Deciphering a global network of functionally associated post‐translational modifications

Various post‐translational modifications (PTMs) fine‐tune the functions of almost all eukaryotic proteins, and co‐regulation of different types of PTMs has been shown within and between a number of proteins. Aiming at a more global view of the interplay between PTM types, we collected modifications for 13 frequent PTM types in 8 eukaryotes, compared their speed of evolution and developed a method for measuring PTM co‐evolution within proteins based on the co‐occurrence of sites across eukaryotes. As many sites are still to be discovered, this is a considerable underestimate, yet, assuming that most co‐evolving PTMs are functionally associated, we found that PTM types are vastly interconnected, forming a global network that comprise in human alone >50 000 residues in about 6000 proteins. We predict substantial PTM type interplay in secreted and membrane‐associated proteins and in the context of particular protein domains and short‐linear motifs. The global network of co‐evolving PTM types implies a complex and intertwined post‐translational regulation landscape that is likely to regulate multiple functional states of many if not all eukaryotic proteins.     

Accumulation and post‐translational modifications of plant tubulins

The microtubular cytoskeleton of plant cells provides support for several functions (including the anchoring of proteins, assembly of the mitotic spindle, cytoplasmic streaming and construction of cell walls). Both α‐ and β‐tubulins are encoded through multigene families that are differentially expressed in different organs and tissues. To increase the variability of expression, both protein subunits are subjected to post‐translational modifications, which could contribute to the assembly of specific microtubule structures. This review aims to highlight the role of specific post‐translational modifications of tubulin in plant cells. We initially describe the expression and accumulation of α‐ and β‐tubulin isoforms in different plants and at different stages of plant development. Second, we discuss the different types of post‐translational modifications that, by adding or removing specific functional groups, increase the isoform heterogeneity and functional variability of tubulin. Modifications are proposed to form a ‘code’ that can be read by proteins interacting with microtubules. Therefore, the subpopulations of microtubules may bind to different associated proteins (motor and non‐motor), thus creating the physical support for various microtubule functions.     

The role of post‐translational modifications in cardiac hypertrophy

Pathological cardiac hypertrophy involves excessive protein synthesis, increased cardiac myocyte size and ultimately the development of heart failure. Thus, pathological cardiac hypertrophy is a major risk factor for many cardiovascular diseases and death in humans. Extensive research in the last decade has revealed that post‐translational modifications (PTMs), including phosphorylation, ubiquitination, SUMOylation, O‐GlcNAcylation, methylation and acetylation, play important roles in pathological cardiac hypertrophy pathways. These PTMs potently mediate myocardial hypertrophy responses via the interaction, stability, degradation, cellular translocation and activation of receptors, adaptors and signal transduction events. These changes occur in response to pathological hypertrophy stimuli. In this review, we summarize the roles of PTMs in regulating the development of pathological cardiac hypertrophy. Furthermore, PTMs are discussed as potential targets for treating or preventing cardiac hypertrophy.     

Towards understanding the crosstalk between protein post‐translational modifications: Homo‐ and heterotypic PTM pair distances on protein surfaces are not random

Post‐translational modifications (PTMs) represent an important regulatory layer influencing the structure and function of proteins. With broader availability of experimental information on the occurrences of different PTM types, the investigation of a potential “crosstalk” between different PTM types and combinatorial effects have moved into the research focus. Hypothesizing that relevant interferences between different PTM types and sites may become apparent when investigating their mutual physical distances, we performed a systematic survey of pairwise homo‐ and heterotypic distances of seven frequent PTM types considering their sequence and spatial distances in resolved protein structures. We found that actual PTM site distance distributions differ from random distributions with most PTM type pairs exhibiting larger than expected distances with the exception of homotypic phosphorylation site distances and distances between phosphorylation and ubiquitination sites that were found to be closer than expected by chance. Random reference distributions considering canonical acceptor amino acid residues only were found to be shifted to larger distances compared to distances between any amino acid residue type indicating an underlying tendency of PTM‐amenable residue types to be further apart than randomly expected. Distance distributions based on sequence separations were found largely consistent with their spatial counterparts suggesting a primary role of sequence‐based pairwise PTM‐location encoding rather than folding‐mediated effects. Our analysis provides a systematic and comprehensive overview of the characteristics of pairwise PTM site distances on proteins and reveals that, predominantly, PTM sites tend to avoid close proximity with the potential implication that an independent attachment or removal of PTMs remains possible. Proteins 2016; 85:78–92. © 2016 Wiley Periodicals, Inc.     

Expanding the ubiquitin code through post‐translational modification

Ubiquitylation is among the most prevalent post‐translational modifications (PTMs) and regulates numerous cellular functions. Interestingly, ubiquitin (Ub) can be itself modified by other PTMs, including acetylation and phosphorylation. Acetylation of Ub on K6 and K48 represses the formation and elongation of Ub chains. Phosphorylation of Ub happens on multiple sites, S57 and S65 being the most frequently modified in yeast and mammalian cells, respectively. In mammals, the PINK1 kinase activates ubiquitin ligase Parkin by phosphorylating S65 of Ub and of the Parkin Ubl domain, which in turn promotes the amplification of autophagy signals necessary for the removal of damaged mitochondria. Similarly, TBK1 phosphorylates the autophagy receptors OPTN and p62 to initiate feedback and feedforward programs for Ub‐dependent removal of protein aggregates, mitochondria and pathogens (such as Salmonella and Mycobacterium tuberculosis). The impact of PINK1‐mediated phosphorylation of Ub and TBK1‐dependent phosphorylation of autophagy receptors (OPTN and p62) has been recently linked to the development of Parkinson's disease and amyotrophic lateral sclerosis, respectively. Hence, the post‐translational modification of Ub and its receptors can efficiently expand the Ub code and modulate its functions in health and disease.     

Functional analysis tools for post‐translational modification: a post‐translational modification database for analysis of proteins and metabolic pathways

Post‐translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high‐resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post‐Translational Modifications (FAT‐PTM) database ( https://bioinformatics.cse.unr.edu/fat-ptm/ ), which currently supports eight different types of PTM and over 49 000 PTM sites identified in large‐scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT‐PTM database currently supports tools to visualize protein‐centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein‐centric metabolic pathways and groups of proteins that are co‐modified by multiple PTMs. Overall, the FAT‐PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.     

Aberrant post‐translational modifications compromise human myosin motor function in old age

Novel experimental methods, including a modified single fiber in vitro motility assay, X‐ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging‐related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force‐generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X‐ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age‐specific myosin post‐translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.     

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post‐translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM‐dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross‐linking‐assisted and stable isotope labeling in cell culture‐based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation‐dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine‐9 (H3K9Me₃)‐dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation‐dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine‐3 (H3T3‐Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3‐Phos‐binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3‐Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.     

Post‐translational modifications of transthyretin affect the triiodonine‐binding potential

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys₁₀), which is frequently affected by oxidative post‐translational modifications. As Cys₁₀ is part of the thyroid hormone‐binding channel within the TTR molecule, PTM of Cys₁₀ may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys₁₀ modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype‐ and site‐specific manner with S‐glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function‐specific patterns of TTR with a substantial decrease in S‐sulphonated, S‐cysteinylglycinated and S‐glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys₁₀ seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.     

Autophagy in plants: Physiological roles and post‐translational regulation

In eukaryotes, autophagy helps maintain cellular homeostasis by degrading and recycling cytoplasmic materials via a tightly regulated pathway.Over the past few decades, significant progress has been made towards understanding the physiological functions and molecular regulation of autophagy in plant cells. Increasing evidence indicates that autophagy is essential for plant responses to several developmental and environmental cues, functioning in diverse processes such as senescence, male fertility, root meristem maintenance, responses to nutrient starvation,and biotic and abiotic stress. Recent studies have demonstrated that, similar to nonplant systems,the modulation of core proteins in the plant autophagy machinery by posttranslational modifications such as phosphorylation, ubiquitination,lipidation, S-sulfhydration, S-nitrosylation, and acetylation is widely involved in the initiation and progression of autophagy. Here, we provide an overview of the physiological roles and posttranslational regulation of autophagy in plants.     

Regulation of connexin‐ and pannexin‐based channels by post‐translational modifications

Connexin (Cx) and pannexin (Panx) proteins form large conductance channels, which function as regulators of communication between neighbouring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signalling, survival and death processes. The functional properties of gap junctions and hemichannels are modulated by different physiological and pathophysiological stimuli. At the molecular level, Cxs and Panxs function as multi‐protein channel complexes, regulating their channel localisation and activity. In addition to this, gap junctional channels and hemichannels are modulated by different post‐translational modifications (PTMs), including phosphorylation, glycosylation, proteolysis, N‐acetylation, S‐nitrosylation, ubiquitination, lipidation, hydroxylation, methylation and deamidation. These PTMs influence almost all aspects of communicating junctional channels in normal cell biology and pathophysiology. In this review, we will provide a systematic overview of PTMs of communicating junction proteins and discuss their effects on Cx and Panx‐channel activity and localisation.     

PTM MarkerFinder,a software tool to detect and validate spectra from peptides carrying post‐translational modifications

Mass spectrometry (MS) analysis of peptides carrying post‐translational modifications is challenging due to the instability of some modifications during MS analysis. However, glycopeptides as well as acetylated, methylated and other modified peptides release specific fragment ions during CID (collision‐induced dissociation) and HCD (higher energy collisional dissociation) fragmentation. These fragment ions can be used to validate the presence of the PTM on the peptide. Here, we present PTM MarkerFinder, a software tool that takes advantage of such marker ions. PTM MarkerFinder screens the MS/MS spectra in the output of a database search (i.e., Mascot) for marker ions specific for selected PTMs. Moreover, it reports and annotates the HCD and the corresponding electron transfer dissociation (ETD) spectrum (when present), and summarizes information on the type, number, and ratios of marker ions found in the data set. In the present work, a sample containing enriched N‐acetylhexosamine (HexNAc) glycopeptides from yeast has been analyzed by liquid chromatography‐mass spectrometry on an LTQ Orbitrap Velos using both HCD and ETD fragmentation techniques. The identification result (Mascot .dat file) was submitted as input to PTM MarkerFinder and screened for HexNAc oxonium ions. The software output has been used for high‐throughput validation of the identification results.     

Histone deacetylases: salesmen and customers in the post‐translational modification market

HDACs (histone deacetylases) are enzymes that remove the acetyl moiety from N‐?‐acetylated lysine residues in histones and non‐histone proteins. In recent years, it has turned out that HDACs themselves are also subject to post‐translational modification. Such structural alterations can determine the stability, localization, activity and protein—protein interactions of HDACs. This subsequently affects the modification of their substrates and the co‐ordination of cellular signalling networks. Intriguingly, physiologically relevant non‐histone proteins are increasingly found to be deacetylated by HDACs, and aberrant deacetylase activity contributes to several severe human diseases. Targeting the catalytic activity of these enzymes and their post‐translational modifications are therefore attractive targets for therapeutical intervention strategies. To achieve this ambitious goal, details on the molecular mechanisms regulating post‐translational modifications of HDACs are required. This review summarizes aspects of the current knowledge on the biological role and enzymology of the phosphorylation, acetylation, ubiquitylation and sumoylation of HDACs.     

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.     

A recent advance in the intracellular and extracellular redox post‐translational modification of proteins in plants

Plants, as sessile organisms, have acquired through evolution sophisticated regulatory signal pathways to overcome external variable factors during each stage of the life cycle. Among these regulatory signals, two pathways in particular, reactive oxygen species and reactive nitrogen species, have become of significant interest in several aspects of plant biology, underpinning these molecules as critical regulators during development, cellular differentiation, and plant‐pathogen interaction. Recently, redox posttranslational modifications (PTM), such as S‐nitrosylation on cysteine residues and tyrosine nitration, have shed light on multiple protein targets, as they are associated with signal networks/downstream metabolic pathways, capable of transducing the imbalance of redox hemostasis and consequently redirecting the biochemical status under stress conditions. However, most of the redox PTM have been studied only in the intracellular compartment, providing limited information concerning redox PTM in the extracellular matrix of plant cells. Nevertheless, recent studies have indicated the plausibility of redox PTM in extracellular proteins, including cell wall associated proteins. Accordingly, in this review, we endeavor to examine evidence of redox PTM supported by mass spectrometry data in the intracellular and extracellular space in plant cells. As a further example, we focus the last section of this review on illustrating, using molecular dynamics simulation, the effect of S‐nitrosylation on the structural conformation of well‐known cell wall‐associated proteins including pectin methylesterase and xyloglucan endo‐transglycosylases.     

Co‐ and post‐translational modifications of the 26S proteasome in yeast

The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co‐ and post‐translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N‐terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N‐terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho‐amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with λ phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N‐ and O‐linked oligosaccharides nor O‐linked β‐N‐acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co‐ and post‐translational modifications, including N^α‐acetylation, N^α‐myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.     

The use of post‐translationally modified peptides for detection of biomarkers of immune‐mediated diseases

Biomarkers are decision‐making tools at the basis of clinical diagnostics and essential for guiding therapeutic treatments. In this context, autoimmune diseases represent a class of disorders that need early diagnosis and steady monitoring. These diseases are usually associated with humoral or cell‐mediated immune reactions against one or more of the body's own constituents. Autoantibodies fluctuating in biological fluids can be used as disease biomarkers and they can be, thus, detected by diagnostic immunoassays using native autoantigens. However, it is now accepted that post‐translational modifications may affect the immunogenicity of self‐protein antigens, triggering an autoimmune response and creating neo‐antigens. In this case, post‐translationally modified peptides represent a more valuable tool with respect to isolated or recombinant proteins. In fact, synthetic peptides can be specifically modified to mimic neo‐antigens and to selectively detect autoantibodies as disease biomarkers. A ‘chemical reverse approach’ to select synthetic peptides, bearing specific post‐translational modifications, able to fishing out autoantibodies from patients' biological fluids, can be successfully applied for the development of specific in vitro diagnostic/prognostic assays of autoimmune diseases. Herein, we report the successful application of this approach to the identification of biomarkers in different autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family

Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.