Sulfide Oxidation and Signaling

硫化物氧化和信号传导

• 批准号: 10543114
• 负责人: RUMA V BANERJEE
• 金额: $ 39万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543114
• 关键词: Ablation; Apical; Binding; Biological; Butyrates; Butyryl-CoA dehydrogenase; Carbon; Cells; Cellular Metabolic Process; Coenzyme A; Collaborations; Colon; Colon Carcinoma; Colorectal Cancer; Complex; Diet; Dietary Fiber; Dietary Proteins; Dioxygenases; Disease; Electron Transport; Environment; Enzymatic Biochemistry; Enzymes; Epithelial Cells; Exposure to; Fermentation; Flavins; Health; Human; Hydrogen Sulfide; In Vitro; Kinetics; Malignant - descriptor; Mediating; Metabolic; Metabolism; Methods; Microbe; Mitochondria; Non-Malignant; Oxidoreductase; Pathway interactions; Physicians; Poison; Poisoning; Post-Translational Protein Processing; Proteins; Proteomics; Quinones; Role; Scientist; Signal Transduction; Structure; Sulfides; Testing; Ubiquinone; cancer cell; charge transfer complex; cofactor; colonic crypt; gut microbiota; host microbiome; inhibitor; insight; lipid metabolism; metabolomics; microbial; microbiota; nucleotide metabolism; overexpression; oxidation; persulfides; protein intake; radiotracer; respiratory; respiratory toxin; sulfurtransferase

The tripartite interaction between dietary fiber and protein intake, microbial fermentation and host cell metabolism, is on display in the colon where epithelial cells are routinely exposed to high concentrations of the respiratory poison, hydrogen sulfide (H2S). H2S is also beneficent; it is used for signaling via persulfidation, a posttranslational modification, and is a substrate for the electron transfer chain, generating ATP. Thiosulfate is the primary product of H2S oxidation in colon and its synthesis involves three mitochondrial enzymes: sulfide quinone oxidoreductase (SQR), a persulfide dioxygenase (PDO), and a sulfurtransferase. We have found that the apical localization of all three enzymes in colonic crypts is influenced by the presence or absence of microbiota and that these enzymes are significantly overexpressed in colon cancer. We now seek to expand from our initial focus on the enzymology of human sulfide oxidation enzymes to the interconnection between the oxidation pathways for H2S and butyrate, which is the preferred fuel for colonocytes and is furnished by microbial metabolism. We posit that prioritization of H2S over butyrate oxidation (to avert poisoning), is achieved via coenzyme A persulfide (CoASSH) produced by SQR and oxidized by PDO. CoASSH inhibits butyryl-CoA dehydrogenase (ACADS), explaining why native ACADS has long been purified as a “green” enzyme due to an inhibitory charge transfer complex between CoASSH and the flavin cofactor. To test this hypothesis, we will determine the kinetics of CoASSH synthesis and its inhibition of ACADS in vitro and of butyrate oxidation in cells with normal or ablated SQR or PDO expression. We will also determine the structures of human SQR and PDO (with a tightly bound inhibitor) to inform our kinetic and cellular studies and will assess the competition between SQR and complexes I and II for a limited coenzyme Q pool. Using a proteomic method, we have identified >1800 persulfide targets and we will investigate the role of H2S signaling in modulating metabolism in nonmalignant and malignant colon epithelial cells. For this, we will use radiolabel tracing and metabolomics analyses to assess the influence of sulfide on central carbon, nucleotide and lipid metabolism, evaluate the role of a sulfurtransferase in catalytic persulfidation of protein targets, and assess the influence of gut microbiota and diet on host sulfide metabolism. The impact of our broad and rigorous attack on the challenging study of sulfide-mediated signaling and metabolic modulation, supported by strong ongoing collaborations (with a physician scientist, cancer cell biologists, a microbiologist and physical chemists), will be to provide fundamentally important biological insights at the relatively unexplored host-microbiome interface.

膳食纤维和蛋白质摄入、微生物发酵和 宿主细胞代谢，在结肠中显示，其中上皮细胞通常暴露于 高浓度的呼吸道毒物硫化氢（H2S）。H2S也是有益的;它 用于通过过硫化（一种翻译后修饰）进行信号传导，并且是 电子传递链，生成ATP。硫代硫酸盐是H2S氧化的主要产物 结肠及其合成涉及三种线粒体酶：硫化物醌氧化还原酶 (SQR)过硫化物双加氧酶（PDO）和硫转移酶。我们发现， 所有三种酶在结肠隐窝中的定位受以下因素的存在或不存在的影响： 这些酶在结肠癌中显著过表达。我们现在 寻求从我们最初对人类硫化物氧化酶酶学的关注扩展到 H2S和丁酸盐的氧化途径之间的相互联系，这是优选的 为结肠细胞提供燃料，并由微生物代谢提供。我们强调H2S的优先级 通过辅酶A过硫化物（CoASSH）实现丁酸氧化（以避免中毒） 由SQR产生并由PDO氧化。CoASSH抑制丁酰辅酶A脱氢酶 （ACADS），解释了为什么天然ACADS长期以来一直被纯化为“绿色”酶，这是由于 CoASSH和黄素辅因子之间的抑制性电荷转移复合物。为了验证这一 假设，我们将确定CoASSH合成的动力学及其对ACADS的抑制作用， 在体外和具有正常或消除的SQR或PDO表达的细胞中的丁酸氧化。我们将 还确定了人类SQR和PDO的结构（与紧密结合的抑制剂）， 我们的动力学和细胞研究，并将评估SQR和复合物I之间的竞争 和II用于有限辅酶Q库。使用蛋白质组学方法，我们已经确定了>1800 我们将研究H2S信号在调节代谢中的作用， 非恶性和恶性结肠上皮细胞。为此，我们将使用放射性标记追踪， 代谢组学分析评估硫化物对中心碳、核苷酸和脂质的影响 代谢，评估硫转移酶在蛋白质靶的催化过硫化中的作用， 并评估肠道微生物群和饮食对宿主硫化物代谢的影响。的影响 我们对硫化物介导的信号传导的挑战性研究的广泛和严格的攻击， 代谢调节，由强有力的持续合作支持（与一位医生科学家， 癌细胞生物学家，微生物学家和物理化学家），将从根本上提供 在相对未开发的宿主-微生物组界面的重要生物学见解。