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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的各种细胞区室中氧化还原环境的干扰与许多病理学有关，包括神经退行性疾病、癌症、心血管疾病和衰老。由于氧化的比例还原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，扰乱这一比率的工程工具将使研究氧化还原失衡在驱动这些比率中的作用成为可能。病理学。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。许多微需氧细菌和原生动物缺乏传统的多重复合呼吸链，并且相反，依靠催化形成 H2O NADH 氧化酶反应的酶来回收 NAD+ 并消除环境中的有毒氧气。由于该反应的产物是良性水而不是过氧化氢 (H2O2)，这些酶是干扰哺乳动物新陈代谢的有吸引力的试剂细胞。在这项工作中，我们建议设计一种细菌 H2O 形成 NADH 氧化酶以实现 NADPH 特异性。然后，这种 NADPH 氧化酶将在哺乳动物细胞的不同区室中表达，其对细胞活力和新陈代谢将得到系统评估。由于之前没有报告过任何工具以隔室特定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 代谢及其调节以及 NAD(P)H 池大小的基本见解不同的细胞区室。我们的工作还探索了微需氧人类如何原生动物寄生虫，如肠贾第鞭毛虫、阴道毛滴虫和溶组织内阿米巴，缺乏传统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 中鉴定出一种天然蛋白质，它代表黄二铁核心蛋白与它的氧化还原伙伴：红氧还蛋白和红氧还蛋白氧化还原酶。该融合蛋白催化四电子使用 NAD(P)H 的还原当量将氧气还原为水。我们对结构的研究和这种融合蛋白的机制将有助于了解这些人类寄生虫如何能够维持其最佳的氧化还原环境。这些机制可能成为治疗干预的有吸引力的目标，使得我们来对抗人类原生动物寄生虫引起的疾病。