Post-translational modifications of poliovirus proteins

The post-translational modifications of poliovirus proteins have been investigated by analysis of glycosylation, sulphation, phosphorylation and acylation of the proteins made in the infected HeLa cells. No glycosylation or sulphation of proteins specific for virus-infected cells was apparent. A number of changes in the pattern of phosphorylated proteins took place. The specific myristylation of the structural protein VP4 and its precursors was clearly apparent. Acylation of viral proteins with oleic or palmitic acid was not detected. Myristylation took place in the presence of the protease inhibitor ZnCl2, but not in the presence of inhibitors of translation, such as cycloneximide and anysomycin.     

Post-translational modifications in the context of therapeutic proteins

The majority of protein-based biopharmaceuticals approved or in clinical trials bear some form of post-translational modification (PTM), which can profoundly affect protein properties relevant to their therapeutic application. Whereas glycosylation represents the most common modification, additional PTMs, including carboxylation, hydroxylation, sulfation and amidation, are characteristic of some products. The relationship between structure and function is understood for many PTMs but remains incomplete for others, particularly in the case of complex PTMs, such as glycosylation. A better understanding of such structural-functional relationships will facilitate the development of second-generation products displaying a PTM profile engineered to optimize therapeutic usefulness.     

Post-translational modifications of mitochondrial outer membrane proteins

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

Post-translational modifications of mitochondrial outer membrane proteins

Abstract

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

Post-translational modifications of histone proteins by monoamine neurotransmitters

Protein monoaminylation is a biochemical process through which biogenic monoamines (e.g., serotonin, dopamine, histamine, etc.) are covalently bonded to certain protein substrates via Transglutaminase 2, an enzyme that catalyzes the transamidation of primary amines to the γ-carboxamides of glutamine residues. Since their initial discovery, these unusual post-translational modifications have been implicated in a wide variety of biological processes, ranging from protein coagulation to platelet activation and G-protein signaling. More recently, histone proteins – specifically histone H3 at glutamine 5 (H3Q5) – have been added to the growing list of monoaminyl substrates in vivo, with H3Q5 monoaminylation demonstrated to regulate permissive gene expression in cells. Such phenomena have further been shown to contribute critically to various aspects of (mal)adaptive neuronal plasticity and behavior. In this short review, we examine the evolution of our understanding of protein monoaminylation events, highlighting recent advances in the elucidation of their roles as important chromatin regulators.     

Post-translational modifications of p21rho proteins.

Post-translational modifications of the ras proteins, which are required for plasma membrane localization and biological function of the proteins, have been shown to include prenylation and carboxymethylation at the carboxyl terminal cysteine residue of the cysteine-aliphatic amino acid-aliphatic amino acid-any amino acid (CAAX) box. In addition, p21Ha-ras and p21N-ras, but not p21K-ras (B), are palmitoylated. The three mammalian rho proteins (A, B, and C) are also members of the ras superfamily but have distinct biological activities and different intracellular distributions from p21ras. Analysis showed all three rho proteins are modified by a COOH-terminal carboxymethylation similar to p21ras, whereas p21rhoC labeled with [3H]mevalonic acid in vivo revealed the presence of a C20 prenoid, similar to that already described for p21rhoA. However, in vivo and in vitro studies of p21rhoB showed this protein to be modified by both C15 and C20 prenoids. Mutation of C193 in the CAAX box abolished prenylation, whereas mutation of the adjacent C192 resulted in a significant reduction in the amount of the C20, but not C15 prenoid, recovered from p21rhoB. In vivo labeling studies with [3H]palmitic acid and mutational analysis showed that both cysteine residues at 189 and 192 upstream of the CAAX box in p21rhoB are sites for palmitoylation. We conclude that there are different populations of post-translationally modified p21rhoB in the cell and that the sequence specificity for geranylgeranyl- and farnesyltransferases may be more complicated than previously proposed.     

Post-translational modifications of proteins: some problems left to solve

Three major questions regarding the post-translational modification of amino acid side chains in proteins are briefly considered: (1) What are the biological functions of the reactions, (2) what is the specificity of the processing reactions in selecting only a few or sometimes even only one residue for modification, and (3) how do we solve the uniqueness of the processing steps in the production of recombinant proteins? The answers to these questions are not obvious at this time.     

Post-translational modifications of lantibiotics

Several newly reported post-translational modification reactions are involved in lantibiotic biosynthesis. A short overview of the present knowledge on the post-translational modifications and on the enzymes involved in lantibiotic biosynthesis is given. The oxidative decarboxylation of the epidermin precursor peptide EpiA is described in detail. The FMN-containing oxidoreductase EpiD is involved in the formation of the C-terminal S-[(Z)-2-aminovinyl]-D-cysteine residue of epidermin: under reducing conditions the side chain of the C-terminal cysteine residue of EpiA is converted to an enethiol. EpiD has no absolute substrate specificity and can be used for modification of peptides having the C-terminal consensus motif [V/I/L/(M)/F/Y/W]-[A/S/V/T/C/(I/L)]-C.Abbreviations Dha 2,3-didehydroalanine - Dhb (Z)-2,3-didehydrobutyrine - ES-MS Electrospray Mass Spectrometry - FAD Flavin Adenine Dinucleotide - FMN Flavin Mononucleotide - MBP Maltose-Binding Protein - TFA TrifluoroAcetic Acid - TLC Thin-Layer Chromatography     

Post-translational modifications of apolipoprotein A-I and Po proteins in the avian peripheral nerve

Apolipoprotein A-I (apo A-I), a soluble lipid transporter, and Po, the major glycoprotein of myelin, are actively synthesized during myelination. To explore the status of post-translational modifications of these proteins in the avian PNS during rapid myelination, endoneurial slices from one day old chick sciatic nerves were incubated with various radioactive precursors that could serve as indicators of such processes. The proteins were isolated from the incubation medium (secreted fraction), the 1% Triton-X-100-soluble intracellular-endoneurial (intracellular) fraction, and myelin-related and purified compact myelin fractions by immunoprecipitation with monospecific anti-apo A-I or anti-Po antisera. Our results demonstrated that secreted apo A-I is fatty acylated, but not phosphorylated or sulfated. Avian Po protein was phosphorylated by a phorbol ester sensitive protein kinase. Sulfation, as well as fatty acylation, of avian Po protein was observed in organ culture using highly sensitive methods of detection. These results indicate that fatty acylation of secreted apo A-I and phosphorylation, sulfation and fatty acylation of Po have been conserved during evolution, and that these post-translational modifications may play a common function in various species.     

Post-translational modifications of superoxide dismutase

Post-translational modifications of proteins control many biological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins and also have shed light on the post-translational modification of superoxide dismutase. In this review, we introduce some covalent modifications of superoxide dismutase, nitration, phosphorylation, glutathionylaion, and glycation. Nitration has been the most extensively analyzed modification both in vitro and in vivo. Reaction of human Cu,Zn superoxide dismutase (SOD) with reactive nitrogen species resulted in nitration of a single tryptophan residue to 6-nitrotryptophan, which could be a new biomarker of a formation of reactive nitrogen species. On the other hand, tyrosine 34 of human MnSOD was exclusively nitrated to 3-nitrotyrosine and almost completely inactivated by the reaction with peroxynitrite. The nitrated MnSOD has been found in many diseases caused by ischemia/reperfusion, inflammation, and others and may have a pivotal role in the pathology of the diseases. Most of the post-translational modifications have given rise to a reduced activity of SOD. Since phosphorylation and nitration of SOD have been shown to have a possible reversible process, these modifications may be related to a redox signaling process in cells. Finally we briefly introduce a metal insertion system of SOD, focusing particularly on the iron misincorporation of nSOD, as a part of post-translational modifications.     

Post-translational modifications during lantibiotic biosynthesis

Recent reports have provided the first insights into the mechanisms of the extensive post-translational modifications involved in the biosynthesis of the lantibiotics, a class of peptide antimicrobial agents. These modifications involve dehydration of several serine and threonine residues followed by intramolecular conjugate additions of cysteines, resulting in extensively cross-linked polycyclic structures. Both in vivo and in vitro studies indicate low substrate specificity of the modification machinery, which has been explored for re-engineering of the structures of a number of members. In addition to these developments in understanding their biosynthesis, studies on the mode of action of several lantibiotics have shown a unique mechanism of binding to lipid II, an intermediate in cell wall biosynthesis.     

Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.     

Post-translational modifications of rat liver mitochondrial outer membrane proteins identified by mass spectrometry

The identification of post-translational modifications is difficult especially for hydrophobic membrane proteins. Here we present the identification of several types of protein modifications on membrane proteins isolated from mitochondrial outer membranes. We show, in vivo, that the mature rat liver mitochondrial carnitine palmitoyltransferase-I enzyme is N-terminally acetylated, phosphorylated on two threonine residues, and nitrated on two tyrosine residues. We show that long chain acyl-CoA synthetase 1 is acetylated at both the N-terminal end and at a lysine residue and tyrosine residues are found to be phosphorylated and nitrated. For the three voltage-dependent anion channel isoforms present in the mitochondria, the N-terminal regions of the protein were determined and sites of phosphorylation were identified. These novel findings raise questions about regulatory aspects of carnitine palmitoyltransferase-I, long chain acyl-CoA synthetase and voltage dependent anion channel and further studies should advance our understanding about regulation of mitochondrial fatty acid oxidation in general and these three proteins in specific.