Mechanisms of Iron and Thiol Redox Regulation in Yeast

酵母中铁和硫醇氧化还原调节机制

• 批准号: 9916760
• 负责人: Caryn E Outten
• 金额: $ 42.68万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-10 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916760
• 关键词: Binding; Biochemical; Biochemical Pathway; Biogenesis; Biological Models; Cell Survival; Cell physiology; Cells; Cellular biology; Coupled; Cysteine; DNA-Protein Interaction; Disease; Disulfides; Enzymes; Equilibrium; Eukaryota; Gene Expression; Gene Proteins; Genetic; Glutathione; Goals; Heme; Homeostasis; Human; In Vitro; Interdisciplinary Study; Iron; Iron Overload; Left; Maintenance; Malignant Neoplasms; Measurement; Metabolism; Metals; Mitochondria; Molecular; Molecular Genetics; Mutagenesis; NMR Spectroscopy; Neurodegenerative Disorders; Organelles; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Prevention strategy; Process; Production; Protein Biochemistry; Proteins; Reaction; Reactive Oxygen Species; Regulation; Research; Signal Transduction; Site; Source; Sulfhydryl Compounds; System; Testing; X-Ray Crystallography; Yeast Model System; Yeasts; absorption; base; biophysical analysis; biophysical techniques; cofactor; design; genetic approach; genetic manipulation; glutaredoxin; human disease; in vivo; innovation; insight; iron metabolism; mitochondrial dysfunction; prevent; programs; protein function; public health relevance; sensor; treatment strategy; yeast genetics

 DESCRIPTION (provided by applicant): Iron and thiol redox homeostasis are intimately connected in cellular metabolism. Iron is an essential cofactor for proteins and enzymes in numerous biochemical pathways, but when left unchecked, excess iron catalyzes formation of reactive oxygen species (ROS) that disrupt thiol redox homeostasis. Intracellular thiol-disulfide balance is critical, in turn, for the activity of proteins with functionally important cysteine residues, which includes many Fe-binding enzymes. Thus, iron homeostasis and maintenance of thiol-disulfide balance are mutually dependent processes that are critical for cell survival. Th tripeptide glutathione (GSH) and glutaredoxin (Grx) proteins function together in both thiol redox control and iron homeostasis by catalyzing thiol-disulfide exchange reactions and participating in Fe-S cluster biogenesis pathways. Maintenance of GSH and iron homeostasis in the mitochondrion is especially important since this organelle is the primary site for Fe- S cluster and heme biogenesis, as well as the main source and target of ROS production. However, there are significant gaps in understanding both iron regulation mechanisms and mitochondrial thiol redox control pathways at the cellular and molecular level that require further study. The long term goals of this research program are: (1) to identify the mechanisms for maintaining adequate intracellular levels of the essential metal iron, and (2) to characterize intracellular factors that control mitochondrial thiol redox balance and GSH flux between subcellular compartments. Providing mechanistic insight into these critical cellular functions is essential for preventing and treating diseases of iron overload, oxidative stress, and mitochondrial redox imbalance. For the iron regulation project, the innovative approach to accomplish these goals is to use a combination of protein biochemistry, mutagenesis, yeast genetics and cell biology, and biophysical methods (UV-visible absorption, CD, resonance Raman, EXAFS, Mössbauer, EPR, NMR spectroscopy, SAXS, and X-ray crystallography). The in vitro biochemical and biophysical studies will probe protein-protein, metal-protein, and protein-DNA interactions in iron sensing pathways to uncover the molecular details of iron signaling, while the genetics and cell biology studies test how these molecular interactions influence the in vivo functions and dynamic localization of iron signaling factors. For the mitochondrial redox project, a molecular genetics approach will be used to manipulate gene expression and protein localization, coupled with in vivo thiol redox measurements using targeted GFP-based redox sensors, to identify factors that influence thiol-disulfide balance and control GSH flux between subcellular compartments. Both projects exploit yeast model systems since these simple eukaryotes are easy to maintain and genetically manipulate in the lab, yet expresses many of the same redox and metal homeostasis systems as human cells. Overall, this multidisciplinary research program is designed to tease out the mechanistic details of both iron regulation and subcellular thiol redox control at the cellular and molecular level.

 描述(申请人提供)：铁和硫醇氧化还原动态平衡在细胞新陈代谢中密切相关。铁是许多生化途径中蛋白质和酶的重要辅因子，但如果不加控制，过量的铁会催化形成活性氧物种(ROS)，从而扰乱硫醇氧化还原动态平衡。细胞内硫醇-二硫键的平衡反过来对具有重要功能的半胱氨酸残基的蛋白质的活性至关重要，半胱氨酸残基包括许多铁结合酶。因此，铁稳态和维持硫醇-二硫键平衡是相互依赖的过程，对细胞生存至关重要。谷胱甘肽和谷氧还蛋白通过催化硫醇-二硫键交换反应和参与铁-S簇生物发生途径，共同参与硫醇氧化还原控制和铁的动态平衡。线粒体中谷胱甘肽和铁的动态平衡的维持尤为重要，因为该细胞器是铁-S簇和血红素生物发生的主要部位，也是ROS产生的主要来源和靶点。然而，在细胞和分子水平上对铁调节机制和线粒体硫醇氧化还原调控途径的理解存在着重大差距，需要进一步研究。这项研究计划的长期目标是：(1)确定维持细胞内足够的必需金属铁水平的机制，(2)表征控制线粒体硫醇氧化还原平衡和亚细胞间GSH通量的细胞内因素。提供对这些关键细胞功能的机械洞察对于 防治铁超载、氧化应激、线粒体氧化还原失衡等疾病。对于铁调节项目，实现这些目标的创新方法是结合使用蛋白质生物化学、诱变、酵母遗传学和细胞生物学，以及生物物理方法(紫外可见吸收、CD、共振拉曼、EXAFS、Mössbauer、EPR、核磁共振光谱、SAXS和X射线结晶学)。体外生化和生物物理研究将探索铁信号通路中蛋白质-蛋白质、金属-蛋白质和蛋白质-DNA的相互作用，以揭示铁信号转导的分子细节，而遗传学和细胞生物学研究将测试这些分子相互作用如何影响体内铁信号因子的功能和动态定位。对于线粒体氧化还原项目，将使用分子遗传学方法来操纵基因表达和蛋白质定位，并使用基于GFP的目标氧化还原传感器在体内进行硫醇氧化还原测量，以确定影响硫醇-二硫键平衡的因素并控制亚细胞间的GSH通量。这两个项目都利用了酵母模型系统，因为这些简单的真核细胞很容易在实验室进行维护和基因操作，但表达许多与人类细胞相同的氧化还原和金属动态平衡系统。总体而言，这一多学科研究计划旨在从细胞和分子水平梳理铁调节和亚细胞硫醇氧化还原控制的机制细节。