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 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 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.

Arabidopsis phospholipase Dδ as an initiator of cytoskeleton-mediated signalling to fundamental cellular processes

2009 ◽  
Vol 36 (2) ◽  
pp. 190 ◽  
Author(s):  
Angela Y. Y. Ho ◽  
David A. Day ◽  
Melissa H. Brown ◽  
Jan Marc
Keyword(s):  
Potential Interaction ◽  
Mass Spectrometry Analysis ◽  
Actin Binding ◽  
Sequence Alignments ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Plant Signal Transduction ◽  
Interaction Partners ◽  
Cytoskeletal Rearrangements ◽  
Partner Proteins

Phospholipase D (PLD), in combination with the cytoskeleton, plays a key role in plant signal transduction. One isotype of the multigene Arabidopsis PLD family, AtPLDδ, has been implicated in binding microtubules, although the molecular details of the mechanism and identities of potential interaction partners are unclear. We constructed a GFP-AtPLDδ reporter gene, stably transformed it into an Arabidopsis suspension cell line, and used epitope-tagged affinity pull-down assays to isolate a complex of co-purifying proteins. Mass spectrometry analysis of the complex revealed a set of proteins including β-tubulin, actin 7, HSP70, clathrin heavy chain, ATP synthase subunits, and a band 7–4/flotillin homologue. Sequence alignments with defined tubulin- and actin-binding regions from human HsPLD2 revealed highly homologous regions in all 12 AtPLD isotypes, suggesting direct interactions of AtPLDδ with tubulin and actin, while interactions with the remaining partners are likely to be mediated by the cytoskeleton. We propose that AtPLDδ acts through a complex of cytoskeletal and partner proteins to modulate fundamental cellular processes such as cytoskeletal rearrangements, vesicular trafficking, assembly of Golgi apparatus, mitosis and cytokinesis.

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 Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation

2021 ◽  
Vol 22 (13) ◽  
pp. 7011
Author(s):  
Barbora Mikolaskova ◽  
Matus Jurcik ◽  
Ingrid Cipakova ◽  
Tomas Selicky ◽  
Jan Jurcik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Processing ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Terminal Region ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Vitro Assay ◽  
Spectrometry Analysis

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.

scholarly journals Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

2020 ◽  
Author(s):  
Onyekachi E. Ononye ◽  
Christopher W. Sausen ◽  
Lata Balakrishnan ◽  
Matthew L. Bochman
Keyword(s):  
Limited Proteolysis ◽  
Strand Break ◽  
Mass Spectrometry Analysis ◽  
Helicase Domain ◽  
Lysine Acetylation ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Substrate Preferences ◽  
C Terminus ◽  
N Terminus

ABSTRACTIn S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.

scholarly journals Defining the Dynamic Regulation of O-GlcNAc Proteome in the Mouse Cortex---the O-GlcNAcylation of Synaptic and Trafficking Proteins Related to Neurodegenerative Diseases

2021 ◽  
Vol 2 ◽  
Author(s):  
Van N Huynh ◽  
Sheng Wang ◽  
Xiaosen Ouyang ◽  
Willayat Y Wani ◽  
Michelle S Johnson ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Synaptic Proteins ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Dynamic Regulation ◽  
Spectrometry Analysis ◽  
Adult Mice ◽  
Mouse Cortex ◽  
Circadian Clock Proteins ◽  
Pathway Analyses

O-linked conjugation of ß-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification process that senses nutrient availability and cellular stress and regulates diverse biological processes that are involved in neurodegenerative diseases and provide potential targets for therapeutics development. However, very little is known of the networks involved in the brain that are responsive to changes in the O-GlcNAc proteome. Pharmacological increase of protein O-GlcNAcylation by Thiamet G (TG) has been shown to decrease tau phosphorylation and neurotoxicity, and proposed as a therapy in Alzheimer’s disease (AD). However, acute TG exposure impairs learning and memory, and protein O-GlcNAcylation is increased in the aging rat brain and in Parkinson’s disease (PD) brains. To define the cortical O-GlcNAc proteome that responds to TG, we injected young adult mice with either saline or TG and performed mass spectrometry analysis for detection of O-GlcNAcylated peptides. This approach identified 506 unique peptides corresponding to 278 proteins that are O-GlcNAcylated. Of the 506 unique peptides, 85 peptides are elevated by > 1.5 fold in O-GlcNAcylation levels in response to TG. Using pathway analyses, we found TG-dependent enrichment of O-GlcNAcylated synaptic proteins, trafficking, Notch/Wnt signaling, HDAC signaling, and circadian clock proteins. Significant changes in the O-GlcNAcylation of DNAJC6/AUXI, and PICALM, proteins that are risk factors for PD and/or AD respectively, were detected. We compared our study with two key prior O-GlcNAc proteome studies using mouse cerebral tissue and human AD brains. Among those identified to be increased by TG, 15 are also identified to be increased in human AD brains compared to control, including those involved in cytoskeleton, autophagy, chromatin organization and mitochondrial dysfunction. These studies provide insights regarding neurodegenerative diseases therapeutic targets.

scholarly journals Redox Regulation of Nonmuscle Myosin Heavy Chain during Integrin Engagement

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Tania Fiaschi ◽  
Giacomo Cozzi ◽  
Paola Chiarugi
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Redox Regulation ◽  
Movement Analysis ◽  
Mass Spectrometry Analysis ◽  
Actin Binding ◽  
Nonmuscle Myosin ◽  
Spectrometry Analysis ◽  
Process Mass Spectrometry

On the basis of our findings reporting that cell adhesion induces the generation of reactive oxygen species (ROS) after integrin engagement, we were interested in identifying redox-regulated proteins during this process. Mass spectrometry analysis led us to identify nonmuscle myosin heavy chain (nmMHC) as a target of ROS. Our results show that, while nmMHC is reduced in detached/rounded cells, it turns towards an oxidized state in adherent/spread cells due to the integrin-engaged ROS machinery. The functional role of nmMHC redox regulation is suggested by the redox sensitivity of its association with actin, suggesting a role of nmMHC oxidation in cytoskeleton movement. Analysis of muscle MHC (mMHC) redox state during muscle differentiation, a process linked to a great and stable decrease of ROS content, shows that the protein does not undergo a redox control. Hence, we propose that the redox regulation of MHC in nonprofessional muscle cells is mandatory for actin binding during dynamic cytoskeleton rearrangement, but it is dispensable for static and highly organized cytoskeletal contractile architecture in differentiating myotubes.

scholarly journals Changes in subclass-specific IgG Fc glycosylation associated with the postnatal maturation of the murine immune system

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Gabriela Barrientos ◽  
Siniša Habazin ◽  
Mislav Novokmet ◽  
Yahia Almousa ◽  
Gordan Lauc ◽  
...  
Keyword(s):  
Immune System ◽  
Critical Time ◽  
Mass Spectrometry Analysis ◽  
Postnatal Life ◽  
Postnatal Maturation ◽  
Spectrometry Analysis ◽  
Time Period ◽  
Neonatal Immune System ◽  
Specific Igg ◽  
Early Postnatal Life

Abstract Early postnatal life is characterized by a critical time period in which the developing neonatal immune system transitions from passive immunity, induced by protective maternal antibodies, to the competence of a fully functioning immune system. The inflammatory capability of both maternal and neonatal antibodies is governed by N-linked glycosylation of the Fc region, and though this has been examined extensively in adults, there is currently little information regarding antibody glycosylation patterns during early postnatal life. To characterize the murine IgG Fc glycosylation profile during early life, we used nano-LC-ESI-Qq-TOF mass spectrometry analysis to assess subclass specific Asn-297 glycosylation patterns in the serum of BALB/c mice from 5–60 days of age. From birth to adulthood, we observed a decline in proinflammatory Fc glycosylation in all IgG subclasses. This was shown by significantly reduced agalactosylated and monogalactosylated structures combined with increased sialylation after weaning at 45 and 60 days of age. This information indicates that the transition between neonatal life and adulthood in mice is accompanied by reduction of inflammatory IgG antibodies. Our study contributes to a growing body of literature indicating the importance of IgG Fc glycosylation and its association with inflammation during different life stages.

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