Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13852号 / 工博第2956号 / 新制||工||1436(附属図書館) / 26068 / UT51-2008-C768 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 森 泰生 / 学位規則第4条第1項該当

Mechanical ventilation (MV) is widely used in spinal injury patients to compensate for respiratory muscle failure. MV is known to induce lung inflammation, while spinal cord injury (SCI) is known to contribute to local inflammatory response. Interaction between MV and SCI was evaluated in order to assess the impact it may have on the pulmonary inflammatory profile. Sprague Dawley rats were anesthetized for 24 h and randomized to receive either MV or not. The MV group included C4–C5 SCI, T10 SCI and uninjured animals. The nonventilated (NV) group included T10 SCI and uninjured animals. Inflammatory cytokine profile, inflammation related to the SCI level, and oxidative stress mediators were measured in the bronchoalveolar lavage (BAL). The cytokine profile in BAL of MV animals showed increased levels of TNF-α, IL-1β, IL-6 and a decrease in IL-10 (P = 0.007) compared to the NV group. SCI did not modify IL-6 and IL-10 levels either in the MV or the NV groups, but cervical injury induced a decrease in IL-1β levels in MV animals. Cervical injury also reduced MV-induced pulmonary oxidative stress responses by decreasing isoprostane levels while increasing heme oxygenase-1 level. The thoracic SCI in NV animals increased M-CSF expression and promoted antioxidant pulmonary responses with low isoprostane and high heme oxygenase-1 levels. SCI shows a positive impact on MV-induced pulmonary inflammation, modulating specific lung immune and oxidative stress responses. Inflammation induced by MV and SCI interact closely and may have strong clinical implications since effective treatment of ventilated SCI patients may amplify pulmonary biotrauma.

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The Sco proteins, present in all kind of organisms, are regarded as one of the key players in the cytochrome c oxidase (COX) assembly. However, experimental and structural data, such as the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis. Our current studies in S. cerevisiae have strongly suggested an antioxidant role to Sco2 protein (ySco2p). While the single deletion of SCO2 does not result in a distinctive phenotype, the concomitant deletion of superoxide dismutase 1 (SOD1) leads to an increased sensitivity to oxidative stress generating agents (paraquat, menadione, plumbagin) compared to the respective single mutants. Since S. cerevisiae is a good model to functionally characterize genes from more complex organisms, identification of such a phenotype has paved the way to test whether the Sco2 homologues from other organisms are able to substitute for the function of ySco2p. The Sco homologues from Homo sapiens, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster and Kluyveromyces lactis were integrated into the genome of the double deletion mutant. The functional complementation was tested by both growth and biochemical ROS assays. All homologues except for K. lactis K07152 and A. thaliana HCC1 were able to complement the phenotype, indicating their role in antioxidant defense. Interestingly, pathogenic human SCO2 point mutations failed to restore this function. The observation of non-functional homologues despite of the high sequence similarity to ySco2p strengthened our hypothesis on the importance of conserved aminoacid(s) for the defensive role. For this purpose, selected homologues were aligned and the conservation was judged not only based on identity but also similarity (e.g. charge, hydrophobicity). Interestingly, alignment results have pointed out an aminoacid site (located 15 aminoacids downstream of CxxxC motif) that a positively charged lysine is found only in the non-functional homologues. Subsequent mutagenesis analyses verified the functional importance of this aminoacid site (gain and loss of functions) and revealed the detrimental effect of positive charge on antioxidant function. In order to explain the observed functional change, further effort will be put into the calculations of the electrostatic potential and identifications of protein-protein interactions.:Contents List of figures x List of tables xii Abbreviations xiii 1 Introduction 1 1.1 ROS production 1 1.2 Oxidative stress 2 1.3 Antioxidant response 3 1.4 The thioredoxin fold: From structure to function 6 1.5 Sco proteins 7 1.5.1 Structural similarity of Sco proteins to antioxidant enzymes 8 1.5.2 Current knowledge about Sco proteins of S. cerevisiae 9 1.6 Background studies 10 1.7 Using yeast as a model 11 1.7.1 Cross-species complementation studies 11 1.7.2 Yeast model for human mitochondria studies 12 1.8 Aim of the study 12 2 Materials & Methods 14 2.1 Materials 14 2.1.1 Chemicals and Reagents 14 2.1.2 Equipments 16 2.1.3 Kits 17 2.1.4 Antibodies 18 2.1.5 Plasmid 18 2.1.6 Primers 19 2.1.7 S. cerevisiae strains 22 2.1.8 Media 22 2.2 Methods 24 2.2.1 Cultivation of S. cerevisiae cells 24 2.2.1.1 Culture conditions 24 2.2.1.2 Preparation of glycerol stocks 24 2.2.2 Molecular Biology Methods 24 2.2.2.1 S. cerevisiae genomic DNA isolation 24 2.2.2.2 RNA isolation 25 2.2.2.2a Cultured mammalian cells (HEK293) 25 2.2.2.2b Drosophila melanogaster 25 2.2.2.3 RNA purity and concentration determination 25 2.2.2.4 Reverse transcription 25 2.2.2.5 Polymerase chain reaction 25 2.2.2.5a Standard PCR 25 2.2.2.5b Overhang PCR 26 2.2.2.5c Overlap extension PCR 27 2.2.2.5d Site-directed mutagenesis by overlap extension PCR 27 2.2.2.6 DNA agarose gel electrophoresis 28 2.2.2.7 DNA gel extraction and clean-up 29 2.2.2.8 DNA sequencing 29 2.2.2.9 Southern blotting 29 2.2.2.9a DNA preparation 29 2.2.2.9b Blotting 30 2.2.2.9c Preparation of a DIG-labelled probe 30 2.2.2.9d Hybridization of the DIG-labelled probe to DNA 30 2.2.2.9e Detection of hybridized DIG-labelled URA3 probe 31 2.2.2.10 Yeast transformation 32 2.2.2.11 Growth assay 32 2.2.3 Protein methods 33 2.2.3.1 Isolation of crude mitochondria from yeast 33 2.2.3.2 SDS-PAGE 33 2.2.3.3 Protein transfer 34 2.2.3.4 Colloidal Coomassie gel staining 34 2.2.3.5 Protein detection 35 2.2.3.6 Stripping the membrane and reprobing 35 2.2.4 Biochemical methods 35 2.2.4.1 Methylene Blue staining 36 2.2.4.2 Quantification of ROS 36 2.2.4.2a Amplex Red staining 36 2.2.4.2b Lipid peroxidation assay 36 2.2.5 Bioinformatics 37 2.2.6 Statistical Analysis 37 3 Results 40 3.1 Selection of homologues by bioinformatic analysis 40 3.2 Generation of recombinant strains 42 3.3 Confirmation of site-specific integration by check PCR 44 3.4 Verification of single site integration by Southern Blotting 44 3.5 Analysis of the functional homology between selected homologues and ySCO2 45 3.5.1 Complementation assay in solid media 45 3.5.2 Complementation assay in liquid media 47 3.6 Determination of cell viability 48 3.7 Quantification of ROS 51 3.7.1 Quantification of extracellular H2O2 51 3.7.2 Quantification of lipid peroxidation 53 3.8 Investigation of the expression and subcellular localization of homologues 55 3.9 Investigation of the impact of pathogenic hSCO2 mutations on its antioxidant role 58 3.10 Mutational analysis of ySCO2 60 3.11 Identification of functionally important residues 61 3.12 Prediction of salt bridges 65 3.13 Alanine mutagenesis 66 4 Discussion 68 4.1 Functional homology between the selected homologues and ySCO2 68 4.1.1 A. thaliana homologues, HCC1 & HCC2 68 4.1.2 H. sapiens homologues, hSCO1 & hSCO2 69 4.1.3 D. melanogaster homologue, SCOX 70 4.1.4 Yeast homologues, K07152 & SpSCO1 70 4.2 The localization and expression pattern of homologues 71 4.3 The impact of pathogenic hSCO2 mutations on its antioxidant role 72 4.4 Mutational analysis of ySCO2 73 4.5 Attempts to understand the underlying reason(s) behind charge-related functional change 74 4.6 Potential mechanisms associated with the antioxidant action of ySco2p 78 5 Summary 81 6 References 84

De manière générale, l'inhalation de particules entraîne des réactions inflammatoires et des réactions d'oxydo-réduction responsables de la dégradation des matrices biologiques qui exercent, de plus, un fort impact cardio-vasculaire. La combustion du bois est une source majeure de composés organiques semi-volatils, parmi lesquels les méthoxyphénols (MPs), tels que le coniféryl aldéhyde (CA), le syringaldéhyde (SR), ou l'acétosyringone (AS). Les MPs sont néanmoins peu étudiés dans la littérature alors que la toxicité d'autres composés également issus de la combustion de la biomasse, tels que le monoxyde de carbone, les suies et les hydrocarbures polyaromatiques est intensivement étudiée. Ce travail a montré par GC/MS que le vieillissement en atmosphère simulée (ozone, rayonnements lumineux) dégrade le CA en produits secondaires moins cytotoxiques, évalués sur des fibroblastes en culture, mais préserve le taux atmosphérique de SR et AS, de toxicité avérée. Un dispositif original de production de MPs particulaires (Ø~50nm, N~7E4particules/cm3, m~5µg/m3) en atmosphère contrôlée a été validé et permet la 1ièreétude in vivo des MPs. L'exposition chez le rat (1-3mois) montre une modification des défenses antioxydantes et des changements cardiaques principalement avec AS, puis CA et un peu moins pour SR. Des processus adaptatifs sont démontrés après 5mois d'exposition.Par ailleurs, il a été montré in vitro sur des adénocacinomes pulmonaires A549 en culture, que le CA induit une destructuration du tapis cellulaire et l'apoptose (caspase 3) mais pas d'effet pro-inflammatoire (IL8, Cox-2). En conclusion, ce travail contribue à étudier l'impact des MPs in vitro et in vivo. / In general, inhalation of particles is at the origin of inflammatory and oxidative reactions who are responsible of the degradation of biological cellular constituents, and could have a strong cardiovascular impact. The wood combustion is a major source of semivolatile organic compounds such as the methoxyphenols (MPs) including coniferyl aldehyde (CA), syringaldehyde (SR), or acetosyringone (AS). The MPs are however few studies into literature while toxicity of other compounds also from biomass combustion, as carbon monoxide, soot and polycyclic aromatic hydrocarbon are intensively studies.This work has shown by GC/MS that aging in simulated atmosphere (ozone + light rays) degraded CA in secondary products less cytotoxic, studies on fribroblastes culture but keep the atmospherical level of SR and AS which have a toxicity proved.A device of MPs particle production original by atomization, with a check system (height, composition, weight) and exposition flow continuous (Ø~50 nm, N~7E4 particles/cm3, m~5 µg/m3) adapted to little animals, was developed and validated, allowed the first study in vivo with these molecules. Between 1 and 3 month of exposition to rat Wistar, show modified antioxidant defences and cardiac modification (ischaemia/reperfusion) principally with AS, then CA and less SR. The adaptatives processes (remodeling) are demonstrated after 5 month of exposition.Furthermore, it is showed in vitro on lung adenocacinum cell lines (A549), CA induced a monolayer destructuration and apoptosis (caspase 3) but no effect proinflammatory (IL8, Cox-2 and iNOS).To conclude, this work contributes to study the impact of MPs in vitro and in vivo.

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.