Biogenesis of human mitochondrial iron-sulfur proteins

人类线粒体铁硫蛋白的生物合成

• 批准号: 10001537
• 负责人: JOHN LUTE MARKLEY
• 金额: $ 35.94万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001537
• 关键词: Acyl Carrier Protein; Address; Affect; Aging; Apoproteins; Binding; Binding Sites; Biochemical; Biogenesis; Biological Assay; Calorimetry; Cells; Chemicals; Complex; Cysteine; Defect; Disease; Dissociation; Environment; Escherichia coli; Exhibits; Exposure to; Face; Fatty Acids; Ferredoxin; Fluorine; Generations; Growth; Hand; Heat-Shock Proteins 70; Human; In Vitro; Investigation; Ions; Iron; Iron-Sulfur Proteins; Label; Length; Link; Methods; Mitochondria; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; NMR Spectroscopy; Organism; Peptides; Play; Process; Property; Proteins; Publishing; Reaction; Reporting; Roentgen Rays; Role; Scaffolding Protein; Signal Transduction; Site; Source; Structure; Sulfides; Sulfur; Sulofenur; Testing; Titrations; X-Ray Crystallography; acyl group; crosslink; cysteine desulfurase; design; experimental study; fatty acid metabolism; frataxin; human disease; improved; overexpression; protein complex

PROJECT SUMMARY/ABSTRACT Defects in iron-sulfur cluster assembly or delivery underlie many human diseases and aging processes, yet the detailed mechanisms for these processes are still unknown. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe-S clusters efficiently in a way that minimizes cellular exposure to their toxic constituents (iron and sulfide ions). Many of these proteins are dynamic and participate in weak complexes that have resisted structural analysis. We are studying these proteins and their interactions in solution by a combination of NMR spectroscopy, small angle X-ray scattering, chemical crosslinking, isothermal titration calorimetry, and functional biochemical assays. We are applying this approach to address important questions regarding the mechanism of assembly of Fe-S clusters in human mitochondria. We are building on the results of recent X-ray structures that provide a framework for our investigations. Last year, two independent groups published X-ray structures of the (NFS1-ISD11-Acp)2 complex that exhibited very different quaternary structures. To determine if these two different structures exist in solution, our approach is to introduce 19F into NFS1 at a site that is different in the two structures. If two structures are present in solution, we expect to see separate 19F NMR signals for each. By using 2D NMR experiments, we can investigate the local environment of the probe and, if separate signals are observed may be able to determine the lifetimes of each state. To date, all structural and functional studies of the mitochondrial cysteine desulfurase complex have utilized complexes produced by overexpressing the human proteins NFS1 and ISD11 in E. coli cells. The resulting (NFS1-ISD11-Acp)2 complex contains the holo-form of E. coli acyl carrier protein (Acp) in place of human mitochondrial acyl carrier protein (ACP). We will determine structural and functional properties of (NFS1-ISD11-Acp)2 and (NFS1-ISD11-ACP)2 without an acyl chain and with acyl chains of different lengths to identify differences between Acp and ACP and to test the published hypothesis that acyl carrier protein in the cysteine desulfurase complex provides a regulatory link coordinating mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis. Another aim is to characterize the conformational changes in the cysteine desulfurase complex that accompany different steps in cluster assembly and the transfer of the assembled cluster on ISCU to the co-chaperone HSC20. Although it is known that ISCU populates two interconverting conformations in solution (one structured and one intrinsically disordered), the functional roles of these two states remain to be elucidated. By separately labeling Trp residues in ISCU and NFS1 with fluorine, we have determined that 19F NMR enables the observation of different conformational states. These preliminary results set the stage for experiments designed to answer questions about the roles of the structured and dynamic states of ISCU and how assembled clusters are transferred from the cysteine desulfurase complex to HSC20.

项目概要/摘要 铁硫簇组装或传递的缺陷是许多人类疾病和衰老过程的基础，然而 这些过程的详细机制仍然未知。生物体已经进化出机械，包括 专门的蛋白质一起运作，以最大限度地减少细胞的方式有效地组装 Fe-S 簇 接触其有毒成分（铁和硫离子）。许多这些蛋白质是动态的并参与 在抵抗结构分析的弱配合物中。我们正在研究这些蛋白质及其相互作用 通过 NMR 光谱、小角 X 射线散射、化学交联的组合，在溶液中 等温滴定量热法和功能生化测定。我们正在应用这种方法来解决 关于人类线粒体中 Fe-S 簇组装机制的重要问题。我们是 以最近的 X 射线结构结果为基础，为我们的研究提供了框架。去年，两 独立小组发表了 (NFS1-ISD11-Acp)2 复合物的 X 射线结构，该结构表现出截然不同的特征 四级结构。为了确定溶液中是否存在这两种不同的结构，我们的方法是 在两个结构不同的站点将 19F 引入 NFS1。如果溶液中存在两种结构， 我们期望看到每个信号单独的 19F NMR 信号。通过使用二维核磁共振实验，我们可以研究 探头的局部环境，如果观察到单独的信号，则可以确定探头的寿命 每个州。迄今为止，线粒体半胱氨酸脱硫酶复合物的所有结构和功能研究 利用在大肠杆菌细胞中过表达人类蛋白质 NFS1 和 ISD11 产生的复合物。这 所得 (NFS1-ISD11-Acp)2 复合物包含大肠杆菌酰基载体蛋白 (Acp) 的完整形式，以代替 人线粒体酰基载体蛋白（ACP）。我们将确定结构和功能特性 (NFS1-ISD11-Acp)2 和 (NFS1-ISD11-ACP)2 不带酰基链且具有不同长度的酰基链 确定 Acp 和 ACP 之间的差异，并检验已发表的假设，即酰基载体蛋白 半胱氨酸脱硫酶复合物提供了协调线粒体脂肪酸合成的调节链接 铁硫簇生物发生。另一个目的是表征半胱氨酸的构象变化 脱硫酶复合物伴随簇组装和组装转移的不同步骤 ISCU 上的簇到辅助伴侣 HSC20。尽管众所周知 ISCU 填充了两个互转换 溶液中的构象（一种是结构化的，一种是本质上无序的），这两种构象的功能作用 各国仍有待阐明。通过分别用氟标记 ISCU 和 NFS1 中的色氨酸残基，我们得到 确定 19F NMR 能够观察不同的构象状态。这些初步结果 为旨在回答有关结构化和动态角色的问题的实验奠定了基础 ISCU 的状态以及组装的簇如何从半胱氨酸脱硫酶复合物转移到 HSC20。