Mitochondrial cysteine desulfurase

线粒体半胱氨酸脱硫酶

• 批准号: 7937766
• 负责人: ANDREW B. DANCIS
• 金额: $ 33.16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937766
• 关键词: Active Sites; Anemia; Bacteria; Biochemical Genetics; Biogenesis; Cell Survival; Cells; Complex; Cysteine; DNA Repair; Data; Defect; Disease; Enzymes; Eukaryotic Cell; Exhibits; Friedreich Ataxia; Functional disorder; Genes; Genome Stability; Human; Iceberg; In Vitro; Inner mitochondrial membrane; Iron; Iron-Sulfur Proteins; Life; Malignant Neoplasms; Mammals; Membrane; Methods; Mitochondria; Mitochondrial Myopathies; Mitochondrial Proteins; Mutation; Nature; Nerve Degeneration; Nuclear; Physiological; Process; Proteins; Regulation; Role; Saccharomyces cerevisiae; Scaffolding Protein; Sideroblastic Anemia; Sulfides; Sulfur; Testing; Time; Transfer RNA; Yeasts; cysteine desulfurase; in vivo; mutant; persulfides; prevent; protein complex; research study; trafficking; yeast protein

Cysteine desulfurases perform essential functions in releasing sulfur from cysteine, forming a covalent persulfide, and channeling the persulfide sulfur to biologically critical recipients e.g. ironsulfur (Fe-S) clusters and thiolated tRNAs. The activity of cysteine desulfurase cannot be replaced by exogenously supplied sulfide or by other sulfur metabolizing enzymes, and thus cysteine desulfurase is essential for cell viability. The eukaryotic cysteine desulfurase is encoded by a single nuclear gene and is found primarily in mitochondria. In Saccharomyces cerevisiae mitochondria, the cysteine desulfurase protein Nfs1 is assembled in a large protein complex with a recently identified special accessory protein called Isd11. We found that Isd11 is required for Nfs1 cysteine desulfurase activity. Thus, Nfs1 and Isd11 expressed together in bacteria are assembled into an active complex, while these components expressed separately are inactive. Here we propose to characterize the active Nfs1/Isd11 complex, and regulation of its cysteine desulfurase activity by Fe-S cluster scaffold proteins, Isu. Aim 1 is to determine the role of yeast Isd11 in the active Nfs1/Isd11 cysteine desulfurase complex. Experiments will be performed using bacterial expressed and purified proteins, and also in a more physiological context using isolated intact mitochondria. Aim 2 is to characterize formation of persulfide sulfur on Nfs1/Isd11 in isolated intact mitochondria, with focus on the regulatory effect of the D37A mutation of Isu. The significance derives from the essential role of cysteine desulfurase for viability of all human cells. The enzyme is found primarily in mitochondria and is required for Fe-S cluster synthesis and tRNA thiolation in mitochondria, both are essential processes. The identification of specific diseases arising from defective Fe-S cluster assembly (e.g. Friedreich's ataxia, sideroblastic anemia, and mitochondrial myopathy) represents the tip of the iceberg. The recent discovery of a key role of Fe-S clusters in DNA repair and genome stability suggests that cysteine desulfurases are involved in these processes.

半胱氨酸脱硫酶在从半胱氨酸中释放硫方面发挥着重要作用，形成 共价过硫化物，并将过硫化物硫引导至生物学关键受体，例如 铁硫 (Fe-S) 簇和硫醇化 tRNA。半胱氨酸脱硫酶的活性不能 被外源提供的硫化物或其他硫代谢酶取代，因此 半胱氨酸脱硫酶对于细胞活力至关重要。编码真核半胱氨酸脱硫酶 由单个核基因产生，主要存在于线粒体中。在酿酒酵母中 线粒体中，半胱氨酸脱硫酶蛋白 Nfs1 组装成一个大蛋白复合物， 最近发现了一种特殊的辅助蛋白，称为 Isd11。我们发现 Isd11 是必需的 Nfs1 半胱氨酸脱硫酶活性。因此，Nfs1 和 Isd11 在细菌中一起表达 组装成活性复合物，而这些单独表达的成分则无活性。 在这里，我们建议表征活性 Nfs1/Isd11 复合物及其半胱氨酸的调节 Fe-S 簇支架蛋白 Isu 的脱硫酶活性。目标 1 是确定酵母的作用 Isd11 位于活性 Nfs1/Isd11 半胱氨酸脱硫酶复合物中。实验将使用 细菌表达和纯化的蛋白质，以及在更生理的背景下使用分离的 完整的线粒体。目标 2 是表征 Nfs1/Isd11 上过硫化物硫的形成 分离出完整的线粒体，重点研究 Isu D37A 突变的调节作用。 其重要性源自半胱氨酸脱硫酶对所有人类生存能力的重要作用 细胞。该酶主要存在于线粒体中，是 Fe-S 簇合成所必需的 线粒体中的tRNA硫醇化，两者都是重要的过程。具体识别 由 Fe-S 簇组装缺陷引起的疾病（例如弗里德赖希共济失调、铁粒幼细胞 贫血和线粒体肌病）代表了冰山一角。最近发现了一个 Fe-S簇在DNA修复和基因组稳定性中的关键作用表明半胱氨酸脱硫酶 参与这些过程。