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（由申请人提供）：铁和硫醇氧化还原稳态在细胞代谢中密切相关。铁是许多生化途径中蛋白质和酶的重要辅助因子，但如果不加以控制，过量的铁会催化活性氧 (ROS) 的形成，从而破坏硫醇氧化还原稳态。反过来，细胞内硫醇-二硫键平衡对于具有重要功能的半胱氨酸残基的蛋白质（包括许多铁结合酶）的活性至关重要。因此，铁稳态和硫醇-二硫化物平衡的维持是相互依赖的过程，对细胞生存至关重要。 Th 三肽谷胱甘肽 (GSH) 和谷氧还蛋白 (Grx) 蛋白通过催化硫醇-二硫键交换反应并参与 Fe-S 簇生物发生途径，在硫醇氧化还原控制和铁稳态中共同发挥作用。线粒体中 GSH 和铁稳态的维持尤为重要，因为该细胞器是 Fe-S 簇和血红素生物合成的主要场所，也是 ROS 产生的主要来源和目标。然而，在细胞和分子水平上理解铁调节机制和线粒体硫醇氧化还原控制途径还存在显着差距，需要进一步研究。 该研究计划的长期目标是：(1) 确定维持细胞内必需金属铁充足水平的机制，以及 (2) 表征控制线粒体硫醇氧化还原平衡和亚细胞区室之间 GSH 通量的细胞内因子。提供对这些关键细胞功能的机制洞察对于 预防和治疗铁过载、氧化应激和线粒体氧化还原失衡疾病。对于铁调控项目，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、穆斯堡尔、EPR、NMR 光谱、SAXS 和 X 射线晶体学）。体外生化和生物物理研究将探讨铁传感通路中的蛋白质-蛋白质、金属-蛋白质和蛋白质-DNA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则测试这些分子相互作用如何影响铁信号转导因子的体内功能和动态定位。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，结合使用基于 GFP 的靶向氧化还原传感器进行体内硫醇氧化还原测量，以确定影响硫醇-二硫化物平衡和控制亚细胞区室之间的 GSH 通量的因素。这两个项目都利用酵母模型系统，因为这些简单的真核生物易于在实验室中维护和基因操作，但表达许多与人类细胞相同的氧化还原和金属稳态系统。总体而言，这个多学科研究计划旨在梳理细胞和分子水平上铁调节和亚细胞硫醇氧化还原控制的机制细节。