Assembly of Biological Iron-Sulfur Clusters

描述(由申请人提供)： 项目概述铁-硫团簇存在于200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。因此，铁-硫的生物合成过程对几乎所有形式的生命都是必不可少的，并且在原核生物和真核生物中非常保守。已经出现了三种不同类型的铁-硫簇组装机制，称为NIF、ISC和SUF系统，在每种情况下，总的机制都涉及半胱氨酸脱硫酶介导的临时簇在支架蛋白上的组装，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，组装或转移机制都不是在分子水平上理解的。该项目的长期目标是在分子水平上了解使用NIF、ISC和SUF系统的铁-硫簇生物合成。阐明铁-硫簇生物合成的机制对于理解细胞铁的动态平衡以及与铁超载和线粒体呼吸链缺陷相关的人类疾病具有重要意义。该方法包括使用分子生物学技术来实现靶酶和蛋白质的大规模表达和/或特定位点的变化，生化和酶分析，X射线结晶学，以及生物物理光谱技术的应用(电子顺磁共振、吸收、磁性和自然圆二色谱、共振拉曼和穆斯堡尔谱)，这些技术可以探测在簇生物合成或转移到受体蛋白质过程中铁或铁-硫中心的性质和详细性质。目的是建立已知的四种铁硫簇支架蛋白的簇结合形式的结构和簇组装和转移的分子机制，确定每种类型的支架蛋白在铁硫蛋白成熟过程中的作用和特异性，并表征用于调控铁硫簇生物合成的机制。公共卫生相关性：铁-硫簇对人类健康的重要性在很大程度上源于它们在铁稳态中的关键作用，以及它们参与大量的酶和蛋白质，特别是线粒体呼吸链中的那些酶和蛋白质。从分子水平上了解铁-硫簇的生物发生对于理解各种人类疾病至关重要，这些疾病包括贫血、肌病和共济失调，这些疾病是由铁-硫簇组装蛋白的缺陷引起的。

DESCRIPTION (provided by applicant): Project Summary Iron-sulfur clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of iron-sulfur biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of iron-sulfur cluster assembly machinery have emerged, termed the NIF, ISC and SUF systems, and 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 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 using the NIF, ISC and SUF systems. Elucidating the mechanism of iron-sulfur cluster biosynthesis is central to understanding cellular iron homeostasis and thereby human diseases associated with iron-overload 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, X-ray crystallography, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, absorption, magnetic and natural circular dichroism, resonance Raman, and M"ssbauer) that can probe the nature and detailed properties of iron or iron-sulfur centers during cluster biosynthesis or transfer to acceptor proteins. The objectives are to establish the structure of cluster-bound forms and the molecular mechanism of cluster assembly and transfer for each the four known types of iron-sulfur cluster scaffold proteins, identify the roles and specificity of each type of scaffold protein in the maturation of iron-sulfur proteins, and characterize the mechanisms used to regulate iron-sulfur cluster biosynthesis. PUBLIC HEALTH RELEVANCE: The importance of iron-sulfur clusters to human health stems largely from their crucial role in iron homeostasis and their involvement in a large number of enzymes and proteins, particularly those in the mitochondrial respiratory chain. A molecular-level understanding of iron-sulfur cluster biogenesis is crucial for understanding a variety of human diseases involving anemias, myopathies and ataxias that arise from defects in iron-sulfur cluster assembly proteins.