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.     

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.     

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.     

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.     

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.     

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.     

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.     

Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2

The Ara h 2 proteins are major determinants of peanut allergens. These proteins have not been fully studied at the molecular level. It has been previously proposed that there are two isoforms of Ara h 2, based on primary structures that were deduced from two reported cDNA sequences. In this report, four isoforms have been purified and characterized individually. Mass spectrometric methods have been used to determine the protein sequences and to define post‐translational modifications for all four isoforms. Two pairs of isoforms have been identified, corresponding to a long‐chain form and a form that is shorter by 12 amino acids. Each pair is further differentiated by the presence or absence of a two amino acid sequence at the carboxyl terminus of the protein. Modifications that were characterized include site‐specific hydroxylation of proline residues, but no glycosylation was found, in contrast to previous reports.     

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.     

Application of mass spectrometry to the identification and quantification of histone post-translational modifications

The core histones are the primary protein component of chromatin, which is responsible for the packaging of eukaryotic DNA. The NH(2)-terminal tail domains of the core histones are the sites of numerous post-translational modifications that have been shown to play an important role in the regulation of chromatin structure. In this study, we discuss the recent application of modern analytical techniques to the study of histone modifications. Through the use of mass spectrometry, a large number of new sites of histone modification have been identified, many of which reside outside of the NH(2)-terminal tail domains. In addition, techniques have been developed that allow mass spectrometry to be effective for the quantitation of histone post-translational modifications. Hence, the use of mass spectrometry promises to dramatically alter our view of histone post-translational modifications.     

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.     

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.     

SwissPIT: An workflow‐based platform for analyzing tandem‐MS spectra using the Grid

The identification and characterization of peptides from MS/MS data represents a critical aspect of proteomics. It has been the subject of extensive research in bioinformatics resulting in the generation of a fair number of identification software tools. Most often, only one program with a specific and unvarying set of parameters is selected for identifying proteins. Hence, a significant proportion of the experimental spectra do not match the peptide sequences in the screened database due to inappropriate parameters or scoring schemes. The Swiss protein identification toolbox (swissPIT) project provides the scientific community with an expandable multitool platform for automated in‐depth analysis of MS data also able to handle data from high‐throughput experiments. With swissPIT many problems have been solved: The missing standards for input and output formats (A), creation of analysis workflows (B), unified result visualization (C), and simplicity of the user interface (D). Currently, swissPIT supports four different programs implementing two different search strategies to identify MS/MS spectra. Conceived to handle the calculation‐intensive needs of each of the programs, swissPIT uses the distributed resources of a Swiss‐wide computer Grid (http://www.swing‐grid.ch).     

Type‐II histone deacetylases: elusive plant nuclear signal transducers

Since the beginning of the 21st century, numerous studies have concluded that the plant cell nucleus is one of the cellular compartments that define the specificity of the cellular response to an external stimulus or to a specific developmental stage. To that purpose, the nucleus contains all the enzymatic machinery required to carry out a wide variety of nuclear protein post‐translational modifications (PTMs), which play an important role in signal transduction pathways leading to the modulation of specific sets of genes. PTMs include protein (de)acetylation which is controlled by the antagonistic activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Regarding protein deacetylation, plants are of particular interest: in addition to the RPD3‐HDA1 and Sir2 HDAC families that they share with other eukaryotic organisms, plants have developed a specific family called type‐II HDACs (HD2s). Interestingly, these HD2s are well conserved in plants and control fundamental biological processes such as seed germination, flowering or the response to pathogens. The aim of this review was to summarize current knowledge regarding this fascinating, but still poorly understood nuclear protein family.     

The extraordinary thermal stability of EstA from S. islandicus is independent of post translational modifications

Enzymes from thermophilic and hyper‐thermophilic organisms have an intrinsic high stability. Understanding the mechanisms behind their high stability will be important knowledge for the engineering of novel enzymes with high stability. Lysine methylation of proteins is prevalent in Sulfolobus, a genus of hyperthermophilic and acidophilic archaea. Both unspecific and temperature dependent lysine methylations are seen, but the significance of this post‐translational modification has not been investigated. Here, we test the effect of eliminating in vivo lysine methylation on the stability of an esterase (EstA). The enzyme was purified from the native host S. islandicus as well as expressed as a recombinant protein in E. coli, a mesophilic host that does not code for any machinery for in vivo lysine methylation. We find that lysine mono methylation indeed has a positive effect on the stability of EstA, but the effect is small. The effect of the lysine methylation on protein stability is secondary to that of protein expression in E. coli, as the E. coli recombinant enzyme is compromised both on stability and activity. We conclude that these differences are not attributed to any covalent difference between the protein expressed in hyperthermophilic versus mesophilic hosts.     

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.     

Mass spectrometry based analysis of human plasma‐derived factor X revealed novel post‐translational modifications

Human coagulation factor X is a central component of the blood coagulation cascade that converts, under its activated form, prothrombin into thrombin. Generation of thrombin is the final step of the clotting cascade that leads to the clot by polymerization of fibrinogen molecules into a fibrin network. Today, research of new by‐passing agents of the coagulation may contribute to an increased interest for human factor X, which may, in consequence, lead to the need of a more exhaustive picture of its structural features. Several post‐translational modifications of human factor X such as γ‐carboxylation/β‐hydroxylation of the N‐terminal light chain and N‐/O‐glycosylation of the activation peptide have been described. But, so far as we know, no comprehensive studies of its post‐translational modifications have been reported. In this article we report an exhaustive structural analysis of human factor X by mass spectrometry using successive protein and peptide mapping. Surprisingly, human factor X was found to be mostly O‐glucosylated on its light chain at Ser₁₀₆ position, Ser₉ of its activation peptide is phosphorylated at about 30% and its C‐terminal heavy chain is fully O‐glycosylated at Thr₂₄₉ by a mucin‐type O‐glycan (HexNAc‐Hex‐NeuAc). The knowledge of these post‐translational modifications is mandatory for the development of recombinant molecules.