Engineered flavin-dependent enzymes for probing redox environment and regulation

摘要 不同细胞隔间中氧化还原环境的紊乱与许多病理有关， 包括神经退行性疾病、癌症、心血管疾病和老龄化。由于氧化态与氧化态的比率 还原的烟酰胺腺嘌呤二核苷酸是细胞氧化还原环境的主要贡献者， 干扰这一比率的工程工具将使这项研究能够研究氧化还原失衡在推动这些变化中的作用 病理学。在这项工作中，我们研究了一些微生物用来控制 它们的最佳氧化还原环境，以及这些机制在哺乳动物细胞中的可能应用。 一些微嗜氧细菌和原生动物缺乏传统的多复合体呼吸链，以及 取而代之的是依赖催化形成H2O的NADH氧化酶反应的酶来循环NAD+并 消除环境中的有毒氧气。因为这个反应的产物是良性的水，而不是 过氧化氢(H_2O_2)，这些酶是干扰哺乳动物代谢的诱人试剂 细胞。在这项工作中，我们建议设计一种针对NADPH特异性的细菌形成H2O的NADH氧化酶。 然后，这种NADPH氧化酶将在哺乳动物细胞的不同隔室中表达，它对 将对细胞活性和新陈代谢进行系统评估。因为之前没有工具被报告给 以隔室特有的方式安全地增加细胞中的NADP+/NADPH比率，我们的工作将提供 对NADPH代谢及其调节的基本见解，以及对NAD(P)H池大小的了解 不同的细胞隔间。我们的工作还探索了微嗜氧性人类 肠贾第鞭毛虫、阴道毛滴虫和溶组织内阿米巴等原生动物寄生虫缺乏 传统的呼吸链，控制它们的氧化还原环境，以支持能量代谢。我们 已经在阴道毛滴虫中发现了一种天然蛋白质，它代表黄铁蛋白核心蛋白与 它的氧化还原伙伴：鲁布鲁还蛋白和鲁布雷还辛氧化还原酶。这种融合蛋白催化一个四电子 用NAD(P)H的还原当量将氧气还原为水。我们对其结构和性质的研究 这种融合蛋白的机制将为这些人类寄生虫如何维持其 最佳的氧化还原环境。这些机制可以成为治疗干预的有吸引力的靶点，允许 美国抗击由人类原生动物寄生虫引起的疾病。

Abstract Disturbances of the redox environment in various cellular compartments are linked to many pathologies, including neurodegenerative diseases, cancer, cardiovascular disease and aging. Since the ratio of oxidized to reduced nicotinamide adenine dinucleotides is a major contributor to the cellular redox environment, engineering tools to perturb this ratio would enable the study the role of redox imbalances in driving these pathologies. In this work we examine the fundamental mechanisms used by some microorganisms to control their optimal redox environment, as well as the possible applications of these mechanisms in mammalian cells. A number of microaerophilic bacteria and protozoa lack a conventional multi-complex respiratory chain, and instead rely on enzymes which catalyze a H2O-forming NADH oxidase reaction to recycle NAD+ and to eliminate toxic oxygen from the environment. Since the product of this reaction is benign water and not hydrogen peroxide (H2O2), these enzymes represent attractive reagents to perturb metabolism in mammalian cells. In this work we propose to engineer a bacterial H2O-forming NADH oxidase towards NADPH specificity. This NADPH oxidase will then be expressed in different compartments of mammalian cells, and its effects on cell viability and metabolism will be systematically evaluated. Since no tool has previously been reported to safely increase the NADP+/NADPH ratio in cells in a compartment specific manner, our work will provide fundamental insights into NADPH metabolism and its regulation, as well as into the sizes of NAD(P)H pools in different cellular compartments. Our work also explores the mechanisms of how microaerophilic human protozoan parasites like Giardia intestinalis, Trichomonas vaginalis and Entamoeba histolytica, which lack conventional respiratory chains, control their redox environments in order to support energy metabolism. We have identified in T.vaginalis a natural protein which represents a fusion between a flavodiiron core protein with its redox partners: rubredoxin and rubredoxin oxidoreductase. This fusion protein catalyzes a four-electron reduction of oxygen to water using reducing equivalents of NAD(P)H. Our studies of the structure and mechanism of this fusion protein will provide insights into how these human parasites are able to maintain their optimal redox environment. These mechanisms can be attractive targets for therapeutic intervention, allowing us to combat diseases caused by human protozoan parasites.