SUMOylome in the Developing Rat Brain

2021 ◽  
Vol 14 ◽  
Author(s):  
Marie Pronot ◽  
Félicie Kieffer ◽  
Anne-Sophie Gay ◽  
Delphine Debayle ◽  
Raphaël Forquet ◽  
...  
Keyword(s):  
Subcellular Fractionation ◽  
Mass Spectrometry Analysis ◽  
Mammalian Brain ◽  
Synaptic Organization ◽  
Post Translational Modifications ◽  
Spectrometry Analysis ◽  
Functional Changes ◽  
Neuronal Communication ◽  
Associated Proteins ◽  
And Function

Synapses are highly specialized structures that interconnect neurons to form functional networks dedicated to neuronal communication. During brain development, synapses undergo activity-dependent rearrangements leading to both structural and functional changes. Many molecular processes are involved in this regulation, including post-translational modifications by the Small Ubiquitin-like MOdifier SUMO. To get a wider view of the panel of endogenous synaptic SUMO-modified proteins in the mammalian brain, we combined subcellular fractionation of rat brains at the post-natal day 14 with denaturing immunoprecipitation using SUMO2/3 antibodies and tandem mass spectrometry analysis. Our screening identified 803 candidate SUMO2/3 targets, which represents about 18% of the synaptic proteome. Our dataset includes neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins as well as vesicular trafficking and cytoskeleton-associated proteins, defining SUMO2/3 as a central regulator of the synaptic organization and function.

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