Thesis advisor: Clare O'Connor / As organisms get older, the proteins in their cells also age, and as this happens, the amino acids that make up these proteins may become chemically modified and begin to lose their integrity. One example of an age-related modification occurs when the amino acid residue methionine is oxidized by a reactive oxygen species to methionine sulfoxide. Methionine sulfoxide reductase is an enzyme that repairs this damage to the protein by catalyzing a reaction that reduces methionine sulfoxide back to methionine. The fission yeast Schizosachharomyces pombe was used as the experimental model to study methionine sulfoxide reductase in vivo, taking advantage of the variety of tools available with which to study the organism. In S. pombe there are two genes encoding methionine reductase activities, msrA and msrB. The first goal of this project was to construct yeast strains in which the endogenous msrA and msrB genes had been inactivated. This was accomplished via homologous recombination reactions in which the msr genes were replaced with a selectable marker for biosynthesis of uracil (ura4+). After the construction and verification of the two knockout strains, the sensitivities of the strains to reactive oxygen species were tested. Both strains showed reduced resistance to oxidative stress. Future experiments will include more detailed analyses of the abilities of the strains to survive oxidative stress. Finally, the two knockout strains of yeast will be mated with one another in order to produce a double msr knockout, in order to examine the effects of a complete lack of methionine sulfoxide reductase activity on the organism. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Methionine sulfoxide reductase (MSR) is an important antioxidant to help mitigate oxidative stress that contributes to age-associated neurodegenerative diseases, such as Alzheimer’s Disease and Parkinson’s Disease. In MSR deficient Drosophila melanogaster (fruit flies), larvae show a developmental delay like that seen when wild-type larvae are reared on nutrient deficit culture medium. These investigators further showed that serotonin levels were depressed in these nutrient deficient larvae. The overarching aim of this study was to better understand the role of serotonin in MSR regulated physiology. Supplementing food with serotonin partially rescued the slower mouth hook movements (MHM) observed in the MSR-deficient flies. However, supplementation with serotonin altering drugs that cross the blood brain barrier (5-hydroxytryptophan, fluoxetine, or paravi chlorophenylalanine) did not rescue MHM and caused impairments to the growth of larvae during development. This study indicates that serotonin regulates feeding behavior partially through the regulation of MSR production but acts independently to regulate development. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while similar to 3.6 x 10(3) CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia colt TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized Glade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

This study has investigated the involvement of photosynthetic electron transport chain on the regulation of gene expression of methionine sulfoxide reductase (UfMSR) in the marine macroalga Ulva fasciata Delile.UfMSRA is from copper stress and UfMSRB ir from hypersalinity stress. UfMSRA is similar to Arabidopsis AtMSRA4 and UfMSRB is similar to AtMSRB1. UfMSRA is specific to the MetSO S-enantiomer and UfMSRB catalytically reduces the MetSO R-enantiomer. Both enzymes are required, since in the cell oxidation of Met residues at the sulfur atom results in a racemic mixture of the two stereoisomers. UfMSRA and UfMSRB transcripts were increased by white light, blue light and red light with the maximum at 1 h following a decline, but kept constant in the dark. The magnitude of UfMSRA and UfMSRB transcript increase showed a positive linear correlation to increasing light intensity from 0-1200 u mole¡Pm-2¡Ps-1. The treatment with linear electron transport chain inhibitors, hydroxylamine, 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and stigmatellin, effectively inhibited PS II activity under 300 u mole¡Pm-2¡Ps-1 irradiance. DBMIB and stigmatellin can increase UfMSRA transcript that was reversed by 2,6-dichlorophenolindophenol (DCPIP), a PS I electron donor. It indicates that the block of electron transport of the downstream of cytochrome b6f indeuces UfMSRA gene expression. Hydroxylamine, DCMU and DBMIB decreased UfMSRB transcript that was not reversed by DCPIP while stigmatellin increased UfMSRB mRNA level, reflecting a role of reduced state with Qo site located at cytochrome b6f on the induction of UfMSRB gene expression. The cyclic electron transport chain inhibitors, antimycin A that inhibited photosynthetic electron transport, can inhibit the increase of UfMSRA and UfMSRB transcripts by irradiance. UfMSRA and UfMSRB gene expression were both modulated by cyclic electron transport chain and linear electron transport chain. These results reveal that photosynthetic electron transport chain modulates UfMSRA and UfMSRB gene expression by change its redox state.

Chlamydomonas reinhardtii can utilize CO2 for autotrophic growth (HSM plus 5% CO2) or acetate for mixotrophic growth (TAP). This study was to elucidate the differential regulation of methionine sulfoxide reductase (MSR) gene expression between HSM plus 5% CO2 and TAP cultured cells, and also to determine the difference of gene expression in response to high light (1,000 £gE m-2 s-1). The role of photosynthetic electron transport (PET) in the regulation of MSR gene expression was also examined by the use of PET inhibitors. High light inhibited PSII activity (Fv/Fm and Fv'/Fm') of HSM plus 5% CO2 and TAP cultured cells., while the responses of CrMSR gene expression in mixotrophically grown cells were different from autotrophically grown cells, High light increased the expression of CrMSRA1, CrMSRA2, CrMSRA3, CrMSRA5, CrMSRB1.2, and CrMSRB2.1, but inhibited the expression of CrMSRA4 and CrMSRB2.2 in autotrophically grown cells. The expression of CrMSRA3, CrMSRA5, and CrMSRB2.1 in mixotrophically grown cells was increased by high light but that of CrMSRA1, CrMSRA4, and CrMSRB2.2 was inhbited. The number of MSR isoform that was up-regulated by high light was greater in autotrophically grown cell than that in mixotrophically grown cells. Using the PET inhibitors (3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)), most of the CrMSRA expression was regulated by reduced QA for autotrophically grown cells while reduced PQ was the main site for mixotrophically grown cells by high light. The expression of CrMSRB in autotrophically grown cells was mainy modulated by QA (-) or Cytb6f (-), while that was not affected by PET, except a role of Cytb6f (-) on the high light-induced CrMSRB2.2 expression. We fouind that CrMSRB gene expression in autotrophically grown cells was highly affected by PET but not for micotrophically grtown cells. The present result that H2O2 did not accumulate in autotrophically and mixotrophically grown cells suggests that H2O2 may be not involved in the regulation of high light regulation of CrMSR gene expression. The present study shows that the mRNA expression of CrMSR isoforms in Chlamydomonas was diffrerentially regulated between autotrophically and mixttrophically grown cells. The relationship between the utilization of different C source and CrMSR gene expression will be discussed.

Bacillus cereus est une bactérie aéro-anaérobie facultative à Gram positif ubiquiste pouvant s’adapter à de nombreux environnements et s’y développer. C’est un agent pathogène de l’homme capable de produire tout un éventail de protéines extracellulaires et de toxines jouant un rôle majeur dans la pathogénicité de ce micro-organisme. B. cereus croit suivant un métabolisme de type respiratoire en aérobiose et fermentaire en anaérobiose en l’absence d’accepteur final d’électrons. En aérobiose, la chaine respiratoire est une source majeure des dérivés réactifs de l'oxygène (ROS) endogènes. En anaérobiose, les ROS endogènes sont générés en réponse au stress oxydant secondaire au stress nutritionnel et au stress réducteur, lorsque les cultures sont réalisées à bas potentiel d’oxydo-réduction (POR). Les résidus méthionines (Met) sont particulièrement sensibles à l’oxydation par les ROS. L’oxydation des Met conduit à la formation de méthionine sulfoxyde (Met(O)), un dérivé oxydé stable détectable par spectrométrie de masse (MS). L'oxydation des résidus Met est réversible : leur réduction est catalysée par des méthionines sulfoxyde réductases (Msr). Pour déterminer le rôle de l’oxydation des résidus Met, nous avons réalisé une étude exhaustive par MS de la dynamique de l’exoprotéome de la souche ATCC 14579 (pBClin 15) de B. cereus en aérobiose (pO2 = 100%) et en anaérobiose (pO2 = 0%) à haut (POR initial = +140 mV) et bas potentiel redox (PORi= -350 mV). Les résultats ont montré que la dynamique des toxines était représentative de la dynamique de l’exoprotéome à la fois en termes d’abondance relative de protéines et d’oxydation des Met dans les trois conditions testées. L’analyse des résultats suggèrent que (i) l’abondance des toxines et leur taux de méthionines oxydés reflètent le niveau d’oxydation cellulaire et (ii) la sécrétion de toxines au cours de la croissance cellulaire contribue au maintien de l'homéostasie redox intracellulaire en piégeant les ROS endogènes, en particulier en phase active de croissance en aérobiose et en fin de croissance en anaérobiose. Pour étayer l’hypothèse selon laquelle, les Met des protéines extracellulaires, et des toxines en particuliers sont des composants de la machinerie cellulaire antioxydante, nous avons construit une souche mutante ne synthétisant plus MsrAB et comparer le protéome et l’exoprotéome de cette souche mutante avec celle de la souche parentale en aérobiose et anaérobiose à haut POR. Cette étude a mis en évidence l’implication de MsrAB mais également du plasmide cryptique pBClin15 dans la sécrétion des toxines et le maintien de l'homéostasie redox intracellulaire / Bacillus cereus is a Gram-positive aerobic or facultative anaerobic worldwide-distributed bacterium. In addition, B. cereus is a human pathogen able to produce a range of extracellular enzymes and toxins playing a major role in the virulence of the bacteria. In presence of oxygen, B. cereus performs respiration. Without oxygen or other electron acceptors, it performs mixed-acid fermentation. Under aerobiosis, the respiratory electron transport chain is a major source of endogenous reactive oxygen species (ROS). Under anaerobiosis, endogenous ROS are generated in response to reductive stress (mainly under high-reductive anaerobiosis) and to starvation (nutrient stress), i.e. in response to secondary oxidative stresses. Methionine residues (Met) of proteins are vulnerable to oxidation by free radicals. Oxidation of Met leads to the formation of methionine sulfoxide (Met (O)), a stable by-product detectable by mass spectrometry (MS). Met(O) can be reduced back to Met by the action of methionine sulfoxide reductase (Msr). To determine the role of oxidation of Met residues, B. cereus exoproteome time courses were monitored by MS under low oxidation-reduction potential (ORP) anaerobiosis (initial ORP = +140 mV and pO2 = 0%), high-ORP anaerobiosis (iORP = -350 mV and pO2 = 0%), and aerobiosis (pO2 = 100%). The results indicated that toxin-related proteins were the most representative of the exoproteome changes, both in terms of protein abundance and their Met(O) content in the presence and in the absence of oxygen. The analysis results suggest that (i) the abundance of toxins and their oxidized methionines rates reflect the cellular oxidation level and (ii) the secretion of toxins during growth helps to maintain redox homeostasis by keeping endogenous ROS at bay, during the exponential growth phase under aerobic conditions and at the end of growth under anaerobiosis. To support our hypothesis that Met residues of extracellular proteins, particulars of toxins are components of the cellular machinery antioxidant, we constructed a mutant strain by deleting the gene of MsrAB and compare the cellular proteome and exoproteome of this mutant strain with the wild-type strain under aerobiosis and high-ORP anaerobiosis. This study highlighted the involvement of MsrAB but also pBClin15 plasmid in the secretion of toxins and maintain of the intracellular redox homeostasis.

Les Méthionine sulfoxyde réductases (Msr) catalysent la réduction spécifique des méthionine sulfoxydes (Met-O) en méthionines (Met). Elles sont impliquées dans la résistance des cellules à un stress oxydant et dans la virulence des bactéries pathogènes du genre Neisseria. Cette famille d'enzyme se compose de trois classes, les MsrA et B, structuralement distinctes, et présentant une stéréosléctivité respectivement pour l'isomère S et R de la fonction sulfoxyde du substrat. Une troisième classe, découverte récemment, et appelée fRMsr, catalyse la réduction spécifique de la forme libre de l'isomère R de la fonction sulfoxyde. La fRMsr appartient à la famille des domaines GAF, généralement impliqués dans la signalisation cellulaire, et les fRMsr représentent le premier domaine GAF présentant une activité enzymatique. Les études réalisées au cours de ma thèse sur la fRMsr de Neisseria meningitidis ont permis de montrer que : 1) fRMsr de N. meningitidis présente un mécanisme catalytique identique à MsrA/B avec la formation d'au moins un pont disulfure intramoléculaire Cys84-Cys118 réduit par la thiorédoxine (Trx) ; 2) La Cys118 est le résidu catalytique sur lequel l'intermédiaire acide sulfénique doit se former ; 3) L'étape réductase est l'étape cinétiquement déterminante du mécanisme à deux étapes conduisant à la formation du pont disulfure Cys84-Cys118. La combinaison de l'analyse des résultats cinétiques, et de la structure tridimensionnelle de la fRMsr de N. meningitidis en complexe avec le substrat ont permis de montrer : 1) L'existence d'un site de reconnaissance oxyanion impliqué dans la stabilisation de la fonction carboxylate ; 2) Un rôle de la fonction carboxylate du résidu Asp143 dans la catalyse de l'étape réductase ; 3) Le résidu Glu125 est impliqué dans la reconnaissance et/ou le positionnement du substrat Met-O probablement via la stabilisation du groupement NH3+ ; 4) Un rôle du résidu Asp141 dans le positionnement des résidus Asp143 et Glu125 ; 5) le noyau indole du Trp62 est impliqué dans la stabilisation du groupe méthyle-[epsilon] / Methionine sulfoxide reductases (Msr) catalyze the specific reduction of methionine sulfoxides (Met-O) into methionine (Met). They are involved in cell defences against oxidative stress and virulence of pathogenic bacteria of Neisseria genius. This family of enzymes consists of three classes, MsrA and MsrB, structurally-unrelated, Specific for the S and the R epimer of the sulfoxide function of the substrate, respectively. A third class, recently discovered and called fRMsr, selectively reduce the free form of the R epimer of the sulfoxide function. The fRMsr belongs to the family of GAF domains, they are usually involved in cell signaling, and fRMsr represent the first GAF domain to show enzymatic activity. The studies of the Neisseria meningitidis fRMsr have shown that: 1) The Neisseria meningitidis fRMsr have a identical catalytic mechanism to MsrA and MsrB with the formation of at least one intramolecular disulfide bond, Cys84-Cys118 reduced by thioredoxin (Trx) ; 2) The Cys118 is demonstrated to be the catalytic Cys on which a sulfenic acid is formed ; 3) The Reductase step is the rate determining step of the mechanism leading to the formation of the disulfide bond Cys84-Cys118. The combination of the biochemical and kinetics data, and the examination of the 3D structure of the N. meningitidis fRMsr in complex with its substrate shown: 1) an oxyanion hole involved in the accommodation of the carboxylate group ; 2) the carboxylate group of the Asp143 residue involved in the catalysis of step reductase, and 3) The Glu125 residue involved in the recognition and/or positioning of the Met-O probably by the stabilization of the NH3+; 4) the Asp141 residue involved in the positioning of Asp143 and Glu125 residues ; 5) the indole ring of the Trp62 residue involved in stabilizing of the epsilon-methyl group