Characterizing Isu Scaffolding Protein and ISC Multiprotein Complex Structure and

Isu 支架蛋白和 ISC 多蛋白复合物结构的表征

• 批准号: 8783325
• 负责人: Stephen Dzul
• 金额: $ 3.85万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783325
• 关键词: Active Sites; Address; Affect; Alzheimer' s Disease; American; Anabolism; Biochemical; Biochemical Pathway; Biological Assay; Cessation of life; Characteristics; Chemistry; Circular Dichroism; Complex; Cysteine; DNA; DNA Sequence Rearrangement; Development; Disease; Drug Design; Eukaryota; Friedreich Ataxia; Future; Genes; Genetic; Goals; Homeostasis; Human; Huntington Disease; Impaired health; Individual; Inherited Spinocerebellar Degenerations; Introns; Iron; Iron Overload; Laboratories; Lead; Link; Metabolism; Metals; Methodology; Mitochondria; Molecular; Molecular Chaperones; Multiprotein Complexes; Mutagenesis; Neurodegenerative Disorders; Oxidative Stress; Oxygen; Parkinson Disease; Pathogenesis; Pathway interactions; Patients; Phenotype; Plasmids; Process; Production; Proteins; Proteus; Public Health; Research; Roentgen Rays; Scaffolding Protein; Site; Spectrum Analysis; Structure; Sulfur; Symptoms; System; Testing; Trinucleotide Repeat Expansion; Work; X-Ray Crystallography; Yeasts; absorption; analog; cysteine desulfurase; disulfide bond; experience; frataxin; in vivo; insight; iron metabolism; mutant; novel; novel therapeutics; persulfides; protein complex; protein expression; public health relevance; treatment strategy; yeast protein

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is a debilitating cardio- and neurodegenerative disease currently affecting 6,000 Americans. Recent progress in FRDA research has linked pathogenesis of FRDA to the absence of Frataxin (Fxn), a protein that regulates iron-sulfur cluster (Fe-S) biosynthesis, possibly by serving as a regulator of the pathway or as an iron chaperone that delivers metal to protein partners in the Fe-S cluster bioassembly (ISC) pathway. In eukaryotes, the predominant ISC assembly pathway is located within the mitochondria and driven by coordinated association of the bioassembly scaffold protein (Isu1), a cysteine desulfurase that provides atomic sulfur (Nfs1), an accessory protein (Isd11) and frataxin (Yfh1). The long-range goal of the Stemmler laboratory is to characterize the molecular details of the Fe-S cluster assembly pathway in eukaryotes. The objective of this proposal is to characterize the active site of the Isu1 scaffolding protein in addition to the overll ISC assembly complex. The findings of this study will help in the development strategies to augment the activity of this pathway that is deficient when frataxin is absent. All studies will be performed in the yeast system, where the genetics of Fe-S cluster assembly have been well established. Our preliminary hypothesis is that Fxn provides two Fe (II) atoms and Nfs provides two sulfur atoms at distinct sites on Isu1. Subsequent Isu1 rearrangement brings both sulfur and Fe (II) species together at Isu1's active site to accomplish cluster assembly. This proposal follows two specific aims. Firstly, Isu1 will be manipulated to identify and characterize critical residues essential for Fe-S assembly. Secondly, a multi-protein expression plasmid, with all 4 ISC complex proteins present, will be utilized to determine how Isu1, Nfs1, Isd11 assemble in the presence/absence of frataxin. As a whole, this proposal will facilitate the development of novel therapeutic strategies for FRDA.

描述（由申请人提供）：弗里德赖希共济失调 (FRDA) 是一种使人衰弱的心脏和神经退行性疾病，目前影响 6,000 名美国人。 FRDA 研究的最新进展将 FRDA 的发病机制与 Frataxin (Fxn) 的缺乏联系起来，Fxn 是一种调节铁硫簇 (Fe-S) 生物合成的蛋白质，可能是通过充当该途径的调节剂或作为铁伴侣来提供Fe-S 簇生物组装 (ISC) 途径中的金属与蛋白质伙伴。在真核生物中，主要的 ISC 组装途径位于线粒体内，并由生物组装支架蛋白 (Isu1) 的协调关联驱动，Isu1 是一种提供原子硫 (Nfs1) 的半胱氨酸脱硫酶、辅助蛋白 (Isd11) 和 frataxin (Yfh1)。 Stemmler 实验室的长期目标是表征真核生物中 Fe-S 簇组装途径的分子细节。该提案的目的是除了整体 ISC 组装复合体之外，还表征 Isu1 支架蛋白的活性位点。这项研究的结果将有助于制定策略，以增强当 frataxin 不存在时缺乏的该途径的活性。所有的研究都将 在酵母系统中进行，其中 Fe-S 簇组装的遗传学已经得到很好的建立。我们的初步假设是 Fxn 提供两个 Fe (II) 原子，Nfs 在 Isu1 的不同位点提供两个硫原子。随后的 Isu1 重排将硫和 Fe (II) 物质聚集在 Isu1 的活性位点以完成簇组装。该提案遵循两个具体目标。首先，将操纵 Isu1 来识别和表征 Fe-S 组装所必需的关键残基。其次，将使用包含所有 4 个 ISC 复合蛋白的多蛋白表达质粒来确定 Isu1、Nfs1、Isd11 在存在/不存在 frataxin 的情况下如何组装。总体而言，该提案将促进 FRDA 新型治疗策略的开发。