scholarly journals Bioinformatics analysis reveals biophysical and evolutionary insights into the 3-nitrotyrosine post-translational modification in the human proteome

Open Biology ◽  
2013 ◽  
Vol 3 (2) ◽  
pp. 120148 ◽  
Author(s):  
John Y. Ng ◽  
Lies Boelen ◽  
Jason W. H. Wong
Keyword(s):  
Protein Function ◽  
Bioinformatics Analysis ◽  
Proteome Analysis ◽  
Human Proteome ◽  
Prediction Algorithm ◽  
Post Translational Modification ◽  
Tyrosine Residues ◽  
Cellular Processes ◽  
Wide Range ◽  
Human Proteins

Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function.

scholarly journals Mechanisms, regulation and consequences of protein SUMOylation

2010 ◽  
Vol 428 (2) ◽  
pp. 133-145 ◽  
Author(s):  
Kevin A. Wilkinson ◽  
Jeremy M. Henley
Keyword(s):  
Protein Function ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Wide Range ◽  
Lysine Residues ◽  
Ubiquitination Pathway ◽  
Regulate Protein ◽  
Substrate Proteins ◽  
Protein Sumoylation

The post-translational modification SUMOylation is a major regulator of protein function that plays an important role in a wide range of cellular processes. SUMOylation involves the covalent attachment of a member of the SUMO (small ubiquitin-like modifier) family of proteins to lysine residues in specific target proteins via an enzymatic cascade analogous to, but distinct from, the ubiquitination pathway. There are four SUMO paralogues and an increasing number of proteins are being identified as SUMO substrates. However, in many cases little is known about how SUMOylation of these targets is regulated. Compared with the ubiquitination pathway, relatively few components of the conjugation machinery have been described and the processes that specify individual SUMO paralogue conjugation to defined substrate proteins are an active area of research. In the present review, we briefly describe the SUMOylation pathway and present an overview of the recent findings that are beginning to identify some of the mechanisms that regulate protein SUMOylation.

scholarly journals Comparison of three glycoproteomic methods for the analysis of CHO cells treated with 1,3,4-O-Bu3ManNAc

2020 ◽  
Author(s):  
Joseph L. Mertz ◽  
Shisheng Sun ◽  
Bojiao Yin ◽  
Michael J. Betenbaugh ◽  
Kevin J. Yarema ◽  
...  
Keyword(s):  
Cho Cells ◽  
Protein Function ◽  
Solid Phase ◽  
Chinese Hamster ◽  
Comprehensive Analysis ◽  
The Other ◽  
Post Translational Modification ◽  
Hydrophilic Interaction ◽  
Analysis Methods ◽  
Human Proteins

AbstractComprehensive analysis of the glycoproteome is critical due to the widespread importance of this post-translational modification to protein function, and difficult because of the tremendous complexity it exhibits. Here we compared three glycoproteomic analysis methods, a recently described chemoenzymatic glycoproteome analysis methods, N-linked glycans and glycosite containing peptides (NGAG), Solid-phase extraction of N-linked glycoproteins (SPEG), and hydrophilic interaction liquid chromatography (HILIC), for the analysis of N-linked glycosites of Chinese hamster ovarian (CHO) cells treated with 1,3,4-O-Bu3ManNAc. The NGAG protocol resulted in substantially increased glycosite identifications over both SPEG and HILIC. Interestingly, while the glycosites identified by SPEG and HILIC overlapped strongly, NGAG identified many glycosites not observed in either of the other two methods. Further, utilizing the enhanced intact glycopeptide identification afforded by the NGAG workflow, we also found that of the sugar analog 1,3,4-O-Bu3ManNAc increases sialylation of proteins secreted by CHO cells, including an ectopically expressed human proteins.

scholarly journals Ubiquitin-Specific Proteases: Players in Cancer Cellular Processes

2021 ◽  
Vol 14 (9) ◽  
pp. 848
Author(s):  
Lucas Cruz ◽  
Paula Soares ◽  
Marcelo Correia
Keyword(s):  
Protein Function ◽  
Deubiquitinating Enzymes ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Cellular Targets ◽  
Cellular Processes ◽  
Protein Ubiquitination ◽  
Damage Repair ◽  
Ubiquitin Molecule

Ubiquitination represents a post-translational modification (PTM) essential for the maintenance of cellular homeostasis. Ubiquitination is involved in the regulation of protein function, localization and turnover through the attachment of a ubiquitin molecule(s) to a target protein. Ubiquitination can be reversed through the action of deubiquitinating enzymes (DUBs). The DUB enzymes have the ability to remove the mono- or poly-ubiquitination signals and are involved in the maturation, recycling, editing and rearrangement of ubiquitin(s). Ubiquitin-specific proteases (USPs) are the biggest family of DUBs, responsible for numerous cellular functions through interactions with different cellular targets. Over the past few years, several studies have focused on the role of USPs in carcinogenesis, which has led to an increasing development of therapies based on USP inhibitors. In this review, we intend to describe different cellular functions, such as the cell cycle, DNA damage repair, chromatin remodeling and several signaling pathways, in which USPs are involved in the development or progression of cancer. In addition, we describe existing therapies that target the inhibition of USPs.

scholarly journals A versatile new ubiquitin detection and purification tool derived from a bacterial deubiquitylase

2021 ◽  
Author(s):  
Mengwen Zhang ◽  
Jason M. Berk ◽  
Adrian B. Mehrtash ◽  
Jean Kanyo ◽  
Mark Hochstrasser
Keyword(s):  
Quantitative Proteomics ◽  
Affinity Purification ◽  
Interacting Proteins ◽  
Post Translational Modification ◽  
Considerable Effort ◽  
E3 Ligases ◽  
Cellular Processes ◽  
Wide Range ◽  
Ubiquitin Binding ◽  
Ubiquitin Binding Domain

AbstractProtein ubiquitylation is an important post-translational modification affecting an wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on developing methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain derived from an Orientia tsutsugamushi deubiquitylase. We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As a proof-of-principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin researchers with specific advantages over other methods, such as in detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

scholarly journals An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

2021 ◽  
Author(s):  
Fangxu Sun ◽  
Suttipong Suttapitugsakul ◽  
Ronghu Wu
Keyword(s):  
Large Scale ◽  
Diazonium Salt ◽  
Human Proteome ◽  
Binding Property ◽  
Tyrosine Residue ◽  
Azo Coupling ◽  
Mcf7 Cells ◽  
Bioorthogonal Chemistry ◽  
Tyrosine Residues ◽  
Wide Range

<p>The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, the two different probes were evaluated, and the probe with the best performance was employed to specifically target the tyrosine residues. After the reaction, tagged tyrosine containing peptides were selectively enriched using bioorthogonal chemistry, and a small tag on the peptides from the cleavage perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5,000 tyrosine sites in MCF7 cells and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.</p>

scholarly journals An advanced strategy for comprehensive profiling of ADP-ribosylation sites using mass spectrometry-based proteomics

2018 ◽  
Author(s):  
Ivo A. Hendriks ◽  
Sara C. Larsen ◽  
Michael L. Nielsen
Keyword(s):  
Electron Transfer ◽  
Electron Transfer Dissociation ◽  
Enrichment Analysis ◽  
Functional Enrichment Analysis ◽  
Functional Enrichment ◽  
Post Translational Modification ◽  
Tyrosine Residues ◽  
Adp Ribosylation ◽  
Cellular Processes ◽  
Nuclear Proteome

ABSTRACTADP-ribosylation is a widespread post-translational modification (PTM) with crucial functions in many cellular processes. Here, we describe an in-depth ADP-ribosylome using our Af1521-based proteomics methodology for comprehensive profiling of ADP-ribosylation sites, by systematically assessing complementary proteolytic digestions and precursor fragmentation through application of electron-transfer higher-energy collisional dissociation (EThcD) and electron transfer dissociation (ETD), respectively. While ETD spectra yielded higher identification scores, EThcD generally proved superior to ETD in identification and localization of ADP-ribosylation sites regardless of protease employed. Notwithstanding, the propensities of complementary proteases and fragmentation methods expanded the detectable repertoire of ADP-ribosylation to an unprecedented depth. This system-wide profiling of the ADP-ribosylome in HeLa cells subjected to DNA damage uncovered >11,000 unique ADP-ribosylated peptides mapping to >7,000 ADP-ribosylation sites, in total modifying over one-third of the human nuclear proteome and highlighting the vast scope of this PTM. High-resolution MS/MS spectra enabled identification of dozens of proteins concomitantly modified by ADP-ribosylation and phosphorylation, revealing a considerable degree of crosstalk on histones. ADP-ribosylation was confidently localized to various amino acid residue types, including less abundantly modified residues, with hundreds of ADP-ribosylation sites pinpointed on histidine, arginine, and tyrosine residues. Functional enrichment analysis suggested modification of these specific residue types is directed in a spatial manner, with tyrosine ADP-ribosylation linked to the ribosome, arginine ADP-ribosylation linked to the endoplasmic reticulum, and histidine ADP-ribosylation linked to the mitochondrion.

scholarly journals Post-translational lysine ac(et)ylation in health, ageing and disease

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Anna-Theresa Blasl ◽  
Sabrina Schulze ◽  
Chuan Qin ◽  
Leonie G. Graf ◽  
Robert Vogt ◽  
...  
Keyword(s):  
Protein Function ◽  
Molecular Mechanisms ◽  
Research Field ◽  
Ageing Process ◽  
Post Translational Modification ◽  
Direct Role ◽  
Translational Machinery ◽  
Beneficial Effects ◽  
Cellular Processes ◽  
Technological Advances

Abstract The acetylation/acylation (ac(et)ylation) of lysine side chains is a dynamic post-translational modification (PTM) regulating fundamental cellular processes with implications on the organisms’ ageing process: metabolism, transcription, translation, cell proliferation, regulation of the cytoskeleton and DNA damage repair. First identified to occur on histones, later studies revealed the presence of lysine ac(et)ylation in organisms of all kingdoms of life, in proteins covering all essential cellular processes. A remarkable finding showed that the NAD+-dependent sirtuin deacetylase Sir2 has an impact on replicative lifespan in Saccharomyces cerevisiae suggesting that lysine acetylation has a direct role in the ageing process. Later studies identified sirtuins as mediators for beneficial effects of caloric/dietary restriction on the organisms’ health- or lifespan. However, the molecular mechanisms underlying these effects are only incompletely understood. Progress in mass-spectrometry, structural biology, synthetic and semi-synthetic biology deepened our understanding of this PTM. This review summarizes recent developments in the research field. It shows how lysine ac(et)ylation regulates protein function, how it is regulated enzymatically and non-enzymatically, how a dysfunction in this post-translational machinery contributes to disease development. A focus is set on sirtuins and lysine acyltransferases as these are direct sensors and mediators of the cellular metabolic state. Finally, this review highlights technological advances to study lysine ac(et)ylation.

scholarly journals Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

2020 ◽  
Author(s):  
Sara C. Buch-Larsen ◽  
Ivo A. Hendriks ◽  
Jean M. Lodge ◽  
Martin Rykær ◽  
Benjamin Furtwängler ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Electron Transfer Dissociation ◽  
Post Translational Modification ◽  
Physiological Regulation ◽  
Physiological Conditions ◽  
Adp Ribosylation ◽  
Cellular Processes ◽  
Wide Range ◽  
Enrichment Strategy

SUMMARYADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profiled 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress, corresponding to 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and EThcD, respectively. Under physiological conditions AI-ETD identified 450 ADPr sites on low-abundant proteins, including in vivo cysteine auto-modifications on PARP8 and tyrosine auto-modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provides new insights into the physiological regulation of ADP-ribosylation.

scholarly journals The functional landscape of the human phosphoproteome

2019 ◽  
Author(s):  
David Ochoa ◽  
Andrew F. Jarnuczak ◽  
Maja Gehre ◽  
Margaret Soucheray ◽  
Askar A. Kleefeldt ◽  
...  
Keyword(s):  
Protein Function ◽  
Molecular Mechanisms ◽  
State Of The Art ◽  
Functional Score ◽  
Post Translational Modification ◽  
Functional Sites ◽  
Cellular Processes ◽  
Major Bottleneck ◽  
Genomic Scale ◽  
Almost All

AbstractProtein phosphorylation is a key post-translational modification regulating protein function in almost all cellular processes. While tens of thousands of phosphorylation sites have been identified in human cells to date, the extent and functional importance of the phosphoproteome remains largely unknown. Here, we have analyzed 6,801 publicly available phospho-enriched mass spectrometry proteomics experiments, creating a state-of-the-art phosphoproteome containing 119,809 human phosphosites. To prioritize functional sites, 59 features indicative of proteomic, structural, regulatory or evolutionary relevance were integrated into a single functional score using machine learning. We demonstrate how this prioritization identifies regulatory phosphosites across different molecular mechanisms and pinpoint genetic susceptibilities at a genomic scale. Several novel regulatory phosphosites were experimentally validated including a role in neuronal differentiation for phosphosites present in the SWI/SNF SMARCC2 complex member. The scored reference phosphoproteome and its annotations identify the most relevant phosphorylations for a given process or disease addressing a major bottleneck in cell signaling studies.

scholarly journals Sirtuins: Enzymes with multidirectional catalytic activity

2021 ◽  
Vol 75 ◽  
pp. 152-174
Author(s):  
Ewa Maria Kratz ◽  
Katarzyna Sołkiewicz ◽  
Agnieszka Kaczmarek ◽  
Agnieszka Piwowar
Keyword(s):  
Reactive Oxygen Species ◽  
Catalytic Activity ◽  
Human Body ◽  
Histone Deacetylases ◽  
Oxygen Species ◽  
Post Translational Modification ◽  
Detailed Understanding ◽  
Cellular Processes ◽  
Wide Range

Sirtuins (SIRT) are NAD+-dependent histone deacetylases that play an important role in the functioning of the human body. They participate in numerous processes taking place in cells, including in the post-translational modification of proteins, silencing gene transcription, inducing repair processes, as well as in the regulation of metabolic processes. Sirtuins have also been shown to play an important role in reducing the level of reactive oxygen species as well as in stimulating cell growth, aging and death. Such a wide range of processes, which are affected by sirtuins, have recently made sirtuins the object of many studies aimed at a detailed understanding of the mechanisms of their action and the role they play. The aim of our study was to collect and systematize information on sirtuins, mainly from the last 10 years, both regarding the human body and based on the results of research on animal models or cell lines. The article discusses the structure, function and biological role of sirtuins in cellular processes.

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