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.

项目总结/摘要 铁硫簇组装或递送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是未知的。生物体进化出了由 专门的蛋白质一起运作，以最小化细胞毒性的方式有效地组装Fe-S簇， 暴露于其有毒成分（铁和硫化物离子）。这些蛋白质中有许多是动态的， 在抵抗结构分析的弱复合物中。我们正在研究这些蛋白质及其相互作用 在溶液中通过NMR光谱、小角X射线散射、化学交联 等温滴定量热法和功能性生物化学测定。我们正在应用这种方法来解决 重要的问题有关的机制组装的Fe-S簇在人类线粒体。我们 建立在最近的X射线结构的结果，为我们的调查提供了一个框架。去年两 独立的小组公布了（NFS 1-ISD 11-Acp）2复合物的X射线结构， 四级结构为了确定这两种不同的结构是否存在于溶液中，我们的方法是 在两种结构中不同的位点将19 F引入NFS 1。如果溶液中存在两种结构， 我们期望看到每一个的单独的19 F NMR信号。通过使用2D NMR实验，我们可以研究 探针的局部环境，并且如果观察到单独的信号，则能够确定 每个州。到目前为止，线粒体半胱氨酸脱硫酶复合物的所有结构和功能研究， 已经利用了通过在大肠杆菌中过表达人蛋白质NFS 1和ISD 11产生的复合物。coli细胞。的 得到的（NFS 1-ISD 11-Acp）2复合物含有E.大肠杆菌酰基载体蛋白（Acp）代替 人线粒体酰基载体蛋白（ACP）。我们将确定的结构和功能特性， （NFS 1-ISD 11-ACP）2和（NFS 1-ISD 11-ACP）2，不具有酰基链和具有不同长度的酰基链， 确定ACP和ACP之间的差异，并测试已发表的假设，即酰基载体蛋白在 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连接， 铁硫簇生物成因另一个目的是表征半胱氨酸的构象变化， 脱硫酶复合物伴随着簇组装和组装的转移的不同步骤， 将ISCU上的簇连接至共伴侣HSC 20。尽管众所周知ISCU存在两种相互转换 溶液中的构象（一种结构化，一种本质上是无序的），这两种构象的功能作用 状态仍有待阐明。通过用氟分别标记ISCU和NFS 1中的Trp残基，我们得到 确定19 F 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的角色的问题的实验奠定基础 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HSC 20。