Auswahl der wissenschaftlichen Literatur zum Thema „Protein N-terminal modifications“

Data from a mass spectrometry experiment of a mouse line developed to study the mechanisms of fibromuscular dysplasia and deposited by d'Escamard et al. in ProteomeXchange (PXD051750) have been analyzed. Identification of peptides with post-translational modifications (PTMs) was repeated using more stringent conditions than in the original work. The following modifications were considered during analysis of changes in the PTM levels in experimental and control groups of mice: acetylation of lysine residue and N-terminal protein peptide, ubiquitination of lysine residue, phosphorylation of serine, threonine and tyrosine residues, and deamination of asparagine and glutamine residues. The multistage analysis resulted in selection of 23 proteins with PTMs for which different levels of modification between experimental and control groups could be assumed. These included six proteins with N-terminal protein acetylation, which were particularly interesting: P80318 (T-complex protein 1 subunit gamma), P43274 (Histone H1.4), P97823 (Acyl-protein thioesterase 1), P63242 (Eukaryotic translation initiation factor 5A-1), Q3UMT1 (Protein phosphatase 1 regulatory subunit 12C), Q9D8Y0 (EF-hand domain-containing protein D2). Thus, repeated bioinformatic analysis of the data deposited in the specialized databases resulted in detection of changes in the level of N-terminal acetylation of proteins that might be functionally significant in the mechanisms underlying the development of fibromuscular dysplasia.

ABSTRACT Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.

The N-degron pathway, formerly the N-end rule pathway, regulates functions of regulatory proteins. It impacts protein half-life and therefore directs the actual presence of target proteins in the cell. The current concept holds that the N-degron pathway depends on the identity of the amino (N)-terminal amino acid and many other factors, such as the follow-up sequence at the N terminus, conformation, flexibility, and protein localization. It is evolutionarily conserved throughout the kingdoms. One possible entry point for substrates of the N-degron pathway is oxidation of N-terminal Cys residues. Oxidation of N-terminal Cys is decisive for further enzymatic modification of various neo–N termini by arginylation that generates potentially neofunctionalized or instable proteoforms. Here, I focus on the posttranslational modifications that are encompassed by protein degradation via the Cys/Arg branch of the N-degron pathway—part of the PROTEOLYSIS 6 (PRT6)/N-degron pathway—as well as the underlying physiological principles of this branch and its biological significance in stress response.

Recombinant human interleukin-5 exists as four major isoforms all possessing N-terminal methionine. Peptide mapping and subsequent analysis by fast-atom-bombardment mass spectrometry (f.a.b.-m.s.) have shown that N-terminal modifications are the cause of the charge heterogeneity. In order of decreasing abundance, these are unmodified methionine, retention of N-terminal formyl group, oxidation of N-terminal methionine to sulphoxide and carbamoylation of the N-terminus. These results were confirmed by analysis of the reduced and alkylated intact protein by electrospray-ionization mass spectrometry. The implications of these findings for the production and characterization of recombinant proteins are briefly discussed.

N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.

Lors de la photosynthèse, l’assimilation sous formes de glucides du dioxyde de carbone atmosphérique (CO₂), le principal gaz à effet de serre anthropique, est catalysée par l'enzyme Rubisco, la protéine la plus abondante sur terre. La grande sous-unité de la Rubisco (RbcL) subit une voie de maturation unique conduisant à des modifications N-terminales inhabituelles. Ce mécanisme conservé chez les plantes, résulte en une proline acétylée N-terminale en position 3. Décrypter la voie de maturation de Rubisco est donc une question clé pour la fixation du CO₂ dans le contexte des changements climatiques et du réchauffement climatique. Mon projet de doctorat visait à découvrir la machinerie à l'acétylation de Pro3 et à comprendre la pertinence fonctionnelle associée. Tout d'abord, deux cadres de lecture ouverts (ORFs) chez Arabidopsis thaliana ont été identifiés comme candidats potentiels pouvant contribuer au rocessus. Les fonctions de deux aminopeptidases conservées ont été testées in vitro et dans des lignées d'Arabidopsis thaliana mutantes dépourvues d’activité. Je démontre que l’une est spécifiquement responsable du clivage du résidu 2, alors qu'e l’autre ne contribue pas à la maturation des protéines N-terminales du plaste. Par ailleurs, mes données démontrent que l'acétylation de Pro3 est catalysée par une seule des isoformes d’acétyltransférases présentes dans le plaste. Ainsi, la machinerie de modification N-terminale unique impliquée dans le traitement de RbcL repose sur deux enzymes dédiées à la maturation de RbcL. J'ai pu aussi reconstituer cette voie de maturation chez E. coli. Enfin, j'ai étudié comment ces modifications RbcL affectent l'assemblage, l'activité et l'accumulation de la Rubisco
During photosynthesis, atmospheric carbon dioxide (CO₂), the prevalent anthropogenic greenhouse gas, is assimilated into carbohydrates by the enzyme Rubisco, the most abundant protein on earth. The large subunit of Rubisco (RbcL) undergoes a unique maturation pathway leading to unusual N-terminal modifications. This mechanism is conserved in plants, resulting in an N-terminal acetylated proline at position 3. Unravelling the maturation pathway of Rubisco is therefore a key challenge for CO₂ fixation in the context of climate change and global warming. My PhD project aimed at discovering the machinery leading to Pro3 acetylation and unmasking the associated functional relevance. First, two open reading frames (ORFs) in Arabidopsis thaliana were identified as putative candidates that might contribute to the proteolytic part of this process. The functions of two conserved aminopeptidases were challenged in vitro assay and in knockout Arabidopsis thaliana lines. I showed that one protease is specifically in charge of residue 2 release, while the second does not contribute to N-terminal protein maturation in the plastid. In addition, my data demonstrates that Pro3 acetylation is catalysed by only one acetyltransferase isoform occurring in the plastid. Together, the unique N-terminal modification machinery involved in RbcL processing relies on two enzymes that are dedicated to RbcL processing. I could reconstitute the maturation pathway in E. coli. Finally, I have investigated how the N-terminal modifications of RbcL affect Rubisco assembly, activity, and accumulation

L'excision de la méthionine N-terminale (NME) est la première modification se produisant au N-terminal des protéines (NPM). Les peptides déformylases (PDF) sont les enzymes impliquées dans ce processus co-traductionnel essentiel et conservé. Les PDFs suppriment le groupe formyle lié à la méthionine initiatrice (iMet) présente au début de toutes les chaînes procaryotes naissantes. Les PDFs agissent au niveau du tunnel de sortie des ribosomes, plaque tournante de nombreux facteurs de biogénèse des protéines associées aux ribosomes (PRB), impliqués non seulement sur les MNP, mais également dans le repliement et la translocation des protéines. La déformylation N-terminale implique 95% du protéome bactérien et contribue directement à la stabilité des protéines. Le récent séquençage à haut débit de milliers de génomes a révolutionné notre perception de la distribution des PDFs dans les différents règnes, révélant des PDFs putatives dans tous les organismes, y compris les virus. En particulier, les études concernant les virus présents dans les échantillons microbiens océaniques ont permis d’identifier des gènes inhabituels de PDF chez les phages, constituant la famille la plus abondante de protéines auxiliaires conservées de ces génomes. La comparaison des séquences identifiées révèle que les PDF virales présentent une forte conservation des trois motifs constituant le site catalytique. Cependant, ces PDFs virales ne présentent pas d'extension C-terminale, région réputée importante des PDFs des autres organismes. Sachant que cette extension est impliquée dans la liaison de la PDF d’E. coli au ribosome et est requise pour son activité déformylase in vivo, il était incertain que les PDFs de phage découvertes avaient une activité déformylase classique. Ainsi, la découverte de ces PDFs virales soulève un certain nombre de questions parmi lesquelles: a) Ces PDFs virales présentent-elles une activité déformylase classique? b) Ces PDFs sont-elles capables de se lier aux ribosomes ? c) Pourquoi autant de virus portent-ils une ou plusieurs déformylases ? Dans ce contexte, l’objectif de ma thèse a été d’entreprendre la caractérisation de ces PDFs de phages marins et en particulier la PDF de Vp16 provenant de bactériophages isolés à l’origine de la souche 16 de Vibrio parahaemolyticus. Nos études révèlent que ces PDFs de phages présentent une activité déformylase à la fois in vitro et in vivo, avec une spécificité de substrat similaire à celle des autres PDFs bactériennes. D'autre part, nous avons montré par des études biochimiques et structurales, combinées à des analyses par mutagenèse dirigée, que les propriétés de la PDF de Vp16 diffèrent significativement de celle des autres PDFs caractérisées précédemment. Il faut aussi noter que l'expression de la PDF Vp16 dans les souches d'E. Coli, même à de faibles concentrations, montre un effet bactéricide marqué à une température inférieure à 37 °C. L’effet bactéricide de la PDF Vp16 est indépendant de la présence de la PDF endogène bactérienne et repose strictement sur son activité déformylase. La caractérisation de ce phénotype a révélé que la létalité induite par Vp16 PDF montrait un lien génétique fort avec des gènes codants pour des facteurs cellulaires impliqués dans le ciblage et le repliement précoce des protéines (Trigger Factor et Sec). Contrairement à ce qui a été montré pour les PDFs bactériennes, Vp16 PDF a une forte affinité pour le ribosome bactérien d’E. coli en cours de traduction, interagissant avec une région ribosomale chevauchant celles des facteurs impliqués dans le transit des protéines vers les voies de sécrétion. Une compétition au niveau du ribosome entre Vp16 PDF et ces RPBs pourrait contribuer à la lyse cellulaire de l’hôte. Mon travail suggère un nouveau mécanisme utilisé par les bactériophages permettant de contrôler la viabilité de l'hôte
N-terminal Methionine Excision (NME) is the first occurring N-terminal Protein Modification (NPMs). Peptide deformylases (PDFs) are the enzymes involved in this essential and conserved co-translational process. PDFs remove the formyl group bound to the iMet present at the beginning of all prokaryotic nascent chains. PDFs act on the nascent chain at the level of the ribosome exit tunnel, a central hub for a number of Ribosome-associated Protein Biogenesis factors (RPBs) involved not only on NPMs but also in protein folding and translocation. Deformylation involves 95% of bacterial proteome and it is suggested to directly contribute to protein stability. Recent high-throughput sequencing of thousands of genomes has strongly contributed to revolutionizing our perception of the distribution of PDFs among kingdoms, revealing putative PDFs in all organisms, including viruses. In particular, studies of viruses within oceanic microbial samples retrieved unusual PDFs genes as the most abundant family in most of phage genomes. Sequence comparisons reveal that viral PDFs show high conservation in the three motifs that build the catalytic site; however, viral PDFs do not display a C-terminal extension when compared to the different active PDFs from other organisms. Since this C-terminal extension was shown to be important for PDF-ribosome binding and is required for the in vivo deformylase activity of E. coli PDF, it was unclear whether the discovered phage PDFs might support a classical deformylase activity. Thus, the discovery of these viral PDFs raises a number of questions among which: a) Have these viral PDFs a classical deformylase activity? b) Are these PDFs able to still bind to the ribosomes? c) Why so many viruses carry a peptide deformylase? In this context, the objective of my thesis was to undertake the characterization of these marine phage PDFs and particularly Vp16 PDF derived from the bacteriophages originally isolated from Vibrio Parahaemolyticus strain 16. Our studies reveal that phage PDFs display deformylase activity both in vitro and in vivo with a substrate specificity similar to that of other bacterial PDFs. On the other hand, we showed by biochemical and structural data, combined with site-directed mutagenesis analyses, that Vp16 PDF significantly differs from previously characterized PDFs in terms of their properties, which can be related to its few uncommon peculiarities. Interestingly, expression of Vp16 PDF in E. coli strains, even at low concentrations, exhibited a severe bactericidal effect at temperature lower than 37 °C. This bactericidal effect of Vp16 PDF was independent of the presence of the bacterial endogenous PDF and strictly relied on its PDF activity. Characterization of this phenotype revealed that Vp16 PDF-induced lethality showed a strong genetic link with genes encoding cellular factors involved in nascent pre-secretory protein targeting and folding (Trigger Factor and Sec). Differently from bacterial PDF, I could show that Vp16 PDF has strong affinity for ribosomes with a specific nascent chain, interacting with a ribosomal region overlapping that of factors involved in pre-secretory protein targeting. A competition between Vp16 PDF and these RPBs at the level of the ribosome may contribute to the host lysis, revealing a possible new unrecognized mechanism developed by viruses to control host viability

Les mitochondries sont des organites complexes impliquant un millier de protéines, la plupart codées dans le génome nucléaire. Leur biogenèse a nécessité au cours de l’évolution la mise en place de systèmes efficaces d’adressage et d’import protéique, et des défaillances de ces systèmes sont associées à des pathologies graves, neuropathies, troubles cardiovasculaires, myopathies, maladies neurodégénératives ainsi que cancers. De nombreuses protéines mitochondriales possèdent en N-terminal une séquence d’adressage appelée MTS (Mitochondrial Targeting sequence) qui forme une hélice alpha amphiphile essentielle pour leur import mitochondrial. La séquence des divers MTSs est néanmoins très variables et leur caractéristiques critiques ne sont pas encore bien comprises. Le point de départ de ma thèse est la découverte, chez les levures, d’une surreprésentation en position 2 des séquences MTSs de 4 acides aminés hydrophobes (F, L, I, W). Au cours de mes années de thèse, j’ai pu confirmer, par des expériences de mutagenèse dirigée, le rôle critique de la nature du résidu en position 2 des MTSs. En effet, grâce au développement d’un système novateur de criblage des défauts d’import basé sur le sauvetage fonctionnel de la toxicité d’une protéine mitochondriale, j’ai pu observer que seuls les résidus surreprésentés en position 2 des protéines mitochondriales permettaient un import efficace. Mon travail a ainsi démontré l'existence de fortes contraintes évolutives s’exerçant au niveau de la position 2 des MTSs dont la compréhension pourrait à terme être utile pour optimiser l’adressage mitochondrial de protéines thérapeutiques chez des patients atteints de maladies mitochondriales
Mitochondria are complex organelles involving a thousand proteins, most of which are encoded in the nuclear genome. Their biogenesis has required the evolutionary development of efficient protein addressing and import systems, and failures of these systems are associated with serious pathologies, neuropathies, cardiovascular disorders, myopathies, neurodegenerative diseases and cancers.Many mitochondrial proteins have an N-terminal addressing sequence called MTS (Mitochondrial Targeting Sequence) which forms an amphiphilic alpha helix essential for their mitochondrial import. However, the sequence of the various MTSs is highly variable and their critical characteristics are not yet well understood. The starting point of my thesis was the discovery in yeast of an overrepresentation of 4 hydrophobic amino acids (F, L, I, W) at position 2 of the MTSs sequences. During my thesis, I was able to confirm the critical role of the nature of the residue in position 2 of the MTSs through directed mutagenesis experiments. Indeed, thanks to the development of an innovative system for screening import defects based on the functional rescue of the toxicity of a mitochondrial protein, I was able to observe that only residues overrepresented at position 2 of mitochondrial proteins allowed efficient import. My work has thus demonstrated the existence of strong evolutionary constraints at position 2 of MTSs, the understanding of which could ultimately be useful for optimising the mitochondrial addressing of therapeutic proteins in patients suffering from mitochondrial diseases

Trichomonas vaginalis is a human pathogen causing trichomoniasis, one of the most common non-viral sexually transmitted diseases in both men and women. Trichomoniasis is currently treated with metronidazole, but the pathogen is known to develop resistance against this drug. However as the pathogen is eukaryotic, the targets for the pathogen elimination without seriously affecting the host are limited. Throughout the evolution Trichomonas vaginalis adapted to anaerobic environments by developing an alternative metabolism resulting in a reduced form of mitochondria named hydrogenosome. Hydrogenosomes lack genetic information, therefore all its proteins are nucleus-encoded and need to be transported inside the hydrogenosome using a targeting N-terminal presequence. The peptidase recognizing and cleaving those presequences at the entrance of the organelle, the hydrogenosomal processing peptidase (HPP), is unique for hydrogenosomes and therefore represents a potential drug target against the pathogen. In this work the HPP's substrate specificity towards the targeting presequences was investigated. To do so a proteomic analysis of the proteome of Trichomonas vaginalis hydrogenosomes was performed using a novel optimized protocol for N-terminal peptide sequencing. N-terminal peptides were captured using a...

Proteins are ubiquitous macromolecules that effect and control all the processes of life from reproduction to respiration to physical motion. These diverse molecules also provide physical structure and defensive mechanisms. The twenty canonical amino acids can be found in virtually every protein; however, in some organisms, the set of endogenous amino acids also contains residues outside the “canon,” such as pyrrolysine, selenocysteine, and formylmethionine. Although a range of chemistries is available through natural side-chain diversity, some functionalities such as halogens, ketones, azides, alkenes, and alkynes are not found in nature. The introduction of a broader range of chemical functionality into proteins and protein-based materials through the use of non-canonical amino acids represents a challenging goal for protein engineering. The persistence of all the amino acids throughout protein sequences also presents a challenge for biochemical modification at a particular location. The insertion of a non-natural amino acid at a single location on a protein can allow specific modification without further affecting the natural protein sequence. The focus of this thesis is on a new method for the post-translational site-specific introduction of non-canonical amino acids to the N-terminus of proteins in vitro and progress made towards developing a complementary in vivo method using the E. coli L,F-transferase. Addition of non-proteinogenic functionality to the coat proteins of M13 bacteriophage using non-canonical amino acids is also explored.

The 3,144–amino acid huntingtin protein (HTT) folds in water into a structure consisting of compact, organized domains interspersed with intrinsically disordered protein (IDP) elements. The IDPs function as sites of post-translational modifications and proteolysis as well as in targeting, binding, and aggregation. Although the dominant structural motif of HTT is the α‎-helix–rich HEAT repeat, the expanded polyglutamine (polyQ) toxicity responsible for Huntington’s disease is most likely played out within intrinsically disordered HTT exon 1–like fragments consisting of the 16– to 17–amino acid N-terminal HTTNT segment, the polyQ segment, and a proline-rich segment. The physical behavior of HTT exon 1 fragments is dominated by interactive, polyQ repeat length–dependent structural transitions responsible for membrane and protein–protein interactions and the formation of tetramers, higher oligomers, amyloid fibrils, and inclusions. Understanding the basis of this solution behavior may be the key to disease mechanisms and molecular therapeutic strategies.

Protein purification is not a simple task. Yet, overexpression at bacterial systems with recombinant modifications brings further difficulties. Adding a tag, an affinity label, and expressing particular domains of the whole protein, especially hydrophobic sections, make purification a challenging process. Protein folding pattern may perturb N- or C-terminal tag and this terminal preference may lead to poor purification yield. Codon optimization, solvent content and type, ionic conditions, resin types, and self-cleavage of recombinant proteins bring further difficulties to protein expression and purification steps. The chapter overviews problems of protein purification through a small peptide overexpression in bacteria (Recombinant anti-SARS Coronavirus 2 (SARS-Cov-2) Spike protein Receptor Binding Domain (RBD) antibody (Clone Sb#14). The chapter also covers troubleshooting at distinct steps and highlights essential points to solve crucial issues of protein purification.

Abstract The SAR1 gene (EMBL accession number X51667) contains an intervening sequence that after splicing predicts an open reading frame of 190 amino acids. SAR1 cDNA sequencing confirms the excision of a 139 bp intron. Sari protein contains the four consensus motifs characteristic of GTPases (Bourne et al. 1991), although there are other notable features contained within the protein sequence. Sari p does not contain a C-terminal cysteine or an N-terminal glycine as potential residues for lipid modification, a feature observed in a number of other small GTPases.

Project Title: "Development of a plasmid-based reverse genetics system for the Bluetongue and Epizootic Hemorrhagic Disease viruses to allow comparative characterization of the function of the NS3 viroporin in viral egress". Project details: No - IS-4192-09; Participants – Ehrlich M. (Tel Aviv University), Parker J.S. (Cornell University), DermodyT.S. (Vanderbilt University); Period - 2009-2013. Orbiviruses are insect-borne infectious agents of ruminants that cause diseases with considerable economical impact in Israel and the United States. The recent outbreaks of BTV in Europe and of Epizootic Hemorrhagic Disease Virus (EHDV) in Israel, underscore the need for: (i) a better comprehension of the infection process of orbiviruses, (ii) the identification of unique vs. common traits among different orbiviruses, (iii) the development of novel diagnosis and treatment techniques and approaches; all aimed at the achievement of more effective control and treatment measures. It is the context of these broad goals that the present project was carried out. To fulfill our long-term goal of identifying specific viral determinants of virulence, growth, and transmission of the orbiviruses, we proposed to: (i) develop reverse genetics systems for BTV and EHDV2-Ibaraki; and (ii) identify the molecular determinants of the NS3 nonstructural protein related to viroporin/viral egress activities. The first objective was pursued with a two-pronged approach: (i) development of a plasmid-based reverse genetics system for BTV-17, and (ii) development of an "in-vitro" transcription-based reverse genetics system for EHDV2-Ibaraki. Both approaches encountered technical problems that hampered their achievement. However, dissection of the possible causes of the failure to achieve viral spread of EHDV2-Ibaraki, following the transfection of in-vitro transcribed genomic segments of the virus, revealed a novel characteristic of EHDV2-Ibaraki infection: an uncharacteristically low fold increase in titer upon infection of different cell models. To address the function and regulation of NS3 we employed the following approaches: (i) development (together with Anima Cell Metrology) of a novel technique (based on the transfection of fluorescently-labeledtRNAs) that allows for the detection of the levels of synthesis of individual viral proteins (i.e. NS3) in single cells; (ii) development of a siRNA-mediated knockdown approach for the reduction in levels of expression of NS3 in EHDV2-Ibaraki infected cells; (iii) biochemical and microscopy-based analysis of the localization, levels and post-translational modifications of NS3 in infected cells. In addition, we identified the altered regulation and spatial compartmentalization of protein synthesis in cells infected with EHDV2-Ibaraki or the mammalian reovirus. In EHDV2-Ibaraki-infected cells such altered regulation in protein synthesis occurs in the context of a cell stress reponse that includes the induction of apoptosis, autophagy and activation of the stressrelated kinase c-Jun N-terminal Kinase (JNK). Interestingly, inhibition of such stress-related cellular processes diminishes the production of infectious virions, suggesting that EHDV usurps these responses for the benefit of efficient infection. Taken together, while the present project fell short of the generation of novel reverse genetics systems for orbiviruses, the development of novel experimental approaches and techniques, and their employment in the analysis of EHDV-infected cells, yielded novel insights in the interactions of orbiviruses with mammalian cells.