Mechanisms of Fungal Iron Regulation and Thiol Redox Metabolism

项目总结 铁和硫醇氧化还原动态平衡在细胞代谢中具有相互依赖的作用。铁是影响人体健康的一个辅因 在基本的生化途径中有各种各样的蛋白质和酶，但过量的铁可能会对 通过催化形成破坏硫醇氧化还原动态平衡的活性氧物种。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是至关重要的，这 包括许多铁结合蛋白。三肽谷胱甘肽(GSH)和谷氧还蛋白(GRX)的功能 通过促进氧化还原反应和参与铁平衡，在硫醇氧化还原控制和铁的动态平衡中共同作用. 硫(铁-S)聚集型生物发生途径。我们之前在非致病酵母S.cerevisiae和S. Pombe已经揭示了Grxs的一个亚类，即单硫醇Grxs结合的分子机制 并将谷胱甘肽连接的铁-S簇传递给转录因子Aft1/Aft2。 S.cerevisiae和Php4调控铁的获取和利用途径。此外，我们还拥有 使用分子遗传学和细胞生物学方法，结合体内氧化还原测量，通过遗传学- 编码的荧光氧化还原传感器用于表征影响两种铁的GSH亚细胞转运途径 酿酒酵母的动态平衡和氧化还原调节。在这里，我们将通过研究以下影响来扩展这些发现 谷胱甘肽和谷胱甘肽对庞氏链霉菌转录因子Fep1铁-S结合特性和亲和力的影响 这抑制了铁补充过程中的铁吸收途径。此外，我们还将定义铁的分子细节 致病酵母(光滑假丝酵母菌、白色念珠菌)表达同源蛋白的调控途径 单硫醇Grxs、Aft1/2、Fep1和Php4，但其机理信息很少。同时，我们 将表征酵母细胞亚细胞间的GSH：GSSG通量，并测量 谷胱甘肽缺乏、过剩或损害对必需金属代谢的运输。我们的创新方法 实现这些目标的关键是将酵母分子遗传学和细胞生物学技术与生化、 结构和生物物理方法(紫外可见吸收和CD光谱、EXAFS、X射线结晶学、 穆斯堡尔、EPR和单细胞电感耦合等离子体质谱(ICPAF-MS)。体外生化、结构和生物物理研究将 用于探测铁感应通路中蛋白质-蛋白质、金属-蛋白质和蛋白质-DNA的相互作用 揭示铁信号的分子细节并监测酵母种群中单细胞金属组的变化 对铁或谷胱甘肽代谢变化的反应。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学变化影响铁的体内功能和动态定位 信号和谷胱甘肽代谢因子。总体而言，这个多学科研究项目旨在梳理出 在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。 通过对致病真菌和非致病真菌的研究，我们将比较和对比不同的策略 适应氧化还原扰动和高铁/低铁环境。

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.