Study Of Post-translational Modifications Research Articles

PEIMAN 1.0: Post-translational modification Enrichment, Integration and Matching ANalysis.

Conventional proteomics has discovered a wide gap between protein sequences and biological functions. The third generation of proteomics was provoked to bridge this gap. Targeted and untargeted post-translational modification (PTM) studies are the most important parts of today’s proteomics. Considering the expensive and time-consuming nature of experimental methods, computational methods are developed to study, analyze, predict, count and compute the PTM annotations on proteins. The enrichment analysis softwares are among the common computational biology and bioinformatic software packages. The focus of such softwares is to find the probability of occurrence of the desired biological features in any arbitrary list of genes/proteins. We introduce Post-translational modification Enrichment Integration and Matching Analysis (PEIMAN) software to explore more probable and enriched PTMs on proteins. Here, we also represent the statistics of detected PTM terms used in enrichment analysis in PEIMAN software based on the latest released version of UniProtKB/Swiss-Prot. These results, in addition to giving insight to any given list of proteins, could be useful to design targeted PTM studies for identification and characterization of special chemical groups.Database URL: http://bs.ipm.ir/softwares/PEIMAN/

Comparative Proteomics for the Evaluation of Protein Expression and Modifications in Neurodegenerative Diseases.

Together with hypothesis-driven approaches, high-throughput differential proteomic analysis performed primarily not only in human cerebrospinal fluid and serum but also on protein content of other tissues (blood cells, muscles, peripheral nerves, etc.) has been used in the last years to investigate neurodegenerative diseases. Even if the goal for these analyses was mainly the discovery of neurodegenerative disorders biomarkers, the characterization of specific posttranslational modifications (PTMs) and the differential protein expression resulted in being very informative to better define the pathological mechanisms. In this chapter are presented and discussed the positive aspects and challenges of the outcomes of some of our investigations on neurological and neurodegenerative disease, in order to highlight the important role of protein PTMs studies in proteomics-based approaches.

New roles for old modifications: emerging roles of N-terminal post-translational modifications in development and disease.

The importance of internal post-translational modification (PTM) in protein signaling and function has long been known and appreciated. However, the significance of the same PTMs on the alpha amino group of N-terminal amino acids has been comparatively understudied. Historically considered static regulators of protein stability, additional functional roles for N-terminal PTMs are now beginning to be elucidated. New findings show that N-terminal methylation, along with N-terminal acetylation, is an important regulatory modification with significant roles in development and disease progression. There are also emerging studies on the enzymology and functional roles of N-terminal ubiquitylation and N-terminal propionylation. Here, will discuss the recent advances in the functional studies of N-terminal PTMs, recount the new N-terminal PTMs being identified, and briefly examine the possibility of dynamic N-terminal PTM exchange.

Mycobacterial glycoproteins: a novel subset of vaccine candidates.

Over the last two decades significant research efforts and resources have been devoted to identifying mycobacterial proteins of value to diagnostic assays and vaccine formulations. These scientific endeavors were often preceded by first identifying a target population of proteins, for example cytoplasmic, cell envelope, or extracellular. However, many of these endeavors have overlooked the posttranslational modifications (PTM) to which these protein subsets may be targets of. Consequently, we may be missing essential molecular information relevant to the function and antigenicity of these effector molecules, and in turn the pathobiology of these bugs. Moreover, heterologous expression of mycobacterial proteins in hosts lacking homologous PTM systems negates any functional and/or antigenic role the PTM may impart. Functional and pathogenic roles of PTMs, such as protein glycosylation, have been reported for other Gram-positive bacteria, especially in reference to mucosal pathogens (reviewed by Szymanski and Wren, 2005). Our objective in this piece is to shed light on the lack of PTM studies so that current and future researchers hunting for diagnostic and vaccine candidates shall: (i) be made aware of PTMs of mycobacterial proteins, and (ii) come to understand their growing importance to both pathobiology and immunogenicity. Interestingly, the last 5 years has only yielded 2 reports on the global analyses of mycobacterial glycoproteins, both for M. tuberculosis (Mtb), and over the last decade only a few studies demonstrated the importance of PTMs to the pathobiology and antigenicity of select proteins in Mtb, M. bovis, and Mycobacterium avium subsp. paratuberculosis (MAP). In this communication we present the opinion that in a world of cross-reactive epitopes, and high amino acid sequence similarity between M. bovis, MAP, and their saprophytic counterparts, PTM diversity amongst these species may confer a new level of epitope-specificity for select antigens. We will also highlight that in the case of protein glycosylation, the type and extent of glycosyl moieties should not been seen as having the same outcome. Furthermore, the presence or absence of the PTM of proteins included in subunit vaccine formulations or attenuated strains, may enhance or mask the processing, presentation, and immunogenicity of relevant epitopes. Collectively, these data call to action critical analyses of the components we use to formulate mycobacterial vaccines. As the vast majority of PTM studies have focused on glycosylation, this PTM will be the focal point of discussion.

Heat‐mediated enrichment of α‐synuclein from cells and tissue for assessing post‐translational modifications

α-Synuclein (α-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of α-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α-syn PTMs. Here, by taking advantage of α-syn thermostability, we applied a method to achieve high enrichment of soluble α-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies.

Dual Coordination of Post Translational Modifications in Human Protein Networks

Post-translational modifications (PTMs) regulate protein activity, stability and interaction profiles and are critical for cellular functioning. Further regulation is gained through PTM interplay whereby modifications modulate the occurrence of other PTMs or act in combination. Integration of global acetylation, ubiquitination and tyrosine or serine/threonine phosphorylation datasets with protein interaction data identified hundreds of protein complexes that selectively accumulate each PTM, indicating coordinated targeting of specific molecular functions. A second layer of PTM coordination exists in these complexes, mediated by PTM integration (PTMi) spots. PTMi spots represent very dense modification patterns in disordered protein regions and showed an equally high mutation rate as functional protein domains in cancer, inferring equivocal importance for cellular functioning. Systematic PTMi spot identification highlighted more than 300 candidate proteins for combinatorial PTM regulation. This study reveals two global PTM coordination mechanisms and emphasizes dataset integration as requisite in proteomic PTM studies to better predict modification impact on cellular signaling.

Comprehending dynamic protein methylation with mass spectrometry

Protein methylation is a post-translational modification (PTM) which modulates cellular and biological processes including transcription, RNA processing, protein interactions and protein dynamics. Methylation, catalyzed by highly specific methyltransferase enzymes, occurs on several amino acids including arginine, lysine, histidine and dicarboxylic amino acids like glutamate. Mass spectrometry (MS)-based techniques continue to be the methods of choice for the study of protein PTMs. These approaches are powerful and sensitive tools that have been used to identify, quantify and characterize protein methylation. In addition, metabolic labeling strategies can be coupled to MS detection in order to measure dynamic and differential in vivo protein methylation rates. In this review, different applications of mass spectrometry technologies and methods to study protein methylation are discussed.

Quantitative Proteomic Approaches to Studying Histone Modifications

Histone post-translational modifications (PTMs) positively and negatively regulate gene expression, and are consequently a vital influence on the genomic profile of all eukaryotic species. The study of histone PTMs using classical methods in molecular biology, such as immunofluorescence and Western blotting, is challenging given the technical issues of the approaches, and chemical diversity and combinatorial patterns of the modifications. In light of these many technical limitations, mass spectrometry (MS) is emerging as the most unbiased and rigorous experimental platform to identify and quantify histone PTMs in a high-throughput manner. This review covers the latest developments in mass spectrometry for the analysis of histone PTMs, with the hope of inspiring the continued integration of proteomic, genomic and epigenetic research.

Dynamic Monitoring of Newly Synthesized Proteomes: Up-Regulation of Myristoylated Protein Kinase A During Butyric Acid Induced Apoptosis

Post-translational modification (PTM) is a highly dynamic yet precisely controlled process by which most eukaryotic proteins are chemically diversified. Many critical cellular responses are mediated through PTMs, which lead to modulation of enzyme activity, protein conformation, protein–protein interaction, and cellular localization. Analysis of these modifications at the proteome level could provide invaluable biological insight but remains a technically challenging undertaking. Traditionally, PTMs have been studied by standard molecular biology techniques involving tedious isolation of individual proteins and subsequent direct detection and analysis of amino acids bearing the modification. Recent advances in mass spectrometry, when combined with stable-isotope or metabolic labeling approaches, have enabled several large-scale studies of PTMs and their dynamics. These methods, however, analyze PTM changes of all proteins (old and new) present in the cell at the time of sampling and thus are only able to evaluate PTM dynamics at the ensemble level. With unnatural metabolic building blocks and in vivo compatible conjugation chemistries becoming increasingly available (Scheme 1), we sought to develop a proteomic strategy for the detection and identification of newly synthesized proteomes and their PTMs (Figure 1). We envisioned several advantages of studying the PTM dynamics of newly synthesized proteomes: 1) this method decreases the complexity of the proteome and enables the identification of PTM changes that occur in a predefined protein synthesis window; 2) it gives an accurate estimate of the time scale of different PTM events in transforming newly synthesized, modification-free proteins into mature functional entities; 3) it permits PTM analysis of primary protein synthesis responses to internal and external cues. To isolate a newly synthesized proteome, wemade use of BONCAT (bio-orthogonal noncanonical amino acid tagging), which uses the known methionine surrogates azidohomoalanine (AHA) and homopropargylglycine (HPG) for metabolic incorporation into newly synthesized proteins (Scheme 1, blue) and the corresponding alkyneor azide-modified biotin reporter (Scheme 1, bottom) for subsequent proteome isolation. To monitor dynamic changes of an PTM event, we fed growing cells with an azideor alkynecontaining sugar, fatty acid, or lipid building block (Scheme 1, orange). It should be noted that while our work was in progress, Hang and co-workers reported a tandem labeling and detection method to monitor the dynamic acylation of LCK (a tyrosine kinase) and its turnover. Their work focused on the study of single PTM events (i.e. protein palmitoylation) of a specific protein (i.e., LCK) in a proteome. The work herein, while conceptually similar, greatly expands the scope of this double metabolic incorporation strategy by successfully demonstrating, for the first Scheme 1. Methionine surrogates (blue) and unnatural metabolite PTM probes (orange) form bio-orthogonal pairs for compatible double metabolic incorporation. Those forming pairs are boxed in the same group. Azideand alkyne-containing fluorophore and biotin reporters are also shown (bottom).

Antibody recognition of histone post-translational modifications: emerging issues and future prospects

Antibody recognition of histone post-translational modifications: emerging issues and future prospects

Role of post translational modifications and novel crosstalk between phosphorylation and O-beta-GlcNAc modifications in human claudin-1, -3 and -4

The precise characterization of post translational modifications (PTMs) is important for the understanding of protein regulatory mechanisms and their role in disease. However, experimental studies on PTMs, especially with multifunctional proteins are difficult to follow and investigate. Bioinformatic tools are therefore helpful in predicting key protein modifications. To study the role of PTMs in claudin proteins, specifically claudin-1, -3 and -4 in the onset or progression of human cancers, we performed an in silico study of various PTMs and investigated their interplay. Given that the activity of claudins is known to be influenced by two types of PTMs, specifically palmitoylation and kinase- dependent phosphorylation, we predicted two conserved regions in the topological domains of claudin-1, -3 and -4 as potential palmitoylation sites. Furthermore, conserved phosphorylation residues, which may be targets for kinases and can alter claudin's ability to maintain the integrity of tight junctions, were identified. To our knowledge, this is the first report to suggest O-glycosylation of claudin proteins, as well as a potential novel interplay between phosphorylation and O-glycosylation at Yin Yang sites. Thus, our findings may facilitate the production of anti-cancer drugs, and suggest that novel therapeutic strategies should target post translational events.

Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays

BackgroundPost-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens.Methodology/Principal FindingsIn our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay.Conclusions/SignificanceThis methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research.

Integrated analytical strategies for the study of phosphorylation and glycosylation in proteins

The post-translational modification (PTM) of proteins is a common biological mechanism for regulating protein localization, function, and turnover. The direct analysis of modifications is required because they are not coded by genes, and thus are not predictable. Different MS-based proteomic strategies are used for the analysis of PTMs, such as phosphorylation and glycosylation, and are composed of a structural simplification step of the protein followed by specific isolation step to extract the classes of modified peptides (also called "sub-proteomes") before mass spectrometry. This specific isolation step is necessary because PTMs occur at a sub-stoichiometric level and signal suppression of the modified fractions in the mass spectrometer occurs in the presence of the more-abundant non-modified counterpart. The request of innovative analytical strategies in PTM studies is the capability to localize the modification sites, give detailed structural information on the modification, and determine the isoform composition with increased selectivity, sensitivity, and throughput. This review focuses on the description of recent integrated analytical systems proposed for the analysis of PTMs in proteins, and their application to profile the glycoproteome and the phosphoproteome in biological samples. Comments on the difficulties and usefulness of the analytical strategies are given.

Post-Translational Modifications of Nuclear Co-repressor RIP140: A Therapeutic Target for Metabolic Diseases

Most proteins undergo post-translational modification (PTM), which is known to play roles in normal physiological processes and the progression of many diseases. In this review, we summarized and discussed the mass spectrometry (MS)-based studies of various PTMs of nuclear co-repressor, receptor interacting protein 140 (RIP140), as well as the significance of these PTMs in modulating the biological activities of RIP140, specifically in adipocytes. Comprehensive analyses of RIP140 by MS identified specific sites of PTMs on RIP140, including that of phosphorylation, acetylation, pyridoxylation, and protein arginine methylation. Studies of these PTMs revealed their combinatorial effects on the activities of RIP140 with respect to the regulation of hormone target genes and fat accumulation in adipocytes. These proteomic studies have presented evidence for the biological significance of specific PTMs of RIP140, and uncovered nutritional and physiological factors that trigger these PTMs in adipocytes. This could provide insights into potential, new therapeutic targets for diseases concerning adipocytes such as metabolic disorders.

Posttranslational modification of proteins: Expanding nature's inventory. Christopher T. Walsh, Roberts & Company Publishers, Greenwood Village, CO, 2005, 576 pp., ISBN 0‐9747077‐3‐2, $98.00

Educators in biochemistry, molecular biology, and other related life sciences are continually searching for resources to help them incorporate rapidly developing topics into their classes. One such hot, cutting-edge topic today is the posttranslational modification (PTM)1 of proteins, the myriad of reactions that chemically alter protein side chains and main-chain peptide bond connectivity after the protein emerges from synthesis on the ribosomes (translation). Most of us have been informing our students of the well known PTM examples of proteolytic cleavage, modification of amino acids (phosphorylation, acetylation, etc.), and attachment of carbohydrates. However, in recent years, the repertoire of known PTM reactions has expanded dramatically. The author, who is an active researcher in this area, wrote Posttranslational Modification of Proteins: Expanding Nature's Inventory because he was unable to find any contemporary or comprehensive survey of the field in the literature. With this text, Professor Walsh not only helped himself, but he has also provided a valuable resource for all of us by finally bringing some order and clarity into the dizzying array of PTMs. The book is comprised of 16 chapters, beginning in the first chapter with a brief review of protein biosynthesis (translation), a general survey of the scope of PTM reactions, and a discussion of the techniques and procedures applied to the study of protein modification processes. Chapters 2–15 describe the varieties of PTM processes and are organized according to the type of chemical reaction and necessary enzymes. The order of topics is: phosphorylation, sulfation, Cys/Met oxidation, methylation, N-acetylation, lipidation, proteolysis, ubiquitylation, glycosylation, ADP-ribosylation, hydroxylation, automodification, carboxylation, and modification with swinging arms (biotin, lipoate, and phosphopantetheine). The final chapter covers the PTM diversity present in the proteome. The chapters range in size from about 20 to 50 pages. Biochemical reactions and processes outlining PTM reactions are explained using rigorous chemical mechanisms, but this is what we would expect from the author of the classic text, Enzymatic Reaction Mechanisms, a textbook we have all used and loved. Especially helpful are the references listed at the end of each chapter. Instead of an inclusive listing of all historical and current citations, Professor Walsh has chosen to select only recent review articles to provide an entry to the various reactions. Most references, which number from about 20 to 50 per chapter, are from the 21st century. The text is appropriate for several pedagogical uses. It may be used in a general one-year biochemistry or cell biology class as a supplement to one of the standard texts. Students with a full year of strong organic chemistry should have no difficulty understanding the mechanistic discussions that are described with excellent clarity. Because the chapters are written so that they stand alone, students could achieve understanding from selected chapters without reading the entire text. It could also be used for a one-semester special topics course for advanced undergraduate students and graduate students in biochemistry, molecular biology, and related life sciences. Even if you are an expert and carry out research on PTMs, you will discover new and interesting information in this book. Biologists will be pleased to find discussions on the biological roles of protein modification. Examples include PTM processes that have influence on signal cascades, neurochemical signaling, protein conformational changes, modulation of enzyme action, changes in cellular location of proteins, and of course, regulation of metabolism. The text is enhanced with the incorporation of excellent illustrations and tables that are present on almost every page. Although they are mainly in black and white (with occasional red highlighting), they are clear and complete. The reviewer quibbles with only one figure; it would be more appropriate to show the amino acids in zwitterionic form rather than in unionized form in Fig. 1.2. The author was especially adept at weaving into the discussion examples of how the development of new procedures in mass spectroscopy, x-ray crystallography, capillary electrophoresis, two-dimensional electrophoresis, and HPLC has been essential in solving complex problems in the study of PTMs. Professor Walsh assures us that we have not seen the end of important discoveries in this area. He predicts that we will continue to uncover new and intriguing PTM processes that will help us explain and understand the molecular intricacies of simple and complex organisms.