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Caractérisation cristallographique d'intermédiaires réactionnels de méthionine sulfoxyde réductases en vue de la compréhension de leur mécanisme catalytique : Les trois domaines de la protéine multifonctionnelle PilB de Neisseria meningitidis et la MsrB de Xanthomonas campestris / Crystallographic characterisation of reactional intermediates of methionine sulfoxide reductases with the aim of understanding their catalytic mechanism : The three domains of the multifunctional protein PilB from Neisseria meningitidis and the MsrB from Xanthomonas campestrisRanaivoson, Fanomezana Moutsé 23 November 2007 (has links)
Les résidus méthionine sont facilement oxydables en sulfoxydes in vivo. Cette oxydation est réversible via la méthionine sulfoxyde réductase (Msr), un enzyme ubiquitaire. Dû aux deux configurations possibles du sulfoxyde, deux classes d'enzyme structuralement différents existent : les MsrA sont spécifiques de l'isomère S, et les MsrB de l'isomère R. Les deux possèdent un même mécanisme catalytique en deux étapes. La première étape est dédiée à la réduction du substrat ; elle aboutit à l’oxydation d’une cystéine en acide sulfénique. Puis le recyclage de l’enzyme s’opère avec la formation d’un pont disulfure intramoléculaire qui est finalement réduit par une thiorédoxine (Trx). Chez Neisseria meningitidis la protéine PilB porte MsrA et MsrB sous forme de deux domaines. Un troisième domaine à activité du type Trx existe en N-terminal. Les trois domaines isolés ont été étudiés par cristallographie. (i) Le domaine N-terminal : la structure confirme son homologie avec la Trx, mais son analyse révèle des éléments probablement à l’origine d’un fonctionnement particulier. (ii) La MsrA : la structure de deux mutants a permis d’observer un complexe avec un substrat et l’acide sulfénique. Leurs études s’additionnent à celles de l’enzyme sauvage sous formes réduite et oxydée. (iii) La MsrB : la structure d’un mutant a également permis d’obtenir un complexe et complète les structures des formes réduite et oxydée du sauvage. En addition, la structure de la MsrB de Xanthomonas campestris met à jour des différences conformationnelles entre MsrB d’organismes différents. Enfin, l’étude structurale de PilB entier a été entamée en solution par la méthode de diffusion aux petits angles. / Methionine residues are easily oxidized to sulfoxides, in vivo. This oxidation is reversed via a ubiquitous enzyme named methionine sulfoxide reductase (Msr). Due to the two possible configurations of the sulfoxide group, two structurally-different classes of enzymes exist: MsrAs are specific for the isomer S, and MsrBs for the isomer R. Both classes act through a two-step mechanism. The first step is dedicated to substrate reduction. It results in the sulfenic form of the catalytic cysteine. Then recycling of the enzyme starts with the formation of an intramolecular disulfide bridge, finally reduced by thioredoxin (Trx). In Neisseria meningitidis, the protein PilB bears MsrA and MsrB as two adjacent domains. A third domain with a Trx-like activity exists at the N-terminal end. The three isolated domains have been studied by X-Ray crystallography. (i) The N-terminal domain: its structure confirmed its homology to Trx and DsbEs, but its analysis revealed elements that probably explain its peculiar properties. (ii) MsrA: the structures of two mutants allowed the observation of a complex with the substrate and of the sulfenic acid. These results add to the structure of the reduced and oxidized forms of the wild type domain so that the catalytic mechanism can be analyzed. (iii) MsrB: the structures of the reduced and oxidized forms were completed by that of a complex with the substrate obtained from a mutant. In addition, comparison with the Xanthomonas campestris MsrB structure enlightened conformational differences between MsrBs from distinct organisms. Finally, structural studies of the whole PilB protein have been initiated in solution using small angle X-Ray scattering.
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Etude des méthionine sulfoxyde réductases d'Escherichia coli : rôle de MsrA/B dans la protection de RecA et identification d'une nouvelle activité MsrHenry, Camille 16 December 2016 (has links)
Les méthionines sulfoxyde réductases (Msr) sont impliquées dans la réparation des protéines. MsrA et MsrB sont ubiquitaires et réduisent les méthionines sulfoxydes en méthionines. Chez les bactéries, MsrA et MsrB sont localisées dans le cytoplasme et sont impliquées dans la résistance au stress oxydant. Durant ma thèse, j'ai étudié le rôle du système MsrA/B dans la physiologie d'Escherichia coli. Mon travail a porté sur l'étude de la recombinase A (RecA) comme cible du système MsrA/B. RecA joue un rôle central dans la réparation de l'ADN via ses fonctions principales : la recombinaison homologue, l'induction de la réponse SOS et l'induction de la mutagénèse SOS. J’ai pu établir un lien génétique entre le système MsrA/B et RecA. L'étude révèle que l'absence des Msr affecte la fonction de recombinaison homologue de RecA. J'ai montré que RecA oxydée perd sa capacité à former des filaments sur l’ADN, à hydrolyser de l’ATP et à effectuer l’échange de brin. De manière intéressante, la réparation de RecA oxydée par le système MsrA/B permet de restituer ses fonctions. D'autres analyses ont révélé que le résidu Met35 est important pour l’activité de RecA. Ces résultats m'ont permis de proposer un modèle dans lequel l'état d'oxydation des Met de RecA modulent son activité. Un autre pan de mon travail a permis la caractérisation d’une Msr périplasmique : MsrP. J'ai montré que MsrP est importante lors d’un stress HOCl et que son expression est induite lors d’un tel stress via le système à deux composantes YedVW. La kinase YedV possédant plusieurs Met au sein de son domaine senseur, j'ai proposé un modèle dans lequel l'activation de YedV se ferait via l'oxydation de ses Met. / Methionine sulfoxide reductase (Msr) are involved in proteins repair. MsrA and MsrB are ubiquitous enzymes which reduce methionine sulfoxide into methionine. In bacteria, MsrA and MsrB are localized in the cytoplasm and are involved in the resistance to oxidative stress. During my PhD, I investigated the role of the MsrA/B system in the physiology of the Escherichia coli. My work was devoted to the study of the recombinase A (RecA) as the target of MsrA/B system. RecA plays a central role in DNA repair via its main functions: homologous recombination, induction of the SOS response and the induction of the SOS mutagenesis. I was able to establish a genetic link between the MsrA/B system and RecA. My study shows that the absence of Msr affects the homologous recombination function of RecA. I have shown that RecA oxidized loses its ability to form filaments on DNA, to hydrolyze ATP and perform strand exchange process. Interestingly, repair RecA oxidized by MsrA/B system restores the functions of RecA. Further analysis revealed that the residue Met 35 is important for the activity of RecA. From these results, I proposed a model in which the oxidation of Met and repair MsrA/B is a dynamic system modulating the activity of RecA. Another part of my work allowed the characterization of periplasmic Msr of E. coli: MsrP. I have shown that MsrP is important under HOCl stress and its expression is induced during such stress via the YedVW two components system. The YedV kinase possessing multiple Met in its sensor domain, I proposed a model in which the activation of YedV would be by oxidation of its Met.
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Basic evidence for epidermal H2O2/ONOO--mediated oxidation/nitration in segmental vitiligo is supported by repigmentation of skin and eyelashes after reduction of epidermal H2O2 with topical NB-UVB-activated pseudocatalase PC-KUSSchallreuter, Karin U., Salem, Mohamed M.A., Holtz, Sarah, Panske, Angela 08 1900 (has links)
No / Nonsegmental vitiligo (NSV) is characterized by loss of inherited skin color. The cause of the disease is still unknown despite accumulating in vivo and in vitro evidence of massive epidermal oxidative stress via H2O2 and peroxynitrite (ONOO−) in affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Strictly segmental vitiligo (SSV) with dermatomal distribution is a rare entity, often associated with stable outcome. Recently, it was documented that this form can be associated with NSV (mixed vitiligo). We here asked the question whether ROS and possibly ONOO− could be players in the pathogenesis of SSV. Our in situ results demonstrate for the first time epidermal biopterin accumulation together with significantly decreased epidermal catalase, thioredoxin/thioreoxin reductase, and MSRA/MSRB expression. Moreover, we show epidermal ONOO− accumulation. In vivo FT-Raman spectroscopy reveals the presence of H2O2, methionine sulfoxide, and tryptophan metabolites; i.e., N-formylkynurenine and kynurenine, implying Fenton chemistry in the cascade (n=10). Validation of the basic data stems from successful repigmentation of skin and eyelashes in affected individuals, regardless of SSV or segmental vitiligo in association with NSV after reduction of epidermal H2O2 (n=5). Taken together, our contribution strongly supports H2O2/ONOO-mediated stress in the pathogenesis of SSV. Our findings offer new treatment intervention for lost skin and hair color.—Schallreuter, K. U., Salem, M. A. E. L., Holtz, S., Panske, A. Basic evidence for epidermal H2O2/ONOO−-mediated oxidation/nitration in segmental vitiligo is supported by repigmentation of skin and eyelashes after reduction of epidermal H2O2 with topical NB-UVB-activated pseudocatalase PC-KUS.
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The redox - biochemistry of human hair pigmentation.Schallreuter, Karin U., Salem, Mohamed M.A., Hasse, Sybille, Rokos, Hartmut 10 1900 (has links)
No / The biochemistry of hair pigmentation is a complex field involving a plethora of protein and peptide mechanisms. The in loco factory for melanin formation is the hair follicle melanocyte, but it is common knowledge that melanogenesis results from a fine tuned concerted interaction between the cells of the entire dermal papilla in the anagen hair follicle. The key enzyme is tyrosinase to initiate the active pigmentation machinery. Hence, an intricate understanding from transcription of mRNA to enzyme activity, including enzyme kinetics, substrate supply, optimal pH, cAMP signaling, is a must. Moreover, the role of reactive oxygen species on enzyme regulation and functionality needs to be taken into account. So far our knowledge on the entire hair cycle relies on the murine model of the C57BL/6 mouse. Whether this data can be translated into humans still needs to be shown. This article aims to focus on the effect of H2O2-redox homeostasis on hair follicle pigmentation via tyrosinase, its substrate supply and signal transduction as well as the role of methionine sulfoxide repair via methionine sulfoxide reductases A and B (MSRA and B).
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Specifika ošetřovatelské péče u pacienta s MRSA na operačním sále z pohledu anesteziologické sestry / Specifics of nurse care at the patient with MRSA on surgery from nurses' point of view.KALÁBOVÁ, Miroslava January 2010 (has links)
Title of the thesis: Specifics of the nursing care of patients with MRSA in the operating room from an anesthesiology nurse perspective. The aim of this thesis was to identify the specifics of nursing care of a patient with the MRSA in the operating room from the perspective of nurses working in the operating room. In the theoretical part focus is given to nursing care and its specifics for a patient with MRSA. Chapters relating to the regime measures, barrier nursing procedures and the use of specific aids, instruments and tools are treated in detail. The nursing care provided by anesthesia nurses and perioperative nurses in the operating room is defined. To reach the goals the quantitative research through questionnaires was chosen. The research sample consisted of anesthesia nurses and perioperative care nurses. Two types of standardized questionnaires were used, consisted of closed questions where respondents chose an appropriate answer, and from semi-open questions where respondents could specify their answers, and in one case open questions. Both questionnaires were completed by 12 test questions, where respondents had the choice of three possible answers, where one answer was correct. The results of the investigation are summarized in two types of graphs. Bar charts were used in questions where more answers were possible to mark, and for a better clarity the results were supplemented by tables. Results to other questions, where options were limited by one answer, are shown in percentages in pie graphs. Our thesis is very narrowly focused, because we concentrated on specifics of nursing care in a patient with MRSA in the operating room. We have determined the specifics of barrier nursing, nursing problems in a patient with MRSA in the operating room. We have discovered barrier nursing procedures in a patient with MRSA in the operating room. We have also succeeded in assessing the knowledge of nurses working in the operating room. The goal was met. An interesting finding was that most of anesthetic nurses and perioperative nurses had responded that the structural and technical arrangement in the operating room was not satisfactory in terms of barrier nursing of a patient with MRSA. This problem was expected, owing to the author´s experience in operating theaters. Thus Hypothesis 1 is confirmed. In total five hypotheses were stated, four of which were confirmed and one was disproved. The aim of the thesis was to draw attention to problems in the operating room during nursing a patient with MRSA. In the course of the research work there were no problems, however, it is necessary to mention the lack of literature on MRSA with regard to nursing care in operating theaters. The theoretical section of this thesis could be recommended as a resource for the education plan created on the basis of information collected and the results of this research. The results of the research survey may serve for general public, anesthesia and perioperative nurses who could benefit from the educational plan in practice while providing nursing care for a patient with MRSA.
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