Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical applications.

Given the complexity of the biosynthetic machinery and the delicate chemical composition of proteins, it is remarkable that cells manage to produce and maintain normally functioning proteins under most conditions. However, it is now well known that proteins are susceptible to various non-enzymatic covalent modifications (NECM) under physiological conditions. Such modifications can be of no or little importance to the protein or they can be absolutely detrimental. Often NECM are difficult to study due to the complex and technically demanding methods required to identify many of these modifications. Thus, the role of NECM has not yet been adequately resolved but recent research has allowed a better understanding of such modifications. The present review outlines the various forms of NECM that involve covalent modifications of proteins, and discusses their relevance, biological impact and potential applications in the study of protein turnover and diagnosis of disease.     

Ubiquitin and ubiquitin-like proteins as multifunctional signals

Protein ubiquitylation is a recognized signal for protein degradation. However, it is increasingly realized that ubiquitin conjugation to proteins can be used for many other purposes. Furthermore, there are many ubiquitin-like proteins that control the activities of proteins. The central structural element of these post-translational modifications is the ubiquitin superfold. A common ancestor based on this superfold has evolved to give various proteins that are involved in diverse activities in the cell.     

Post-aggregation Oxidation of Mutant Huntingtin Controls the Interactions between Aggregates

Aggregation of protein molecules is a pathological hallmark of many neurodegenerative diseases. Abnormal modifications have often been observed in the aggregated proteins, supporting the aggregation mechanism regulated by post-translational modifications on proteins. Modifications are in general assumed to occur in soluble proteins before aggregation, but actually it remains quite obscure when proteins are modified in the course of the aggregation. Here we focus upon aggregation of huntingtin (HTT), which causes a neurodegenerative disorder, Huntington disease, and we show that oxidation of a methionine residue in HTT occurs in vitro and also in vivo. Copper ions as well as added hydrogen peroxide are found to oxidize the methionine residue, but notably, this oxidative modification occurs only in the aggregated HTT but not in the soluble state. Furthermore, the methionine oxidation creates additional interactions among HTT aggregates and alters overall morphologies of the aggregates. We thus reveal that protein aggregates can be a target of oxidative modifications and propose that such a “post-aggregation” modification is a relevant factor to regulate properties of protein aggregates.     

Histone modifications in DNA damage response

DNA damage is a relatively common event in eukaryotic cell and may lead to genetic mutation and even cancer. DNA damage induces cellular responses that enable the cell either to repair the damaged DNA or cope with the damage in an appropriate way. Histone proteins are also the fundamental building blocks of eukaryotic chromatin besides DNA, and many types of post-translational modifications often occur on tails of histones. Although the function of these modifications has remained elusive, there is ever-growing studies suggest that histone modifications play vital roles in several chromatin-based processes, such as DNA damage response. In this review, we will discuss the main histone modifications, and their functions in DNA damage response.     

Post-translational modification of 14-3-3 isoforms and regulation of cellular function

14-3-3 is now well established as a family of dimeric proteins that can modulate interaction between proteins involved in a wide range of functions. In many cases, these proteins show a distinct preference for a particular isoform(s) of 14-3-3 and in many cases a specific repertoire of dimer formation influences the particular proteins that 14-3-3 interact. Well over 200 proteins have been shown to interact with 14-3-3. The purpose of this review is to give an overview of the recently identified post-translational modifications of 14-3-3 isoforms and how this regulates function, interaction, specificity of dimerisation between isoforms and cellular location of target proteins. The association between 14-3-3 and its targets usually involves phosphorylation of the interacting protein which has been the subject of many reviews and discussion of this is included in other reviews in this series. However, it is now realised that in some cases the phosphorylation and a number of other, novel covalent modifications of 14-3-3 isoforms may modulate interaction and dimerisation of 14-3-3. Since this aspect is now emerging to be of major importance in the mechanism of regulation by 14-3-3 isoforms and has not been the focus of previous reviews, this will be detailed here.     

Murburn posttranslational modifications of proteins: Cellular redox processes and murzyme-mediated metabolo-proteomics

Murburn concept constitutes the thesis that diffusible reactive species or DRS are obligatorily involved in routine metabolic and physiological activities. Murzymes are defined as biomolecules/proteins that generate/modulate/sustain/utilize DRS. Murburn posttranslational modifications (PTMs) result because murburn/murzyme functionalism is integral to cellular existence. Cells must incorporate the inherently stochastic nature of operations mediated by DRS. Due to the earlier/inertial stigmatic perception that DRS are mere agents of chaos, several such outcomes were either understood as deterministic modulations sponsored by house-keeping enzymes or deemed as unregulated nonenzymatic events resulting out of “oxidative stress”. In the current review, I dispel the myths around DRS-functions, and undertake systematic parsing and analyses of murburn modifications of proteins. Although it is impossible to demarcate all PTMs into the classical or murburn modalities, telltale signs of the latter are evident from the relative inaccessibility of the locus, non-specificities and mechanistic details. It is pointed out that while many murburn PTMs may be harmless, some others could have deleterious or beneficial physiological implications. Some details of reversible/irreversible modifications of amino acid residues and cofactors that may be subjected to phosphorylation, halogenation, glycosylation, alkylation/acetylation, hydroxylation/oxidation, etc. are listed, along with citations of select proteins where such modifications have been reported. The contexts of these modifications and their significance in (patho)physiology/aging and therapy are also presented. With more balanced explorations and statistically verified data, a definitive understanding of normal versus pathological contexts of murburn modifications would be obtainable in the future.     

Identification of sumoylated proteins by systematic immunoprecipitation of the budding yeast proteome

The identification of post-translational modifications to proteins is critical for understanding many important aspects of biology. Utilizing a collection of epitope-tagged yeast strains, we developed a novel approach to determine which proteins are modified by the small ubiquitin-related modifier (SUMO). We crossed traits useful for the detection of SUMO conjugation into 4246 tandem affinity purification-tagged strains and successfully immunoprecipitated and screened 2893 of these proteins for association with SUMO ( approximately 70% of the expressed proteome detectable by immunoblot analysis). We found 82 proteins associated with SUMO, including many of low abundance. Because our screen was performed under non-denaturing conditions, we were able to identify multiple members of four complexes that were associated with SUMO: the RSC chromatin remodeling complex, the mediator complex, the TFIID complex, and the septin complex. In addition, we describe five new direct conjugates of SUMO, and we mutated SUMO conjugation sites in four proteins. This is the first attempt to immunoprecipitate a large fraction of the proteome of a eukaryote, and it demonstrates the utility of this method to identify post-translational modifications in the yeast proteome.     

Post-translational modification of proteins

Many proteins, especially those produced by eukaryotic cells, undergo extensive, essentially irreversible, modifications after their synthesis. This review focuses on three classes of such reactions: proteolytic cleavages, formation of S-S cystine bonds, and formation of asparagine-linked carbohydrate chains. Emphasis is placed on the mechanism of these reactions, and on the importance of these modifications for the proper structure, function and stability of the affected proteins. Using recombinant DNA techniques, it is now possible to synthesize the polypeptide portion of many proteins, such as mammalian peptide hormones and enzymes, in bacterial and yeast cells. These host cells, however, may be unable to carry out essential post-translational modifications. Ways in which the properly modified form of these ‘engineered’ proteins can be produced are considered.     

Inhibitors of glycosyl-phosphatidylinositol anchor biosynthesis

Glycosyl-phosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa such as Trypanosoma brucei, Leishmania major, Plasmodium falciparum and Toxoplasma gondii, and represent the major carbohydrate modification of many cell-surface parasite proteins. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. Therefore, the characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. In vitro and in vivo studies using sugars and substrate analogues as well as natural compounds have shown that it is possible to interfere with GPI biosynthesis at different steps in a species-specific manner. Here we review the recent and promising progress in the field of GPI inhibition.     

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.     

Immediate protein targets of photodynamic treatment in carcinoma cells

Oxidative stress induced in tumor cells undergoing photodynamic treatment (PDT) leads to extensive modification of many proteins in these cells. Protein oxidation mainly gives rise to formation of carbonyls and oxidized thiols. The immediate targets of PDT-induced protein oxidation in A431 tumor cells have been identified using a proteomic approach involving selective biotinylation, affinity purification and mass spectrometric identification of modified proteins. In all, 314 proteins were shown to undergo PDT-mediated oxidative modifications. While abundant structural proteins and chaperones represented a significant fraction of the carbonylated proteins, labeling of proteins containing oxidized thiols allowed identification of many proteins at low abundance and those involved in signaling and redox homeostasis. On the basis of the identification of these proteins, several likely mechanisms of PDT-induced triggering of apoptosis were put forward. This may not only lead to a further understanding of the complex network of cellular responses to oxidative stress, but it may also help in detailed targeting of photodynamic treatment applied to cancer.     

Mapping protein post-translational modifications with mass spectrometry

Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.     

A genetic map of Xenopus tropicalis

Posttranslational modifications constitute a major field of emerging biological significance as mounting evidence demonstrates their key role in multiple physiological processes. Following in the footsteps of protein phosphorylation studies, new modifications are being shown to regulate protein properties and functions in vivo. Among such modifications, an important role belongs to protein arginylation — posttranslational tRNA-mediated addition of arginine, to proteins by arginyltransferase, ATE1. Recent studies show that arginylation is essential for embryogenesis in many organisms and that it regulates such important processes as heart development, angiogenesis, and tissue morphogenesis in mammals. This review summarizes the key data in the protein arginylation field since its original discovery to date.     

The on-off story of protein palmitoylation

Palmitoylation is one of the most frequent post-translational modifications found on proteins. It contributes to membrane association, protein sorting and many other processes. Through its reversibility, palmitoylation also provides mechanisms to regulate the functional activities of integral and peripheral membrane proteins. Here we discuss evidence that proteins can be palmitoylated at different locations in the cell, how targeting to these locations might be directed, and aspects of the proposed functions of palmitoylation.     

Proteomics reveals evidence of cross-talk between protein modifications in bacteria: focus on acetylation and phosphorylation

Recent advances in gel-free, mass spectrometry-based proteomics have firmly established existence of serine phosphorylation, threonine phosphorylation, tyrosine phosphorylation and lysine acetylation on many bacterial proteins. Intriguingly, numerous proteins have been shown to be modified by both modifications, leading to the emerging concept of cross-talk between posttranslational modifications in bacteria. This concept is further supported by biological follow-up studies that are starting to reveal bacterial proteins and processes regulated by multiple modifications. In this review, we provide an overview of the large-scale studies involving protein phosphorylation and acetylation in bacteria and discuss some of the current examples of cross-talk between these and other bacterial modifications.     

Proteomics: a powerful tool in the post-genomic era

Genomics is having a profound impact on biological research, including photosynthesis investigations. Genomes of many photosynthetic organisms have been sequenced. The information about ALL genes that govern and execute photoautotrophic metabolism provides many opportunities to understand genome function and details of known and uncharted pathways. Proteomics, analysis of the protein complement of the genome, is a powerful tool in understanding which proteins are present in a particular tissue under given conditions. Proteomics also allows us to estimate relative levels of proteins and to determine post-translational modifications of the gene products. In this minireview, we discuss the technology and its applications in plant sciences.     

Gel-free proteomic methodologies to study reversible cysteine oxidation and irreversible protein carbonyl formation

Abstract

Oxidative modifications in proteins have been traditionally considered as hallmarks of damage by oxidative stress and aging. However, oxidants can generate a huge variety of reversible and irreversible modifications in amino acid side chains as well as in the protein backbones, and these post-translational modifications can contribute to the activation of signal transduction pathways, and also mediate the toxicity of oxidants. Among the reversible modifications, the most relevant ones are those arising from cysteine oxidation. Thus, formation of sulfenic acid or disulfide bonds is known to occur in many enzymes as part of their catalytic cycles, and it also participates in the activation of signaling cascades. Furthermore, these reversible modifications have been usually attributed with a protective role, since they may prevent the formation of irreversible damage by scavenging reactive oxygen species. Among irreversible modifications, protein carbonyl formation has been linked to damage and death, since it cannot be repaired and can lead to protein loss-of-function and to the formation of protein aggregates. This review is aimed at researchers interested on the biological consequences of oxidative stress, both at the level of signaling and toxicity. Here we are providing a concise overview on current mass-spectrometry-based methodologies to detect reversible cysteine oxidation and irreversible protein carbonyl formation in proteomes. We do not pretend to impose any of the different methodologies, but rather to provide an objective catwalk on published gel-free approaches to detect those two types of modifications, from a biologist's point of view.