Over 400 different types of post-translational modifications (PTMs) have been reported and over 200 various types of PTMs have been discovered using mass spectrometry (MS)-based proteomics. MS-based proteomics has proven to be a powerful method capable of global PTM mapping with the identification of modified proteins/peptides, the localization of PTM sites and PTM quantitation. PTMs play regulatory roles in protein functions, activities and interactions in various heart related diseases, such as ischemia/reperfusion injury, cardiomyopathy and heart failure. The recognition of PTMs that are specific to cardiovascular pathology and the clarification of the mechanisms underlying these PTMs at molecular levels are crucial for discovery of novel biomarkers and application in a clinical setting. With sensitive MS instrumentation and novel biostatistical methods for precise processing of the data, low-abundance PTMs can be successfully detected and the beneficial or unfavorable effects of specific PTMs on cardiac function can be determined. Moreover, computational proteomic strategies that can predict PTM sites based on MS data have gained an increasing interest and can contribute to characterization of PTM profiles in cardiovascular disorders. More recently, machine learning- and deep learning-based methods have been employed to predict the locations of PTMs and explore PTM crosstalk. In this review article, the types of PTMs are briefly overviewed, approaches for PTM identification/quantitation in MS-based proteomics are discussed and recently published proteomic studies on PTMs associated with cardiovascular diseases are included.
- Keywords
- MS‐based proteomics, cardiovascular disease, post‐translational modifications, proteins,
- MeSH
- Phosphorylation MeSH
- Mass Spectrometry methods MeSH
- Cardiovascular Diseases * metabolism genetics pathology MeSH
- Humans MeSH
- Protein Processing, Post-Translational * MeSH
- Proteomics * methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Nitric oxide (NO) is perfectly suited for the role of a redox signalling molecule. A key route for NO bioactivity occurs via protein S-nitrosation, and involves the addition of a NO moiety to a protein cysteine (Cys) thiol (-SH) to form an S-nitrosothiol (SNO). This process is thought to underpin a myriad of cellular processes in plants that are linked to development, environmental responses and immune function. Here we collate emerging evidence showing that NO bioactivity regulates a growing number of diverse post-translational modifications including SUMOylation, phosphorylation, persulfidation and acetylation. We provide examples of how NO orchestrates these processes to mediate plant adaptation to a variety of cellular cues.
- Keywords
- S-nitrosation, S-nitrosylation, SUMOylation, nitric oxide (NO), persulfidation, phosphorylation, reactive nitrogen species (RNS), reactive oxygen species (ROS),
- MeSH
- Nitrosation MeSH
- Nitric Oxide * metabolism MeSH
- Oxidation-Reduction MeSH
- Protein Processing, Post-Translational MeSH
- Plants metabolism MeSH
- S-Nitrosothiols * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- Nitric Oxide * MeSH
- S-Nitrosothiols * MeSH
We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual.
- Keywords
- EPIGENETICS, HISTONES, HUMAN SPERM, MICROSCOPY, PROTAMINE P2,
- MeSH
- Acetylation MeSH
- Chromatin genetics MeSH
- Histone-Lysine N-Methyltransferase biosynthesis genetics MeSH
- Histone Methyltransferases MeSH
- Histones genetics metabolism MeSH
- Humans MeSH
- Methylation MeSH
- Protein Processing, Post-Translational genetics MeSH
- Amino Acid Sequence MeSH
- Spermatozoa growth & development metabolism MeSH
- Tandem Mass Spectrometry MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Chromatin MeSH
- Histone-Lysine N-Methyltransferase MeSH
- Histone Methyltransferases MeSH
- Histones MeSH
Post-translational modifications (PTMs) of biomacromolecules can be useful for understanding the processes by which a relatively small number of individual genes in a particular genome can generate enormous biological complexity in different organisms. The proteomes of barley and the brewing process were investigated by different techniques. However, their diverse and complex PTMs remain understudied. As standard analytical approaches have limitations, innovative analytical approaches need to be developed and applied in PTM studies. To make further progress in this field, it is necessary to specify the sites of modification, as well as to characterize individual isoforms with increased selectivity and sensitivity. This review summarizes advances in the PTM analysis of barley proteins, particularly those involving mass spectrometric detection. Our focus is on monitoring phosphorylation, glycation, and glycosylation, which critically influence functional behavior in metabolism and regulation in organisms.
- Keywords
- barley, mass spectrometry, post-translational modification, protein,
- MeSH
- Phosphorylation MeSH
- Glycosylation MeSH
- Hordeum * genetics MeSH
- Protein Processing, Post-Translational MeSH
- Proteome chemistry MeSH
- Proteomics methods MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Proteome MeSH
Vitamin D receptor (VDR) is a member of the nuclear receptor (NR) superfamily of ligand-activated transcription factors. Activated VDR is responsible for maintaining calcium and phosphate homeostasis, and is required for proper cellular growth, cell differentiation and apoptosis. The expression of both phases I and II drug-metabolizing enzymes is also regulated by VDR, therefore it is clinically important.Post-translational modifications of NRs have been known as an important mechanism modulating the activity of NRs and their ability to drive the expression of target genes. The aim of this mini review is to summarize the current knowledge about post-transcriptional and post-translational modifications of VDR.
- Keywords
- miRNA, phosphorylation, ubiquitination, sumoylation, VDRB1, VDRA,
- MeSH
- Phosphorylation MeSH
- Transcription, Genetic MeSH
- Humans MeSH
- MicroRNAs genetics MeSH
- Protein Processing, Post-Translational * MeSH
- Receptors, Calcitriol genetics metabolism MeSH
- Gene Expression Regulation MeSH
- Sumoylation MeSH
- Ubiquitination MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- MicroRNAs MeSH
- MIRN125 microRNA, human MeSH Browser
- MIRN27 microRNA, human MeSH Browser
- Receptors, Calcitriol MeSH
- VDR protein, human MeSH Browser
AIM: In this study, we analysed the post-translational modification of receptor tyrosine kinase-like orphan receptor (Ror1). Ror1 is highly upregulated in B cells of patients with chronic lymphocytic leukaemia (CLL). Molecularly, Ror1 acts as the Wnt receptor in the non-canonical Wnt pathway. METHODS: The level of Ror1 glycosylation in HEK293 cells and in primary human CLL cells was analysed by treatment of inhibitors interfering with different steps of glycosylation process and by direct treatment of cell lysates with N-glycosidase. Ror1 ubiquitination was determined by ubiquitination assay. Functional consequences of post-translational modifications were analysed by immunohistochemistry and by analysis of cell surface proteins. Differences in Ror1 glycosylation were confirmed by analysis of 14 samples of B cells from CLL patients. RESULTS: We demonstrate that Ror1 is extensively modified by N-linked glycosylation. Glycosylation produces several variants of Ror1 with electrophoretic migration of approx. 100, 115 and 130 kDa. Inhibition of glycosylation interferes with cell surface localization of the 130-kDa variant of Ror1 and prevents Ror1-induced formation of filopodia. Moreover, we show that 130-kDa Ror1 is mono-ubiquitinated. Furthermore, individual CLL patients show striking differences in the electrophoretic migration of Ror1, which correspond to the level of glycosylation. CONCLUSION: Our data show that Ror1 undergoes complex post-translational modifications by glycosylation and mono-ubiquitination. These modifications regulate Ror1 localization and signalling, and are highly variable among individual CLL patients. These may suggest that Ror1 signals only in a subset of CLL patients despite Ror1 levels are ubiquitously high in all CLL patients.
- MeSH
- B-Lymphocytes metabolism MeSH
- CHO Cells MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell metabolism MeSH
- Cricetulus MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Glycosylation MeSH
- HEK293 Cells MeSH
- Immunohistochemistry MeSH
- Microscopy, Confocal MeSH
- Cricetinae MeSH
- Humans MeSH
- Molecular Weight MeSH
- Protein Processing, Post-Translational * drug effects MeSH
- Flow Cytometry MeSH
- Pseudopodia metabolism MeSH
- Signal Transduction * drug effects MeSH
- Receptor Tyrosine Kinase-like Orphan Receptors chemistry genetics metabolism MeSH
- Transfection MeSH
- Protein Transport MeSH
- Ubiquitination MeSH
- Blotting, Western MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- ROR1 protein, human MeSH Browser
- Receptor Tyrosine Kinase-like Orphan Receptors MeSH
Nitric oxide (NO) is considered as a signalling molecule involved in a variety of important physiological and pathological processes in plant and animal systems. The major pathway of NO reactions in vivo represents S-nitrosation of thiols to form S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is the key enzyme in the degradation pathway of S-nitrosoglutathione (GSNO), a low-molecular weight adduct of NO and glutathione. GSNOR indirectly regulates the level of protein S-nitrosothiol in the cells. This study was focused on the dynamic regulation of the activity of plant GSNORs through reversible S-nitrosation and/or oxidative modifications of target cysteine residues. Pre-incubation with NO/NO- donors or hydrogen peroxide resulted in a decreased reductase and dehydrogenase activity of all studied plant GSNORs. Incubation with thiol reducing agent completely reversed inhibitory effects of nitrosative modifications and partially also oxidative inhibition. In biotin-labelled samples, S-nitrosation of plant GSNORs was confirmed after immunodetection and using mass spectrometry S-nitrosation of conserved Cys271 was identified in tomato GSNOR. Negative regulation of constitutive GSNOR activity in vivo by nitrosative or oxidative modifications might present an important mechanism to control GSNO levels, a critical mediator of the downstream signalling effects of NO, as well as for formaldehyde detoxification in dehydrogenase reaction mode.
- Keywords
- Nitric oxide, Post-translational modifications, Redox regulation, S-nitrosation, S-nitrosoglutathione reductase,
- MeSH
- Aldehyde Oxidoreductases antagonists & inhibitors chemistry metabolism MeSH
- Cysteine chemistry metabolism MeSH
- Nitric Oxide Donors pharmacology MeSH
- Nitrosation MeSH
- Nitric Oxide metabolism MeSH
- Oxidation-Reduction MeSH
- Hydrogen Peroxide pharmacology MeSH
- Protein Processing, Post-Translational MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Plant Proteins antagonists & inhibitors chemistry metabolism MeSH
- S-Nitrosoglutathione metabolism MeSH
- S-Nitrosothiols metabolism MeSH
- Signal Transduction MeSH
- Solanum lycopersicum genetics growth & development metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Aldehyde Oxidoreductases MeSH
- Cysteine MeSH
- Nitric Oxide Donors MeSH
- formaldehyde dehydrogenase, glutathione-independent MeSH Browser
- Nitric Oxide MeSH
- Hydrogen Peroxide MeSH
- Recombinant Proteins MeSH
- Plant Proteins MeSH
- S-Nitrosoglutathione MeSH
- S-Nitrosothiols MeSH
This study focuses on the intrinsically disordered regulatory domain of p53 and the impact of post-translational modifications. Through fully atomistic explicit water molecular dynamics simulations, we show the wealth of information and detailed understanding that can be obtained by varying the number of phosphorylated amino acids and implementing a restriction in the conformational entropy of the N-termini of that intrinsically disordered region. The take-home message for the reader is to achieve a detailed understanding of the impact of phosphorylation with respect to (1) the conformational dynamics and flexibility, (2) structural effects, (3) protein interactivity, and (4) energy landscapes and conformational ensembles. Although our model system is the regulatory domain p53 of the tumor suppressor protein p53, this study contributes to understanding the general effects of intrinsically disordered phosphorylated proteins and the impact of phosphorylated groups, more specifically, how minor changes in the primary sequence can affect the properties mentioned above.
- MeSH
- Entropy MeSH
- Phosphorylation MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 * chemistry metabolism MeSH
- Protein Processing, Post-Translational * MeSH
- Protein Domains MeSH
- Molecular Dynamics Simulation * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Tumor Suppressor Protein p53 * MeSH
Pregnane X receptor (PXR) is a member of the nuclear receptor (NR) superfamily of ligand-activated transcription factors and is activated by a huge variety of endobiotics and xenobiotics, including many clinical drugs. PXR plays key roles not only as a xenosensor in the regulation of both major phase I and II drug metabolism and transporters but also as a physiological sensor in the modulation of bile acid and cholesterol metabolism, glucose and lipid metabolism, and bone and endocrine homeostasis. Post-translational modifications such as phosphorylation have been shown to modulate the activity of many NRs, including PXR, and constitute an important mechanism for crosstalk between signaling pathways and regulation of genes involved in both xenobiotic and endobiotic metabolism. In addition, microRNAs have recently been shown to constitute another level of PXR activity regulation. The objective of this review is to comprehensively summarize current understanding of post-transcriptional and post-translational modifications of PXR in regulation of xenobiotic-metabolizing cytochrome P450 (CYP) genes, mainly in hepatic tissue. We also discuss the importance of PXR in crosstalk with cell signaling pathways, which at the level of transcription modify expression of genes associated with some physiological and pathological stages in the organs. Finally, we indicate that these PXR modifications may have important impacts on CYP-mediated biotransformation of some clinically used drugs.
- MeSH
- Biotransformation MeSH
- Enzyme Induction drug effects MeSH
- Protein Interaction Domains and Motifs MeSH
- Liver drug effects enzymology metabolism MeSH
- Humans MeSH
- RNA, Messenger metabolism MeSH
- RNA Processing, Post-Transcriptional * drug effects MeSH
- Protein Processing, Post-Translational * drug effects MeSH
- Pregnane X Receptor MeSH
- Receptors, Steroid chemistry genetics metabolism MeSH
- Cytochrome P-450 Enzyme System genetics metabolism MeSH
- Xenobiotics metabolism pharmacokinetics toxicity MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- RNA, Messenger MeSH
- Pregnane X Receptor MeSH
- Receptors, Steroid MeSH
- Cytochrome P-450 Enzyme System MeSH
- Xenobiotics MeSH
There are two strategies applicable to revealing non-enzymatic post-translational modifications of proteins; while assaying of the hydrolytically stable adducts was the subject of our previous communication [1], here we attempted to review separation technologies for the unfragmented modified proteins. There are a few standard procedures used for this purpose, namely Laemmli gel electrophoresis, different modes of gel permeation chromatography and boronate affinity chromatography. The latter approach makes use of the vicinal hydroxy groups present in glycated proteins. Some (but not all) arising adducts exhibit typical fluorescence which can be exploited for detection. In most cases fluorescence is measured at 370/440 nm for the so-called advanced glycation products or at 335/385 nm for the only so far well characterized glycation marker (pentosidine). Some indication exists that, e.g., synchronous fluorescence detection will probably in the future add to the selectivity and allow the distinction of the different adducts arising during non-enzymatic post-translational modifications (glycation). The proteins reviewed are serum albumin, collagen and lens proteins while glycation of hemoglobin is the subject of another review within the present volume.
- MeSH
- Chromatography, Liquid methods MeSH
- Electrophoresis methods MeSH
- Glycosylation MeSH
- Collagen chemistry MeSH
- Crystallins chemistry MeSH
- Lipid Peroxides chemistry MeSH
- Protein Processing, Post-Translational * MeSH
- Proteins chemistry metabolism MeSH
- Serum Albumin chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- Collagen MeSH
- Crystallins MeSH
- Lipid Peroxides MeSH
- Proteins MeSH
- Serum Albumin MeSH
