Novel components of iron sulfur cluster biosynthesis pathways

描述(申请人提供)：铁硫(FeS)簇是古老的普遍存在的金属蛋白辅因子，其起源被认为位于早期厌氧生物圈的还原环境中。向有氧生命的转变给FeS团簇带来了几个问题，既限制了由于氧化而产生的铁的生物可利用性，又直接促进了具有氧物种的团簇的破坏，如超氧化物和过氧化氢，这些氧物种是有氧呼吸的副产品。这种敏感性已经被宿主防御机制所利用，宿主防御机制将FES簇作为促进细胞死亡的组件。FeS簇的组成成分铁和硫对细胞有很强的毒性，FeS簇是通过特定的生物合成途径形成的，如大肠杆菌中的iscRSUhscBAfdx操纵子和sufABCDSE操纵子。我们的长期目标是确定在模式原核生物中参与FeS簇生物合成的一整套因素，并从功能上表征它们在FeS簇形成中的作用。这项研究的目的是鉴定一种新的因子，CGFS型谷氧还蛋白，GrxD，它与大肠杆菌Suf FeS生物合成途径有关，并确定其在大肠杆菌中的作用。Suf途径似乎适应了胁迫条件下FeS簇的合成，如氧化胁迫和铁限制，并被证明是植物病原菌Erwinia chrysanhemi完全毒力所必需的。此外，FeS簇的生物合成过程是一个基本特征，在一些细菌物种中，Suf系统已被确定为唯一的FeS簇生物合成途径，如结核分枝杆菌，结核病的病原体。结核病每年导致大约200万人死亡，主要发生在发展中国家。近年来，对Suf体系的研究已经比较详细，然而，对ISC和Suf体系的研究尚未确定使Suf能够在应力条件下合成FeS团簇的特定适应性。此外，最近对参与FeS簇生物合成的这两个核心操纵子以外的因子的发现表明，其他未编码在Suf操纵子中的因子，如GrxD，可能与Suf系统一起作用，促进在胁迫条件下FeS簇的合成。我们的目标是结合生化分析、功能基因组学、蛋白质化学和分子生物学，(I)确定grxD和ISC系统突变体之间合成致死性的基础，以及在特定条件下这些遗传相互作用是否能被缓解，(Ii)表征GrxD的物理相互作用并确定其参与的蛋白质复合体的组成，以及结合伙伴对GrxD生化性质的影响，(Iii)具体评估GrxD转移其FES簇的能力，以及(Iv)确定GrxD与Suf和ISC系统的功能关联。 公共卫生相关性：项目叙述铁硫簇对生命是必不可少的，但容易受到氧化损伤，并成为哺乳动物系统促进细菌死亡的目标。Suf FES簇生物合成途径已适应于在胁迫条件下合成，并可能在细菌毒力期间的生存中发挥作用，是新型抗微生物药物的潜在靶点。我们的建议旨在描述一种新的因子，该因子可能与SuF系统一起工作，并可能负责其在应力条件下合成FeS的能力。

DESCRIPTION (provided by applicant): Iron-sulfur (FeS) clusters are ancient ubiquitous metalloprotein cofactors whose origins are thought to lay in the reducing environment of the early anaerobic biosphere. The transition to aerobic life has created several problems for FeS clusters by both limiting the bio-availability of iron due to oxidation and directly promoting the destruction of clusters with oxygen species such as superoxide and peroxide, which are by-products of aerobic respiration. This susceptibility has been leveraged by host defense mechanisms, which target FeS clusters as components through which to promote cell death. The components of FeS clusters, iron and sulfur, are highly toxic to cells and FeS clusters are formed by specific biosynthesis pathways such as the iscRSUhscBAfdx operon and sufABCDSE operon in Escherichia coli. Our long term goal is to determine a complete set of factors involved in FeS cluster biosynthesis in the model prokaryote E. coli and to functionally characterize their roles in FeS cluster formation. The objective of the proposed research is the characterization of a novel factor, the CGFS-type monothiol glutaredoxin, GrxD, which has been implicated as functioning with the E. coli Suf FeS biosynthesis pathway, and the determination of its role in E. coli. The Suf pathway appears to have adapted to a role in FeS cluster synthesis under stress conditions, such as oxidative stress and iron limitation, and has been shown to be necessary for complete virulence of the plant pathogen Erwinia chrysanthemi. Moreover, the process of FeS cluster biosynthesis is an essential trait, and the Suf system has been identified as the only FeS cluster biosynthesis pathway in some bacterial species, such as the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Tuberculosis kills approximately 2 million people a year, mainly in the developing world. The Suf system has been characterized in some detail in recent years, however, studies of both Isc and Suf systems have yet to identify specific adaptations which make Suf capable of FeS cluster synthesis under stress conditions. Moreover, recent identification of factors outside of these two core operons, which participate in FeS cluster biosynthesis, suggests that other factors not encoded in the Suf operon, such as GrxD, may work with the Suf system and facilitate the synthesis of FeS clusters under stress conditions. Using a combination of biochemical assays, functional genomics, protein chemistry and molecular biology our aims are to (i) determine the basis of synthetic lethality between grxD and Isc system mutants, and if these genetic interactions can be alleviated under specific conditions, (ii) characterize the physical interactions of GrxD and determine the composition of protein complexes in which it participates, and the effect of binding partners on the biochemical properties of GrxD, (iii) specifically assess the ability of GrxD to transfer its FeS cluster, and (iv) determine the functional association of GrxD to both Suf and Isc systems. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Iron sulfur clusters are essential for life, but are prone to oxidative damage and are targeted by mammalian systems to promote bacterial death. The Suf FeS cluster biosynthesis pathway has been adapted for synthesis under conditions of stress and may play a role in bacterial survival during virulence and is a potential target for novel antimicrobials. Our proposal is designed to characterize a novel factor which may work with the Suf system and may be responsible for its capacity for FeS synthesis under stress conditions.