Elucidating the mechanisms of [4Fe-4S] cluster insertions into the cytosolic iron-sulfur protein assembly component Nar1

阐明 [4Fe-4S] 簇插入胞质铁硫蛋白组装成分 Nar1 的机制

• 批准号: 428147805
• 负责人: Dr. Joseph James Braymer, Ph.D.
• 金额: --
• 依托单位: Institut für Klinische Zytobiologie und Zytopathologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/428147805?language=en
• 关键词: Elucidating; mechanisms; 4Fe; 4S; cluster

Dedicated cofactor trafficking and insertion pathways are an important aspect in a multitude of biosynthetic and metabolic reactions. The cell has various mechanisms for specifically determining which cofactor gets inserted into which protein, even in structurally similar proteins with different cofactor occupancies. A majority of metal or bioinorganic cofactor transfer reactions are governed by protein interactions, yet mechanistic detail remains unclear for many metalloproteins in how they pass cofactors from one protein to the next, especially at sites buried within proteins. This applies to one of the most ubiquitous types of cofactors found in bacteria, archaea, and eukaryotes, namely iron sulfur (Fe/S) clusters. In the model eukaryotic organism Saccharomyces cerevisiae, 18 mitochondrial Fe/S cluster (ISC) assembly proteins and 11 cytosolic Fe/S protein assembly (CIA) components create an extensive cellular protein network, which synthesizes, traffics, and inserts Fe/S clusters into target apoproteins. An interesting aspect of the CIA machinery in eukaryotes is that many of the trafficking factors themselves bind more than one Fe/S cluster and it remains poorly understood how the CIA components themselves are matured. For example, Nar1 is peculiar because it has been proposed to be a [4Fe-4S] cluster trafficking mediator of the CIA pathway via a labile [4Fe-4S] cluster and at the same time a target apoprotein that requires a buried, non-transferable [4Fe-4S] cluster. Furthermore, Nar1 is homologous to bacterial and algal [FeFe]-hydrogenases, but doesn’t have the characteristic hydrogenase function. Why a hydrogenase-like protein has evolved as an essential eukaryotic Fe/S cluster trafficking protein remains one of the most challenging questions to address in the Fe/S cluster biogenesis field. The aim of this proposal is to use a combination of in vivo, in vitro, and biophysical studies to assess mechanisms of [4Fe-4S] cluster insertions into Nar1 and to use this information to determine the physiological function of Nar1 in the CIA pathway. S. cerevisiae will be used as a tractable organism to study the function of Nar1 and the importance of partner binding proteins in Nar1 maturation. Details into Nar1 maturation will guide reconstitution reactions involving Fe/S cluster transfer to and from Nar1, quantified via isotopically-enriched Fe/S clusters. Biophysical characterization of native Nar1 via protein-protein interaction assays, spectroscopic Fe/S cluster analyses, and X-ray crystallographic structure determination will also be pursued, in part, through essential collaborations in the SPP. Assigning the molecular function of Nar1 will advance our understanding of the multiple ways that Fe/S clusters can be transferred or inserted into apoproteins. Subsequently, this will also enhance our understanding of how disruptions in Fe/S cofactor trafficking lead to Fe/S protein assembly diseases.

专门的辅因子运输和插入途径是许多生物合成和代谢反应中的重要方面。细胞具有多种机制来特异性地确定哪种辅因子插入哪种蛋白质，即使在结构相似的蛋白质中具有不同的辅因子占据。大多数金属或生物无机辅因子转移反应受蛋白质相互作用的控制，但许多金属蛋白如何将辅因子从一种蛋白质传递到下一种蛋白质的机制细节仍然不清楚，特别是在蛋白质内埋藏的位点。这适用于细菌、古细菌和真核生物中最普遍存在的辅因子类型之一，即铁硫（Fe/S）簇。在模式真核生物酿酒酵母（Saccharomyces cerevisiae）中，18个线粒体Fe/S簇（ISC）组装蛋白和11个胞质Fe/S蛋白组装（CIA）组件创建了广泛的细胞蛋白网络，该网络合成、运输和插入Fe/S簇到靶脱辅基蛋白中。真核生物中CIA机制的一个有趣的方面是，许多运输因子本身结合一个以上的Fe/S簇，并且CIA组分本身是如何成熟的仍然知之甚少。例如，Nar 1是独特的，因为它被认为是CIA途径的[4Fe-4S]簇运输介体，通过不稳定的[4Fe-4S]簇，同时是需要埋藏的不可转移的[4Fe-4S]簇的靶脱辅基蛋白。此外，Nar 1与细菌和藻类的[FeFe]-氢化酶同源，但不具有特征性的氢化酶功能。为什么氢化酶样蛋白已经进化为真核生物Fe/S簇运输蛋白的一个重要组成部分，仍然是Fe/S簇生物成因领域最具挑战性的问题之一。本提案的目的是使用体内，体外和生物物理学研究的组合，以评估机制的[4Fe-4S]簇插入到Nar 1和使用此信息来确定Nar 1的CIA途径中的生理功能。S.酿酒酵母将被用作一种易处理的生物体，以研究Nar 1的功能和伴侣结合蛋白在Nar 1成熟中的重要性。Nar 1成熟的细节将指导涉及Fe/S簇转移到Nar 1和从Nar 1转移的重建反应，通过同位素富集的Fe/S簇进行定量。通过蛋白质-蛋白质相互作用测定，光谱Fe/S簇分析和X-射线晶体结构测定的天然Nar 1的生物物理特性也将部分通过SPP中的重要合作来进行。阐明Nar 1的分子功能将有助于我们理解Fe/S簇转移或插入脱辅基蛋白的多种方式。随后，这也将增强我们对Fe/S辅因子运输中断如何导致Fe/S蛋白组装疾病的理解。