Engineered flavin-dependent enzymes for probing redox environment and regulation

用于探测氧化还原环境和调节的工程黄素依赖性酶

• 批准号: 9223586
• 负责人: Valentin Cracan
• 金额: $ 9万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9223586
• 关键词: Aerobic; Affect; Aging; Amino Acid Substitution; Automobile Driving; Bacteria; Benign; Biochemical; Carbon; Cardiovascular Diseases; Cell Line; Cell Survival; Cell physiology; Cells; Cellular Metabolic Process; Chimeric Proteins; Communicable Diseases; Complex; Core Protein; Data; Defect; Disease; Disulfides; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Energy Metabolism; Engineering; Entamoeba histolytica; Environment; Enzymes; Family; Flavins; Giardia lamblia; Giardiasis; Glycolysis; Goals; Human; Hydrogen Peroxide; Impairment; Lactobacillus brevis; Life; Link; Malignant Neoplasms; Mammalian Cell; Metabolic; Metabolism; NADH; NADH oxidase; NADP; NADPH Oxidase; Neurodegenerative Disorders; Niacinamide; Nicotinamide adenine dinucleotide; Organism; Oxidases; Oxidation-Reduction; Oxides; Oxidoreductase; Oxygen; Parasites; Pathology; Pentosephosphate Pathway; Phase; Process; Production; Prokaryotic Cells; Proteins; Protozoa; Reaction; Reagent; Recycling; Regulation; Reporting; Research Personnel; Resistance; Respiratory Chain; Role; Rubredoxins; Sequence Alignment; Specificity; Structure; Substrate Specificity; System; Techniques; Therapeutic; Therapeutic Intervention; Time; Training; Trichomonas Infections; Trichomonas vaginalis; Variant; Water; Work; X-Ray Crystallography; biophysical techniques; career; combat; insight; member; metabolomics; microorganism; mutant; novel; pathogen; polypeptide; prevent; targeted treatment; tool

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.

抽象的 各种细胞室中氧化还原环境的干扰与许多病理有关， 包括神经退行性疾病，癌症，心血管疾病和衰老。由于氧化与 减少烟酰胺腺嘌呤二核苷酸是细胞氧化还原环境的主要因素， 扰动该比率的工程工具将使氧化还原失衡在推动这些方面的作用 病理。在这项工作中，我们研究了一些微生物用于控制的基本机制 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。 许多微型细菌和原生动物缺乏常规的多复合呼吸链，并且 取而代之的是依靠催化H2O形成的NADH氧化酶反应以回收NAD+的酶 消除环境中的有毒氧。因为该反应的产物是良性水而不是 过氧化氢（H2O2），这些酶代表了在哺乳动物中扰动代谢的有吸引力的试剂 细胞。在这项工作中，我们建议将细菌构成H2O的NADH氧化酶针对NADPH特异性进行设计。 然后，该NADPH氧化酶将在哺乳动物细胞的不同隔室中表达，其对 细胞活力和代谢将系统评估。由于以前没有任何工具报告 安全地以特定方式以隔室方式增加细胞中的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NAD（P）H池的大小 不同的细胞室。我们的作品还探讨了微型人类人类如何的机制 原生动物寄生虫，如贾第鞭毛虫，阴道毛毛虫和溶血虫，缺乏 常规的呼吸链控制其氧化还原环境，以支持能量代谢。我们 在t.vaginalis中已经确定了一种天然蛋白 它的氧化还原伴侣：Rubredoxin和Rubredoxin氧化还原酶。该融合蛋白催化四电子 使用NAD（P）h的还原等效物将氧气还原为水。我们对结构和 这种融合蛋白的机制将提供有关这些人类寄生虫如何维持其如何维持其的见解 最佳氧化还原环境。这些机制可能是治疗干预的有吸引力的目标，允许 我们要打击由人类原生动物寄生虫引起的疾病。