The Proteomics of N-terminal Methionine Cleavage

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1′), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (\u3e120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1′ were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2′, P3′, and P4′ strongly slowed the reaction, and some proteins with Val and Thr at P1′ could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought

EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples

Mascot Distiller processing method for protein N-terminal acetylation quantitation. (PDF 24 kb

New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors

International audienceBACKGROUND : Multi-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol. METHODOLOGY/PRINCIPAL FINDINGS : To determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the latest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step CONCLUSIONS/SIGNIFICANCE : Our results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound

Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases

The acetylation-dependent (Ac/)N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3 ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multiomics approaches to characterize potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3 ligases in the Nt-acetylation-(NTA)-dependent turnover of proteins at global- and protein-specific scales. Arabidopsis has two endoplasmic reticulum (ER)-localized DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wild type, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localized SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes

A European network to fine-tune the proteome

Publisher Copyright: © 2024 The Author(s)Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tuning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s).publishersversioninpres

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-alpha-acetylation (NTA) and epsilon-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs andKATs, respectively), although the possibility that the sameGCN5-relatedN-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localizedGNATs, which possess a dual specificity. All characterizedGNATfamily members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinctKAand relaxedNTAspecificities. Furthermore, inactivation ofGNAT2 leads to significantNTAorKAdecreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation processin vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryoticGNATs may also possess these previously underappreciated broader enzymatic activities

Mapping the myristoylome through a complete understanding of protein myristoylation biochemistry

International audienceProtein myristoylation is a C14 fatty acid modification found in all living organisms. Myristoylation tags either the N-terminal alpha groups of cysteine or glycine residues through amide bonds or lysine and cysteine side chains directly or indirectly via glycerol thioester and ester linkages. Before transfer to proteins, myristate must be activated into myristoyl coenzyme A in eukaryotes or, in bacteria, to derivatives like phosphatidylethanolamine. Myristate originates through de novo biosynthesis (e.g., plants), from external uptake (e.g., human tissues), or from mixed origins (e.g., unicellular organisms). Myristate usually serves as a molecular anchor, allowing tagged proteins to be targeted to membranes and travel across endomembrane networks in eukaryotes. In this review, we describe and discuss the metabolic origins of protein-bound myristate. We review strategies for in vivo protein labeling that take advantage of click-chemistry with reactive analogs, and we discuss new approaches to the proteome-wide discovery of myristate-containing proteins. The machineries of myristoylation are described, along with how protein targets can be generated directly from translating precursors or from processed proteins. Few myristoylation catalysts are currently described, with only N-myristoyltransferase described to date in eukaryotes. Finally, we describe how viruses and bacteria hijack and exploit myristoylation for their pathogenicity

Evolution-driven versatility of N-terminal acetylation in photoautotrophs

International audienceN-terminal protein α-acetylation (NTA) is a pervasive protein modification that has recently attracted renewed interest. Early studies on NTA were mostly conducted in yeast and metazoans, providing a detailed portrait of the modification, which was indirectly applied to all eukaryotes. However, new findings originating from photosynthetic organisms have expanded our knowledge of this modification, revealing strong similarities as well as idiosyncratic features. Here, we review the most recent advances on NTA and its dedicated machinery in photosynthetic organisms. We discuss the cytosolic and unique plastid NTA machineries and their critical biological roles in development, stress responses, protein translocation and stability. These new findings suggest that the multitasking plastid and cytosolic machineries evolved to support the specific needs of photoautotrophs

Analyse intégrative du rôle de l'excision de la méthionine N-terminale dans le cytoplasme des eucaryotes supérieurs

Le premier acide aminé incorporé dans une chaîne polypeptidique naissante est toujours la méthionine. On identifie donc toujours ce premier résidu à la méthionine N-terminale. Cependant, les deux tiers des protéines accumulées à l état stationnaire ne présentent plus leur méthionine initiatrice. Cet enlèvement résulte essentiellement d une maturation protéolytique affectant chaque protéine. Ainsi, l Excision de la Méthionine N-terminale (NME) concerne la majorité des protéines et ce dès que les premiers résidus émergent du ribosome. Ce mécanisme est retrouvé dans tous les compartiments cellulaires où une synthèse protéique a lieu : le cytoplasme, les plastes et les mitochondries. Les enzymes responsables du clivage de la méthionine initiatrice sont les METhionine AminoPeptidases (METAPs) ; les METAPs sont conservées dans le Règne vivant. Des études fonctionnelles de délétions géniques ont montré le caractère létal du maintien de la première méthionine dans tous les organismes. Il y a plus de dix ans, les METAPs ont été identifiées comme étant la cible de composés naturels ayant des effets anticellulaires. Aujourd hui un nombre croissant d études rapportent que la NME est une cible prometteuse pour le traitement de nombreuses pathologies. Néanmoins, les bases moléculaires qui expliquent le caractère essentiel de la NME restent très peu comprises, en particulier dans le cytoplasme des eucaryotes supérieurs. Grâce à un système inductible permettant de moduler finement la NME cytoplasmique dans la plante modèle Arabidopsis thaliana et différentes approches incluant des analyses protéomiques et métabolomiques, j ai pu étudier les événements moléculaires précoces associés à l inhibition de la NME cytoplasmique. J ai également caractérisé la contribution relative des deux types de METAP cytoplasmiques au processus. Dans ce contexte, j ai pu démontrer chez A. thaliana que la NME cytoplasmique agit sur deux voies de signalisation fréquemment dérégulées lors de conditions pathologiques : le statut des composés thiolés et la protéolyse. La diminution de la NME cytoplasmique induit une protéolyse accrue principalement via une augmentation du nombre de protéines destinées à une dégradation rapide. Ainsi, l activité de la NME, en modulant la sensibilité de nombreuses protéines à subir la protéolyse, est un élément fondamental de la régulation de la demi-vie protéique. Finalement, mes résultats simialires obtenus également chez les Archées, levures et les lignées de cellules humaines suggèrent l existence d un mécanisme ubiquitaire associé à la NME.The first amino acid incorporated in nascent polypeptide chain is always methionine so called N-terminale methionine. However, in a given proteome, more than fifty percent of proteins have not this first methionine. Indeed, the early proteolytic event affecting a majority of proteins is N-terminal Methionine Excision (NME) as soon as few residues exit from the ribosome. Enzymes ensuring NME process are conserved along species. This mechanism takes place in all compartments where protein synthesis occurs including cytoplasm, plastids and mitochondria and the enzymes responsible of N-methionine excision are METhionine AminoPeptidases (METAP). Early functional studies of gene deletion has quickly showed that NME is an essential process. Ten years ago, METAPs have been identified as the molecular target of natural compounds with anticancer activities. Now, a growing number of studies suggest that NME is a promising target for treatment of various deseases. Nevertheless, molecular mechanisms making NME an essential process is poorly understood in particular in higher eukaryote cytoplasms.Using a dedicated inducible system in the model organism Arabidopsis thaliana and multiple approaches, including proteomics and metabolomics, I examined the earliest molecular events associated with the inhibition of this process and the contribution of both METAP to NME process. In this context, I demonstrated that cytoplasmic NME in A. thaliana orchestrates a cross-talk between two fundamental signaling pathways frequently deregulated in pathological conditions: thiol status and proteolysis. In these studies, we demonstrated that developmental defects induced by cytoplasmic NME inhibition are associated with an increase of the proteolytic activity due to an increase of the proteins available for rapid degradation. Thus, NME activity that modifies the availability of several proteins for degradation is an integral and fundamental element protein turnover regulation. Finally my preliminary results obtained in Archea, Fungi and human cells seem to suggest the existence of a ubiquitous mechanism associated with NME process.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF