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Exploring the Role of Intracellular Aminopeptidases in <em>Staphylococcus aureus</em> PathogenesisMarking, Devon Nicole 01 January 2015 (has links)
Staphylococcus aureus is a remarkably pathogenic bacterium that is widely prevalent among the human population. It is the leading agent of skin and soft tissue infections, and is also responsible for causing an array of severe and life threatening diseases. The invasiveness of the pathogen, coupled with increasing antibiotic resistance seen for S. aureus infections, makes this bacterium a prominent public health concern. The extended pathogenicity of S. aureus is largely due to its repertoire of virulence factors, which are typically characterized by being bound to the cell wall, or secreted into the extracellular environment. Previously, our lab identified a leucine aminopeptidase mutant (pepZ) as being strongly attenuated in virulence for both systemic and localized models of infection. Importantly, PepZ marked the first intracellular bacterial aminopeptidase found to be involved in pathogenesis in Gram-positive bacteria. In this study, we set out to explore the role of the remaining eleven, non-essential, uncharacterized aminopeptidase enzymes in S. aureus disease causation, and present two additional aminopeptidase genes, pepT1 and pepT2, which are important for virulence in both human ex vivo models, and murine in vivo models of disease. Interestingly, these enzymes do not appear to be necessary for the utilization of free peptides for cellular nutrition and metabolism, which is a typical characteristic of aminopeptidases. Transcriptional analysis reveals maximal expression of pepT1 and pepT2 during early exponential growth phase, while localization mapping demonstrates that the PepT1 and PepT2 enzymes are found in the bacterial cytoplasm during all stages of growth. To explore these findings on a global level, an in-depth proteomic investigation of cleavage properties and cellular substrates identified several proteins as having significant changes in N-terminal peptide abundance in pepT1 and pepT2 mutant proteomes. We identified a number of putative independent and shared targets for the PepT1 and PepT2 enzymes that are known to impact cellular fitness and pathogenesis, including: DnaK, a heat shock protein conserved throughout all organisms, which functions to help deal with mis-folded and aggregated proteins that have accumulated as a result of cellular stress; ArlR, the response regulator of the ArlRS two-component system, which is an important regulator of agglutination and virulence in S. aureus; and of special interest, ClpC, an ATP-dependent protease chaperone which is a component of the primary machinery by which protein degradation occurs in S. aureus. Collectively, our data proves the importance of the PepT aminopeptidase enzymes in S. aureus pathogenesis, and indicates these enzymes are likely involved in bacterial stress response and virulence by functioning through the bioactivation/inactivation of key cellular proteins.
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Proteomic studies on protein N-terminus and peptide ion mobility by nano-scale liquid chromatography/tandem mass spectrometry / ナノスケール液体クロマトグラフィー/タンデム質量分析によるタンパク質N末端およびペプチドイオンモビリティーに関するプロテオミクス研究Chang, Chih-Hsiang 23 March 2021 (has links)
京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23135号 / 薬科博第134号 / 新制||薬科||15(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 石濱 泰, 教授 松﨑 勝巳, 教授 加藤 博章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM
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Innovations pour l'annotation protéogénomique à grande échelle du vivant / Innovations for proteogenomic annotation on a large scale for microorganismsBland, Céline 23 September 2013 (has links)
La protéogénomique consiste à affiner l'annotation du génome d'organismes modèles pour lesquels des données protéomiques sont générées à haut-débit. Des erreurs d'annotation structurale ou fonctionnelle sont encore fréquentes. Innover dans les méthodologies permettant de lever ces ambiguïtés est essentiel. L'étude spécifique du N-terminome permet de vérifier expérimentalement l'identification du codon d'initiation de la traduction et de certifier les données obtenues. Pour cela, deux stratégies innovantes ont été développées basées sur : i) le marquage sélectif du N-terminal des protéines, ii) une digestion multienzymatique en parallèle, et ii) l'enrichissement spécifique des peptides N-terminaux marqués par chromatographies liquides successives ou immunocapture dirigée contre le groupement N-terminal ajouté. L'efficacité de ces méthodologies a été démontrée à partir du modèle bactérien Roseobacter denitrificans. Après enrichissement par chromatographie, 480 protéines ont été validées et 46 ré-annotées. Plusieurs sites d'initiation de la traduction ont été décelés et l'annotation par similarité a été remise en cause dans certains cas. Après immunocapture, 269 protéines ont été caractérisées dont 40% ont été identifiées spécifiquement après enrichissement. Trois gènes ont également été annotés pour la première fois. Les résultats complémentaires obtenus après analyse par spectrométrie de masse en tandem facilitent l'interprétation des données pour révéler les sites d'initiation réels de la synthèse des protéines et identifier de nouveaux produits d'expression des gènes. La ré-annotation peut devenir automatique et systématique pour améliorer les bases de données protéiques. / Proteogenomics is a recent field at the junction of genomics and proteomics which consists of refining the annotation of the genome of model organisms with the help of high-throughput proteomic data. Structural and functional errors are still frequent and have been reported on several occasions. Innovative methodologies to prevent such errors are essential. N-terminomics enables experimental validation of initiation codons and certification of the annotation data. With this objective in mind, two innovative strategies have been developed combining: i) selective N-terminal labeling of proteins, ii) multienzymatic digestion in parallel, and iii) specific enrichment of most N-terminal labeled peptides using either successive liquid chromatography steps or immunocapture directed towards the N-terminal label. Efficiency of these methodologies has been demonstrated using Roseobacter denitrificans as bacterial model organism. After enrichment with chromatography, 480 proteins were validated and 46 re-annotated. Several start sites for translation initiation were detected and homology driven annotation was challenged in some cases. After immunocapture, 269 proteins were characterized of which 40% were identified specifically after enrichment. Three novel genes were also annotated for the first time. Complementary results obtained after tandem mass spectrometry analysis allows easier data interpretation to reveal real start sites of translation initiation of proteins and to identify novel expressed products. In this way, the re-annotation process may become automatic and systematic to improve protein databases.
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Développement de méthodologies protéomiques pour l’étude des maladies infectieuses / Development of proteomic methodologies for the study of infectious diseasesWestermann, Benoît 14 December 2016 (has links)
La thématique des maladies infectieuses représente un réel enjeu politique, économique et de santé publique. Les objectifs de mes travaux de thèse étaient de développer des approches protéomiques pour identifier, détecter, caractériser et/ou quantifier des protéines et de les appliquer à l’étude de maladies infectieuses pour lesquelles des données de protéomique pourraient ouvrir à de nouvelles approches thérapeutiques et/ou diagnostiques. Les stratégies d’identification et de détection des protéines développées pour l’étude de la maladie de Lyme ont permis de prouver la faisabilité du diagnostic par spectrométrie de masse et de proposer des protéines candidates-vaccin. Les stratégies de quantification mises en place pour l’étude de la toxoplasmose ont permis d’identifier et de quantifier un complexe protéique clef. Les stratégies de caractérisation du N-terminome pour l’étude du paludisme ont permis d’apporter des preuves expérimentales des processus de maturation et d’adressage des protéines. / Infectious diseases represent a real challenge in terms of politic, economic and public health. The purposes of my thesis works were to develop proteomic approaches able to identify, to detect, to characterize and/or to quantify proteins and to apply these approaches to the study of infectious diseases for which proteomic data cou Id open to new therapeutic and/or diagnostic strategies. Identification and specific detection strategies developed in the study of Lyme's disease allowed to prove the feasibility of diagnosis by mass spectrometry and to propose new vaccine-candidate proteins. Quantification strategies developed in the study of toxoplasmosis allowed us to identify and to quantify a key protein complex of the parasite. N-terminome characterization strategy developed in the study of malaria allowed us to bring experimental proofs of proteins processing and trafficking.
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