Assembly and Repair of Biological Iron-Sulfur Clusters

描述(申请人提供)：铁-硫团簇存在于300多种不同类型的酶或蛋白质中，构成了最古老、最普遍和结构最多样化的生物假体基团之一。然而，最常见的簇合物，立方烷型[Fe4S4]簇合物，对氧化降解特别敏感。因此，铁硫的生物合成和修复过程对几乎所有的有氧生命形式都是必不可少的，并且在原核生物和真核生物中非常保守。细菌中已经出现了三种不同类型的铁-硫簇组装机制，分别称为NIF、ISC和SUF系统，ISC和SUF系统分别构成了真核细胞线粒体和质体铁-硫簇组装机制的基础。在每种情况下，整个机制都涉及半胱氨酸脱硫酶介导的支架蛋白上的瞬时簇组装，以及随后预先形成的簇或簇片段转移到载脂蛋白。然而，在任何情况下，组装、修复或转移机制都不是在分子水平上理解的。这个项目的长期目标是从分子水平理解铁-硫团簇 使用NIF、ISC和SUF系统的生物合成和修复，以及辅助蛋白，如单硫醇谷氧还蛋白和硫氧还蛋白样NFU蛋白。阐明铁-硫簇生物合成和修复的机制对于理解细胞铁稳态以及与铁超载、氧化应激和线粒体呼吸链缺陷相关的人类疾病至关重要。这种方法包括使用分子生物学技术来实现目标酶和蛋白质的大规模表达和/或特定部位的变化，生化和酶分析，以及生物物理光谱技术的应用(电子顺磁共振、紫外可见吸收、圆二色谱和磁诱导圆二色谱、共振拉曼、穆斯堡尔谱和质谱学)，这些技术可以探测在簇生物合成、降解、修复或转移到受体蛋白质过程中铁或铁硫中心的性质、连接和详细性质。目标是建立 铁-硫簇的组装、降解、修复和转移的分子机制，簇转移相对于受体蛋白的特异性，以及铁-硫蛋白调节细胞铁稳态的途径。

DESCRIPTION (provided by applicant): Iron-sulfur clusters are present in more than 300 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. However, the most common type of cluster, the cubane-type [Fe4S4] cluster, is particularly sensitive to oxidative degradation. Hence, the process of iron-sulfur biosynthesis and repair is essential to almost all aerobic life forms and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of iron-sulfur cluster assembly machinery have emerged in bacteria, termed the NIF, ISC and SUF systems, and the ISC and SUF systems form the basis of the eukaryotic mitochondrial and plastid iron-sulfur cluster assembly machineries, respectively. In each case the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of preformed clusters or cluster fragments to apo proteins. However, in no case is the assembly, repair or transfer mechanism understood at the molecular level. The long-term goal of this project is a molecular-level understanding of iron-sulfur cluster biosynthesis and repair using the NIF, ISC and SUF systems, and accessory proteins such as monothiol glutaredoxins and thioredoxin-like Nfu proteins. Elucidating the mechanism of iron-sulfur cluster biosynthesis and repair is central to understanding cellular iron homeostasis and thereby human diseases associated with iron-overload, oxidative stress and defects in the mitochondrial respiratory chain. The approach involves using molecular biology techniques to effect large scale expression and/or site-specific changes in the target enzymes and proteins, biochemical and enzymatic assays, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, UV-visible absorption, circular dichroism, and magnetically-induced circular dichroism, resonance Raman, M¿ssbauer, and mass spectrometry) that can probe the nature, ligation and detailed properties of iron or iron-sulfur centers during cluster biosynthesis, degradation, repair or transfer to acceptor proteins. The objectives are to establish the molecular mechanisms of assembly, degradation, repair, and transfer of iron-sulfur clusters, the specificity of cluster transfer with respect to acceptor proteins, and the means by which iron-sulfur proteins regulate cellular iron homeostasis.