京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第18379号 / 人博第692号 / 新制||人||165(附属図書館) / 25||人博||692(吉田南総合図書館) / 31237 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 土屋 徹, 教授 小松 賢志, 教授 宮下 英明 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

1 Abstract In this thesis I have focused on the role of lipids in the pathogenesis of liver diseases, specifically on cholestasis and non-alcoholic fatty liver disease (NAFLD). The first major aim was to clarify the changes in liver ganglioside metabolism in various types of cholestasis and to elucidate the role of heme oxygenase-1 (HMOX1) and associated oxidative stress. The second objective was to determine the effects of n-3 polyunsaturated fatty acids (n-3 PUFA) administration on NAFLD development in a rodent dietary model of NAFLD and in patients with metabolic syndrome and NAFLD. Our results suggest that increased ganglioside biosynthesis and their re-distribution might represent a general protective mechanism of hepatocytes under cholestatic conditions (both estrogen-induced and obstructive aetiology). These changes are closely related to oxidative stress and might protect hepatocytes against deleterious effect of accumulated bile acids. The lack of HMOX1 activity and subsequent oxidative stress potentiate pathological changes in the liver and resulted in tissue-specific modulation of synthesis and re-distribution of gangliosides (in vivo and in vitro). Contrary to it, HMOX1 activation has an opposite effect and may represent a general hepatoprotective mechanism. We have proven that observed changes...

Heme oxygenase (HMOX) catalyzes first and rate-limiting step in heme degradation. By its action, carbon monoxide (CO), ferrous iron and biliverdin which is subsequently reduced to bilirubin are produced. Before discovery of HMOX reaction mechanism, CO was considered only a toxic waste product without any significant importance for human organism. Bilirubin, marker of liver dysfunction, has been also exposed to similar perception. But results from past decades show that HMOX and its metabolic products play an important role in number of physiological as well as defense against pathophysiological processes. The aim of this thesis was to clarify the role of HMOX and its metabolic products, presumably CO and bilirubin, in vivo and in vitro. We focused on the role of CO in a rat model of lipopolysaccharide-induced cholestasis. We were first to describe tissue distribution and pharmacokinetics of inhaled CO in this animal model and found out that CO inhalation is associated with anti-inflammatory and hepatoprotective effects. In a rat model of ethinylestradiol-induced cholestasis, we demonstrated the anticholestatic effect of HMOX. The induction of HMOX by its substrate heme increased the expression of liver transporters thereby increasing bile flow and simultaneously facilitated effective clearance of...

No description available.

No description available.

Understanding how plants respond to stress is of importance, considering the increasing need to feed a growing population and supply its energy. Plants have complex systems for detecting, and responding to stresses. One stress-responsive system involves myo-inositol (Ins). Ins is a precursor for cell wall components, inositol trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol phosphate signaling molecules, and an alternate ascorbic acid (AsA) synthesis pathway. The enzyme, myo-inositol oxygenase (MIOX) is encoded by four genes in Arabidopsis and catalyzes the first step of Ins catabolism producing D-glucuronic acid (DGlcA). This research focuses on MIOX metabolism of Ins during plant growth and stress responses. I have examined miox mutants for alterations in metabolism and signaling. MIOX2 and MIOX4 expression patterns correlate with miox mutant root growth in varying nutrient conditions, and changes in flowering time. In miox2 mutants, I found an increase in Ins in most tissues, which was accompanied by cold- and abscisic (ABA)- sensitivity; however, miox4 mutants are ABA- insensitive, and have a small increase of Ins in flowers. MIOX2:GFP fusion protein accumulates in the cytoplasm and MIOX4:GFP accumulates in the cytoplasm and nucleus. Overexpresser MIOX4+ plants provide a model system to examine how directing carbon from Ins into DGlcA impacts Ins levels and Ins signaling. I have examined MIOX4+ plants for alterations in MIOX4 RNA and protein, and measured Ins by gas chromatography (GC). My results indicate that MIOX4+ tissues are impacted differently by the MIOX4 transgene, with decreases in Ins after seed imbibition, and increased Ins levels later in development. Ins depletion in seedlings was correlated with a decrease in Ins(1,4,5)P3. To determine the impact of reducing Ins and Ins(1,4,5)P3 in MIOX4+ seedlings, I examined processes known to involve Ins(1,4,5)P3 signaling. MIOX4+ seed have increased seed dormancy, NaCl-sensitivity, and ABA-insensitivity. These results suggest MIOX affects Ins signaling in response to ABA. Together, these data indicate that transcriptional control of MIOX2 and MIOX4 results in distinct roles in plant growth, and that MIOX2 and MIOX4 function in metabolic and signaling processes critical for growth, nutrient sensing, and stress responses. / Ph. D.

Folate is an essential cofactor required for several processes including DNA and amino acid biosynthesis. Folate molecules are made up of three parts: a pteridine ring, p-aminobenzoate (pABA), and a variable number of glutamate residues. Chlamydia trachomatis synthesizes folate de novo; however, several genes encoding enzymes required for the canonical folate biosynthesis pathway are missing, including pabA/B and pabC, which are normally required for pABA biosynthesis from chorismate. Previous studies have found that a single gene in C. trachomatis, CT610, functionally replaces the canonical pABA biosynthesis genes. Interestingly, CT610 does not use chorismate as a substrate. Instead, the CT610-route for pABA biosynthesis incorporates isotopically labeled tyrosine into the synthesized pABA molecule. However, in vitro experiments revealed that CT610 produces pABA without any added substrates (including tyrosine) in the presence of a reducing agent and molecular oxygen. CT610 shares low sequence similarity to non-heme diiron oxygenases and the previously solved crystal structure revealed a diiron active site. Taken together, CT610 is proposed to be a novel self-sacrificing enzyme that uses one of its active site tyrosine residues as a precursor to pABA in a reaction that requires O2 and a reduced metallocofactor. Here, we discuss our progress towards understanding CT610-catalyzed pABA synthesis. Upon investigation of the pABA production and oxygenase activities of several active site tyrosine to phenylalanine variants, we found that Y27 and/or Y43 are the most likely precursors to the resulting pABA molecule. Further, activity was nearly completely abolished with a K152R variant, suggesting that this conserved lysine may be the required amino group donor. We also developed an in vitro Fe(II) reconstitution procedure, where the reconstituted enzyme exhibited a drastic increase in oxygenase activity but, surprisingly, a significant decrease in pABA synthase activity. Interestingly, a significant increase in pABA synthase activity was observed when the enzyme was reconstituted with manganese as opposed to iron, suggesting that the diiron active site of this enzyme might not be directly involved in CT610-dependent production of pABA and instead Mn may be the actual cofactor. Finally, we show that two 18O atoms from molecular oxygen are incorporated into the pABA molecule when synthesized by Mn-reconstituted CT610, providing further evidence for the oxygenase activity of CT610 and supporting our proposed mechanism that involves two monooxygenase reactions. / Master of Science in Life Sciences / Folate is an essential molecule that is required for all cells to survive. Folate is usually made in the cell with the help from proteins known as enzymes. Enzymes help biochemical reactions happen by speeding up the rate of their specific chemical reaction. In order for this to occur, an enzyme binds to a very specific molecule, called a substrate, and facilitates the reaction transforming the substrate into a new product while not altering the enzyme in the process, allowing for the protein to continuously facilitate this reaction. Chlamydia trachomatis is the strain of bacteria that causes one of the most common sexually transmitted infections in the US, Chlamydia. These bacteria make folate themselves but have been shown to make this molecule in a very different way from an average folate-synthesizing organism. One enzyme in C. trachomatis known as CT610 has been shown to participate in this unusual route to produce folate. Interestingly, CT610 is thought to remove part of itself to donate to the molecule it produces, effectively killing the enzyme after only one reaction. In this study we show that CT610 performs very unique chemistry to ultimately facilitate the production of folate to allow C. trachomatis to survive. This knowledge could be used in the future for the design of antibiotics specifically targeting C. trachomatis and thus treating the infections caused by this organism.

Tetrahydrofolate (THF) is an essential cofactor for one-carbon transfer reactions in various biochemical pathways including DNA and amino acid biosynthesis. This cofactor is made up of three distinct moieties: a pteridine ring, p-aminobenzoate (pABA), and glutamate residues. Most bacteria and plants can synthesize folate de novo, unlike animals that obtain folate from their diet. An established pathway for THF biosynthesis exists in most bacteria, but there is evidence of some organisms such as Chlamydia trachomatis and Nitrosomonas europaea which do not contain the canonical THF biosynthesis genes, despite still being able to synthesize THF de novo. Previous studies have shown that these organisms do not contain the pabABC genes, normally required to synthesize the pABA portion of THF, and can circumvent their presence with just a single gene: ct610 and ne1434 from C. trachomatis and N. europaea, respectively. Interestingly, these novel enzymes for pABA synthesis do not use the canonical substrates, chorismate or other shikimate pathway intermediates. The gene product of ct610 was named Chlamydia Protein Associating with Death Domains (CADD) due to its established role in host mediated apoptosis, while the crystal structure showed an architecture similar to know diiron oxygenases. However, we provide evidence of a moonlighting function in pABA synthesis. Isotopic labeling experiments to understand what substrate might be used by CADD found that isotopically labeled tyrosine was incorporated into the final pABA product. Compellingly, CADD was able to produce pABA in the presence of molecular oxygen and a reducing agent alone without the addition of any exogenous substrate, implicating this unusual enzyme as a self-sacrificing pABA synthase from C. trachomatis. Here, we provide strong evidence for Tyr27 being a sacrificial residue that is cleaved from the protein backbone to serve as the pABA scaffold. Furthermore, we also provide evidence that K152 is an internal amino donor for this pABA synthase reaction performed by CADD. In the case of NE1434, we have conducted initial experiments such as site-directed mutagenesis and our findings suggest that these self-sacrificing residues are conserved between two distantly related organisms. Finally, the pABA synthase activity is reliant on an oxygenated dimetal cofactor and despite the crystal structure of CADD depicting a diiron active site, we have demonstrated that CADD's pABA synthase activity is dependent on a heterodinuclear Mn/Fe cofactor. Conversely, NE1434 demonstrates no preference for manganese and likely employs a more traditional Fe/Fe cofactor for catalysis. Our results implicate the CADD and NE1434 as self-sacrificing pABA synthases that have diverging metal requirements for catalysis. / Master of Science in Life Sciences / Folate is a molecule used by all organisms that is necessary for survival. Many kinds of bacteria are able to make this molecule with proteins called enzymes, which help by quickening the rate of a reaction. Enzymes are catalysts that usually work by binding a molecule, called a substrate, and will act on this substrate to generate a product; the enzyme remains unchanged in this process, which allows it to facilitate many more of these reactions. Chlamydia trachomatis, which is a leading cause of sexually transmitted infections (STIs) in the United States, and Nitrosomonas europaea, an environmental bacterium, are able to use enzymes to make their own folate, but not in the way that many other bacteria do. These organisms contain enzymes that use a part of their own structure as a substrate, making them "sacrificial lambs". Our study provides evidence of how these organisms carry out an abnormal chemical reaction to make folate which can help scientists target this pathway for the development of antibiotics.

No / T-type Ca2+ channels regulate proliferation in a number of tissue types, including vascular smooth muscle and various cancers. In such tissues, up-regulation of the inducible enzyme heme oxygenase-1 (HO-1) is often observed, and hypoxia is a key factor in its induction. HO-1 degrades heme to generate carbon monoxide (CO) along with Fe2+ and biliverdin. Since CO is increasingly recognized as a regulator of ion channels (Peers et al. 2015), we have explored the possibility that it may regulate proliferation via modulation of T-type Ca2+ channels. Whole-cell patch-clamp recordings revealed that CO (applied as the dissolved gas or via CORM donors) inhibited all 3 isoforms of T-type Ca2+ channels (Cav3.1-3.3) when expressed in HEK293 cells with similar IC50 values, and induction of HO-1 expression also suppressed T-type currents (Boycott et al. 2013). CO/HO-1 induction also suppressed the elevated basal [Ca2+ ]i in cells expressing these channels and reduced their proliferative rate to levels seen in non-transfected control cells (Duckles et al. 2015). Proliferation of vascular smooth muscle cells (both A7r5 and human saphenous vein cells) was also suppressed either by T-type Ca2+ channel inhibitors (mibefradil and NNC 55-0396), HO-1 induction or application of CO. Effects of these blockers and CO were non additive. Although L-type Ca2+ channels were also sensitive to CO (Scragg et al. 2008), they did not influence proliferation. Our data suggest that HO-1 acts to control proliferation via CO modulation of T-type Ca2+ channels.

Yes / The crotonases comprise a widely distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole-mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) both with nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by α-helices, which typically oligomerizes to form a trimer or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis and synthetic biology, as supported by pioneering protein engineering studies on them. In this Perspective, we give an overview of crotonase diversity and structural biology and then illustrate the scope of crotonase catalysis and potential for biocatalysis. / Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Wellcome Trust