Mechanisms of Fungal Iron Regulation and Thiol Redox Metabolism

真菌铁调节和硫醇氧化还原代谢的机制

• 批准号: 10544771
• 负责人: Caryn E Outten
• 金额: $ 37.25万
• 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 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10544771
• 关键词: Aerobic; Affinity; Antioxidants; Binding; Binding Proteins; Biochemical; Biochemical Pathway; Biogenesis; Biological Availability; Candida albicans; Candida glabrata; Cells; Cellular biology; Communication; Coupled; Cysteine; DNA Binding; DNA-Protein Interaction; Disulfides; Environment; Enzymes; Equilibrium; Fission Yeast; Genetic; Glutathione; Glutathione Disulfide; Glutathione Metabolism Pathway; Goals; Homeostasis; Homologous Gene; Human; Impairment; In Vitro; Interdisciplinary Study; Iron; Ligation; Measurement; Measures; Metabolism; Metals; Microbe; Molecular; Molecular Genetics; Monitor; Mycoses; Organism; Oxidation-Reduction; Oxidative Stress; Pathogenicity; Pathway interactions; Peptides; Population; Property; Proteins; Reaction; Reactive Oxygen Species; Regulation; Repression; Role; Saccharomyces cerevisiae; Signal Transduction; Spectrum Analysis; Sulfhydryl Compounds; Sulfur; System; Techniques; Testing; Work; X-Ray Crystallography; Yeasts; absorption; biophysical analysis; biophysical techniques; cell injury; cofactor; combat; design; fungus; glutaredoxin; in vivo; innovation; iron metabolism; metal metabolism; programs; protein function; response; sensor; trafficking; transcription factor; uptake

PROJECT SUMMARY Iron and thiol redox homeostasis have interdependent roles in cellular metabolism. Iron serves as a cofactor for a wide variety of proteins and enzymes in essential biochemical pathways, but excess iron can be damaging to cells by catalyzing formation of reactive oxygen species 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 proteins. The tripeptide glutathione (GSH) and glutaredoxin (Grx) proteins function together in both thiol redox control and iron homeostasis by facilitating redox reactions and participating in iron- sulfur (Fe-S) cluster biogenesis pathways. Our previous work in the non-pathogenic yeast S. cerevisiae and S. pombe have revealed the molecular mechanisms by which a subclass of Grxs, known as monothiol Grxs, bind and deliver GSH-ligated Fe-S clusters to communicate iron bioavailability to the transcription factors Aft1/Aft2 in S. cerevisiae and Php4 in S. pombe that regulate iron acquisition and utilization pathways. Furthermore, we have used molecular genetics and cell biology approaches coupled with in vivo redox measurements via genetically- encoded fluorescent redox sensors to characterize GSH subcellular trafficking pathways that impact both iron homeostasis and redox regulation in S. cerevisiae. Here we will extend these findings by studying the impact of GSH and Grxs on the Fe-S binding properties and DNA binding affinity of the S. pombe transcription factor Fep1 that represses Fe uptake pathways during iron repletion. Furthermore, we will define the molecular details of iron regulation pathways in pathogenic yeast (Candida glabrata, Candida albicans) that express homologs of monothiol Grxs, Aft1/2, Fep1, and Php4, but for which little mechanistic information is available. In parallel, we will characterize GSH:GSSG flux between subcellular compartments in yeast cells and measure the impact of GSH deficiency, excess, or impaired trafficking on essential metal metabolism. Our innovative approach to accomplish these goals is to combine yeast molecular genetics and cell biology techniques with biochemical, structural, and biophysical methods (UV-visible absorption and CD spectroscopy, EXAFS, X-ray crystallography, Mössbauer, EPR, and single cell ICP-TOF-MS). The in vitro biochemical, structural, and biophysical studies will be used to probe protein-protein, metal-protein, and protein-DNA interactions in iron sensing pathways to uncover the molecular details of iron signaling and to monitor single cell metallomic changes in yeast populations in response to alterations in iron or GSH metabolism. The genetics and cell biology studies test how these molecular interactions and metallome changes influence the in vivo functions and dynamic localization of iron signaling and GSH metabolism factors. Overall, this multidisciplinary research program is designed to tease out the mechanistic details of iron regulation and subcellular thiol redox control at the cellular and molecular level. By studying both pathogenic and non-pathogenic fungi we will compare and contrast different strategies for adapting to redox perturbations and high/low iron environments.

项目概要 铁和硫醇氧化还原稳态在细胞代谢中具有相互依赖的作用。铁作为辅助因子 重要生化途径中的多种蛋白质和酶，但过量的铁可能会损害 通过催化活性氧的形成来破坏硫醇氧化还原稳态。细胞内硫醇- 反过来，二硫键平衡对于具有重要功能的半胱氨酸残基的蛋白质的活性至关重要，这 包括许多铁结合蛋白。三肽谷胱甘肽 (GSH) 和谷氧还蛋白 (Grx) 蛋白的功能 通过促进氧化还原反应和参与铁-硫醇氧化还原控制和铁稳态 硫（Fe-S）簇生物发生途径。我们之前在非致病性酿酒酵母和酿酒酵母方面的工作。 pombe 揭示了 Grxs 的一个亚类（称为单硫醇 Grxs）结合的分子机制 并传递 GSH 连接的 Fe-S 簇，将铁的生物利用度传递给转录因子 Aft1/Aft2 酿酒酵母和粟酒裂殖酵母中的 Php4 调节铁的获取和利用途径。此外，我们还有 使用分子遗传学和细胞生物学方法以及通过遗传进行的体内氧化还原测量 编码荧光氧化还原传感器来表征影响铁的 GSH 亚细胞运输途径 酿酒酵母的稳态和氧化还原调节。在这里，我们将通过研究影响来扩展这些发现 GSH 和 Grxs 对裂殖酵母转录因子 Fep1 的 Fe-S 结合特性和 DNA 结合亲和力的影响 在铁补充过程中抑制铁的吸收途径。此外，我们将定义铁的分子细节 致病性酵母（光滑念珠菌、白色念珠菌）中表达同源物的调节途径 单硫醇 Grxs、Aft1/2、Fep1 和 Php4，但其机制信息很少。与此同时，我们 将表征酵母细胞中亚细胞区室之间的 GSH:GSSG 通量，并测量 谷胱甘肽缺乏、过量或必需金属代谢运输受损。我们的创新方法 实现这些目标的方法是将酵母分子遗传学和细胞生物学技术与生物化学相结合， 结构和生物物理方法（紫外可见吸收和 CD 光谱、EXAFS、X 射线晶体学、 穆斯堡尔、EPR 和单细胞 ICP-TOF-MS）。体外生化、结构和生物物理研究将 用于探测铁传感途径中的蛋白质-蛋白质、金属-蛋白质和蛋白质-DNA 相互作用 揭示铁信号传导的分子细节并监测酵母群体中单细胞金属组学的变化 响应铁或谷胱甘肽代谢的变化。遗传学和细胞生物学研究测试了这些 分子相互作用和金属组变化影响铁的体内功能和动态定位 信号传导和 GSH 代谢因素。总体而言，这个多学科研究计划旨在梳理出 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。 通过研究致病性和非致病性真菌，我们将比较和对比不同的策略 适应氧化还原扰动和高/低铁环境。