The Plant PTM Viewer,a central resource for exploring plant protein modifications

Post‐translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site‐specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370 000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows the submission of mass spectrometry‐based PTM data to remain at pace with future PTM plant studies.     

Rampant purifying selection conserves positions with posttranslational modifications in human proteins

Posttranslational modifications (PTMs) are chemical alterations that are critical to protein conformation and activation states. Despite their functional importance and reported involvement in many diseases, evolutionary analyses have produced enigmatic results because only weak or no selective pressures have been attributed to many types of PTMs. In a large-scale analysis of 16,836 PTM positions from 4,484 human proteins, we find that positions harboring PTMs show evidence of higher purifying selection in 70% of the phosphorylated and N-linked glycosylated proteins. The purifying selection is up to 42% more severe at PTM residues as compared with the corresponding unmodified amino acids. These results establish extensive selective pressures in the long-term history of positions that experience PTMs in the human proteins. Our findings will enhance our understanding of the historical function of PTMs over time and help in predicting PTM positions by using evolutionary comparisons.     

Regulatory effects of protein S-acylation on insulin secretion and insulin action

Post-translational modifications (PTMs) such as phosphorylation and ubiquitination are well-studied events with a recognized importance in all aspects of cellular function. By contrast, protein S-acylation, although a widespread PTM with important functions in most physiological systems, has received far less attention. Perturbations in S-acylation are linked to various disorders, including intellectual disability, cancer and diabetes, suggesting that this less-studied modification is likely to be of considerable biological importance. As an exemplar, in this review, we focus on the newly emerging links between S-acylation and the hormone insulin. Specifically, we examine how S-acylation regulates key components of the insulin secretion and insulin response pathways. The proteins discussed highlight the diverse array of proteins that are modified by S-acylation, including channels, transporters, receptors and trafficking proteins and also illustrate the diverse effects that S-acylation has on these proteins, from membrane binding and micro-localization to regulation of protein sorting and protein interactions.     

Enzymology and significance of protein histidine methylation

Cells synthesize proteins using 20 standard amino acids and expand their biochemical repertoire through intricate enzyme-mediated post-translational modifications (PTMs). PTMs can either be static and represent protein editing events or be dynamically regulated as a part of a cellular response to specific stimuli. Protein histidine methylation (Hme) was an elusive PTM for over 5 decades and has only recently attracted considerable attention through discoveries concerning its enzymology, extent, and function. Here, we review the status of the Hme field and discuss the implications of Hme in physiological and cellular processes. We also review the experimental toolbox for analysis of Hme and discuss the strengths and weaknesses of different experimental approaches. The findings discussed in this review demonstrate that Hme is widespread across cells and tissues and functionally regulates key cellular processes such as cytoskeletal dynamics and protein translation. Collectively, the findings discussed here showcase Hme as a regulator of key cellular functions and highlight the regulation of this modification as an emerging field of biological research.     

An atlas of posttranslational modifications on RNA binding proteins

RNA structure and function are intimately tied to RNA binding protein recognition and regulation. Posttranslational modifications are chemical modifications which can control protein biology. The role of PTMs in the regulation RBPs is not well understood, in part due to a lacking analysis of PTM deposition on RBPs. Herein, we present an analysis of posttranslational modifications (PTMs) on RNA binding proteins (RBPs; a PTM RBP Atlas). We curate published datasets and primary literature to understand the landscape of PTMs and use protein–protein interaction data to understand and potentially provide a framework for understanding which enzymes are controlling PTM deposition and removal on the RBP landscape. Intersection of our data with The Cancer Genome Atlas also provides researchers understanding of mutations that would alter PTM deposition. Additional characterization of the RNA–protein interface provided from in-cell UV crosslinking experiments provides a framework for hypotheses about which PTMs could be regulating RNA binding and thus RBP function. Finally, we provide an online database for our data that is easy to use for the community. It is our hope our efforts will provide researchers will an invaluable tool to test the function of PTMs controlling RBP function and thus RNA biology.     

Evolution and functional cross‐talk of protein post‐translational modifications

Protein post‐translational modifications (PTMs) allow the cell to regulate protein activity and play a crucial role in the response to changes in external conditions or internal states. Advances in mass spectrometry now enable proteome wide characterization of PTMs and have revealed a broad functional role for a range of different types of modifications. Here we review advances in the study of the evolution and function of PTMs that were spurred by these technological improvements. We provide an overview of studies focusing on the origin and evolution of regulatory enzymes as well as the evolutionary dynamics of modification sites. Finally, we discuss different mechanisms of altering protein activity via post‐translational regulation and progress made in the large‐scale functional characterization of PTM function.     

Consequences of post-translational modifications on amyloid proteins as revealed by protein semisynthesis

Alterations to the global levels of certain types of post-translational modifications (PTMs) are commonly observed in neurodegenerative diseases. The net influence of these PTM changes to the progression of these diseases can be deduced from cellular and animal studies. However, at the molecular level, how one PTM influences a given protein is not uniform and cannot be easily generalized from systemic observations, thus requiring protein-specific interrogations. Given that protein aggregation is a shared pathological hallmark in neurodegeneration, it is important to understand how these PTMs affect the behavior of amyloid-forming proteins. For this purpose, protein semisynthesis techniques, largely via native chemical and expressed protein ligation, have been widely used. These approaches have thus far led to our increased understanding of the site-specific consequences of certain PTMs to amyloidogenic proteins’ endogenous function, their propensity for aggregation, and the structural variations these PTMs induce toward the aggregates formed.     

Post-translational modification of plant-made foreign proteins; glycosylation and beyond

The complex and diverse nature of the post-translational modification (PTM) of proteins represents an efficient and cost-effective mechanism for the exponential diversification of the genome. PTMs have been shown to affect almost every aspect of protein activity, including function, localisation, stability, and dynamic interactions with other molecules. Although many PTMs are evolutionarily conserved there are also important kingdom-specific modifications which should be considered when expressing recombinant proteins. Plants are gaining increasing acceptance as an expression system for recombinant proteins, particularly where eukaryotic-like PTMs are required. Glycosylation is the most extensively studied PTM of plant-made recombinant proteins. However, other types of protein processing and modification also occur which are important for the production of high quality recombinant protein, such as hydroxylation and lipidation. Plant and/or protein engineering approaches offer many opportunities to exploit PTM pathways allowing the molecular farmer to produce a humanised product with modifications functionally similar or identical to the native protein. Indeed, plants have demonstrated a high degree of tolerance to changes in PTM pathways allowing recombinant proteins to be modified in a specific and controlled manner, frequently resulting in a homogeneity of product which is currently unrivalled by alternative expression platforms. Whether a recombinant protein is intended for use as a scientific reagent, a cosmetic additive or as a pharmaceutical, PTMs through their presence and complexity, offer an extensive range of options for the rational design of humanised (biosimilar), enhanced (biobetter) or novel products.     

Cell signaling, post-translational protein modifications and NMR spectroscopy

Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.     

Modulating Microtubules: A Molecular Perspective on the Effects of Tail Modifications

Microtubules (MTs), an essential component of the eukaryotic cytoskeleton, are a lattice of polymerized tubulin dimers and are crucial for various cellular processes. The genetic and chemical diversity of tubulin and their disordered tails gives rise to a “tubulin code”. The functional role of tubulin post-translational modifications (PTMs), which contribute to the chemical diversity of the tubulin code, is gradually being unraveled. However, variation in the length and spatial organization of tubulin poly-modifications leads to an enormous combinatorial PTM space, which is difficult to study experimentally. Hence, the impact of the combinatorial tubulin PTM space on the biophysical properties of tubulin tails and their interactions with other proteins remains elusive.Here, we combine all-atom and coarse-grained molecular dynamics simulations to elucidate the biophysical implications of the large combinatorial tubulin PTM space in the context of an MT lattice. We find that tail–body interactions are more dominant in the tubulin dimer than in an MT lattice, and are more significant for the tails of α compared with β tubulin. In addition, polyglutamylation, but not polyglycylation, expands the dimensions of the tubulin tails. Polyglutamylation also leads to a decrease in the diffusion rate of MT-associated protein EB1 on MTs, while polyglycylation often increases diffusion rate. These observations are generally not sensitive to the organization of the polymodifications. The effect of PTMs on MT charge density and tail dynamics are also discussed. Overall, this study presents a molecular quantification of the biophysical properties of tubulin tails and their polymodifications, and provides predictions on the functional importance of tubulin PTMs.     

Engineering chromatin states: Chemical and synthetic biology approaches to investigate histone modification function

Patterns of histone post-translational modifications (PTMs) and DNA modifications establish a landscape of chromatin states with regulatory impact on gene expression, cell differentiation and development. These diverse modifications are read out by effector protein complexes, which ultimately determine their functional outcome by modulating the activity state of underlying genes. From genome-wide studies employing high-throughput ChIP-Seq methods as well as proteomic mass spectrometry studies, a large number of PTMs are known and their coexistence patterns and associations with genomic regions have been mapped in a large number of different cell types. Conversely, the molecular interplay between chromatin effector proteins and modified chromatin regions as well as their resulting biological output is less well understood on a molecular level. Within the last decade a host of chemical approaches has been developed with the goal to produce synthetic chromatin with a defined arrangement of PTMs. These methods now permit systematic functional studies of individual histone and DNA modifications, and additionally provide a discovery platform to identify further interacting nuclear proteins. Complementary chemical- and synthetic-biology methods have emerged to directly observe and modulate the modification landscape in living cells and to readily probe the effect of altered PTM patterns on biological processes. Herein, we review current methodologies allowing chemical and synthetic biological engineering of distinct chromatin states in vitro and in vivo with the aim of obtaining a molecular understanding of histone and DNA modification function. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.     

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.     

Uncovering post-translational modification-associated protein–protein interactions

In living systems, the chemical space and functional repertoire of proteins are dramatically expanded through the post-translational modification (PTM) of various amino acid residues. These modifications frequently trigger unique protein–protein interactions (PPIs) – for example with reader proteins that directly bind the modified amino acid residue – which leads to downstream functional outcomes. The modification of a protein can also perturb its PPI network indirectly, for example, through altering its conformation or subcellular localization. Uncovering the network of unique PTM-triggered PPIs is essential to fully understand the roles of an ever-expanding list of PTMs in our biology. In this review, we discuss established strategies and current challenges associated with this endeavor.     

Proteomic databases and tools to decipher post-translational modifications

Post-translational modifications (PTMs) are vital cellular control mechanism, which affect protein properties, including folding, conformation, activity and consequently, their functions. As a result they play a key role in various disease conditions, including cancer and diabetes. Proteomics as a rapidly growing field has witnessed tremendous advancement during the last decade, which has led to the generation of prodigious quantity of data for various organisms' proteome. PTMs being biologically and chemically dynamic process, pose greater challenges for its study. Amidst these complexities connecting the modifications with physiological and cellular cascade of events are still very challenging. Advancement in proteomic technologies such as mass spectrometry and microarray provides HT platform to study PTMs and help to decipher role of some of the very essential biological phenomenon. To enhance our understanding of various PTMs in different organisms, and to simplify the analysis of complex PTM data, many databases, software and tools have been developed. These PTM databases and tools contain crucial information and provide a valuable resource to the research community. This article intends to provide a comprehensive overview of various PTM databases, software tools, and analyze critical information available from these resources to study PTMs in various biological organisms.     

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.     

Site-selective glycosylation of proteins: creating synthetic glycoproteins

In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). Glycosylation is by far the most diverse of the PTM processes. Natural protein production methods typically produce PTM or glycoform mixtures within which function is difficult to dissect or control. Chemical tagging methods allow the precise attachment of multiple glycosylation modifications to bacterially expressed (bare) protein scaffolds, allowing reconstitution of functionally effective mimics of glycoproteins in higher organisms. In this way combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein-PTM interactions. This protocol describes the modification of Cys residues in proteins using glycomethanethiosulfonates and glycoselenenylsulfides and the modification of azidohomoalanine residues, introduced by Met replacement using auxotrophic Met(-) Escherichia coli strains, with glycoalkynes and the combination of these techniques for the creation of dual-tagged proteins. Each glycosylation procedure outlined in this protocol can be achieved in half a day.     

Analysis of the role of protein phosphorylation in the development of diseases

During recent decades significant progress in studies of the molecular basis of socially significant diseases has been achieved due to introduction of high-throughput methods of genomics and proteomics. Numerous studies, performed within the global program “Human Proteome,” were aimed at identifying all possible proteins in various (including cancer) cell cultures and tissues. One of the aims was to identify socalled biomarkers—the proteins, specific for certain pathologies. However, many studies have shown that the development of the disease is not associated with appearance of new proteins, but it depends on the expression level of certain genes or specific proteoforms representing splice variants, single amino acid polymorphism (SAP) and post-translational modifications (PTM) of proteins. PTMs can play a key role in the development of pathology, because they activate various regulatory or structural proteins in most cellular processes. Among such modifications, phosphorylation appears to be the most significant PTM. This review considers methods of analysis of protein phosphorylation used in studies of the molecular basis of oncological diseases; it contains examples illustrating contribution of modified proteins directly involved in their development as well as examples of screening of such crucial PTMs in diagnostics and selection of methods for treatment.     

Influence of combinatorial histone modifications on antibody and effector protein recognition

Increasing evidence suggests that histone posttranslational modifications (PTMs) function in a combinatorial fashion to regulate the diverse activities associated with chromatin. Yet how these patterns of histone PTMs influence the adapter proteins known to bind them is poorly understood. In addition, how histone-specific antibodies are influenced by these same patterns of PTMs is largely unknown. Here we examine the binding properties of histone-specific antibodies and histone-interacting proteins using peptide arrays containing a library of combinatorially modified histone peptides. We find that modification-specific antibodies are more promiscuous in their PTM recognition than expected and are highly influenced by neighboring PTMs. Furthermore, we find that the binding of histone-interaction domains from BPTF, CHD1, and RAG2 to H3 lysine 4 trimethylation is also influenced by combinatorial PTMs. These results provide further support for the histone code hypothesis and raise specific concerns with the quality of the currently available modification-specific histone antibodies.