Role of the mitochondrial Bol1 and Bol3 proteins in iron-sulfur cluster delivery to diverse recipient proteins

线粒体 Bol1 和 Bol3 蛋白在铁硫簇递送至各种受体蛋白中的作用

• 批准号: 311061741
• 负责人: Professor Dr. Roland Lill
• 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: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/311061741?language=en
• 关键词: Role; mitochondrial; Bol1; Bol3; proteins

The biogenesis of mitochondrial iron-sulfur (Fe/S) proteins is catalyzed by the conserved ISC assembly machinery and can be dissected into three major steps. First, a [2Fe-2S] cluster is synthesized de novo on the scaffold protein Isu1 by the sulfur donor Nfs1-Isd11-Acp1 and four other ISC factors. Second, the Fe/S cluster is released from Isu1 by a dedicated Hsp70 chaperone system, and transferred via the monothiol glutaredoxin Grx5 to [2Fe-2S] target proteins. Third, a transiently Grx5-bound [2Fe-2S] cluster is converted by the Isa1-Isa2-Iba57 proteins to a [4Fe-4S] cluster, which is transiently bound by the specific ISC targeting factors Nfu1 and Ind1, and finally inserted into recipient [4Fe-4S] proteins. Work within this project has identified the two yeast mitochondrial proteins Bol1 and Bol3 as additional ISC targeting factors that cooperatively facilitate specific maturation of lipoyl synthase and respiratory complex II. Human BOLA3 (Bol3 homolog) but not BOLA1 performs a similar targeting function. The solution structure of the two BOLA proteins was resolved in cooperation with L. Banci. Biochemical studies revealed rather different affinities of the two BOLA complexes with [2Fe-2S] cluster-bound GLRX5 (human Grx5 homolog), yet the physiological meaning of this observation remains unclear. Mutational analysis indicated that the [2Fe-2S] cluster is bound to the BOLA1-GLRX5 complex independently of three conserved His residues in BOLA1, leaving the exact architecture of the holo-complex open. Our work has raised the central question of why a [2Fe-2S] cluster-containing Bol-Grx5 complex is needed for [4Fe-4S] protein assembly. Further, based on the radically different BOL and GRX5 deletion phenotypes in both yeast and human cells (and in diseases), it became evident that the precise site of action of Bol-Grx5 within the complex Fe/S protein assembly pathway remains open. In the next funding period, we aim to reach a physiologically meaningful mechanistic understanding of Bol protein function that explains the involvement in [4Fe-4S] cluster insertion into mitochondrial apoproteins. We will test whether the Bol proteins exhibit an increased importance under oxidative stress conditions by examining their function and interacting partner proteins. We will reconstitute lipoyl synthase maturation in vitro to precisely define the mechanistic function of the Bol and Grx5 proteins. In support of this aim, the crystal structure of the Bol-Grx5 complexes will be solved. Finally, we will perform cell biological and biochemical studies to elucidate whether the Bol-Grx5 complexes act before or after the Isa-Iba57 proteins. A comprehensive mechanistic understanding of Bol protein function will be important for a molecular view of the BOLA3-associated mitochondrial disease MMDS2.

线粒体铁-硫(Fe/S)蛋白的生物发生由保守的ISC组装机制催化，可分为三个主要步骤。首先，由硫供体Nfs1-Isd11-Acp1和其他四个ISC因子在支架蛋白Isu1上从头合成[2Fe-2S]簇。其次，铁/S簇通过一个专门的HSP70伴侣系统从Isu1释放出来，并通过单硫醇谷氧还蛋白Grx5转移到[2Fe-2S]靶蛋白上。第三，瞬时结合Grx5的[2Fe-2S]簇被Isa1-Isa2-Iba57蛋白转化为[4Fe-4S]簇，该簇被特定的ISC靶向因子Nfu1和Ind1瞬时结合，最后插入受体[4Fe-4S]蛋白中。在这个项目中的工作已经确定了两个酵母线粒体蛋白Bol1和Bol3是额外的ISC靶向因子，它们协同促进硫酰合成酶和呼吸复合体II的特异性成熟。人BOLA3(Bol3同源物)而不是BOLA1具有类似的靶向功能。与L.Banci合作，对这两种BoLA蛋白的溶液结构进行了解析。生化研究表明，这两个BOLA络合物与[2Fe-2S]簇结合的GLRX5(人Grx5同系物)具有相当不同的亲和力，但这一观察到的生理意义尚不清楚。突变分析表明，[2Fe-2S]簇与BOLA1-GLRX5复合体结合，独立于BOLA1中三个保守的His残基，从而使全息复合体的确切结构开放。我们的工作提出了一个中心问题，即为什么[4Fe-4S]蛋白质组装需要一个含有[2Fe-2S]簇的BOL-Grx5络合物。此外，基于在酵母和人类细胞中(以及在疾病中)BOL和GRX5缺失表型的根本不同，很明显BOL-Grx5在复杂的Fe/S蛋白组装途径中的确切作用部位仍然是开放的。在下一个资金阶段，我们的目标是对BOL蛋白的功能达成具有生理意义的机械理解，以解释[4Fe-4S]簇插入线粒体载脂蛋白的过程。我们将通过检查BOL蛋白的功能和与伙伴蛋白的相互作用来测试BOL蛋白在氧化应激条件下是否显示出更高的重要性。我们将重建脂酰合成酶的体外成熟，以准确地确定BOL和Grx5蛋白的机制功能。为了支持这一目标，BOL-Grx5络合物的晶体结构将得到解决。最后，我们将进行细胞生物学和生化研究，以阐明BOL-Grx5复合体是在Isa-Iba57蛋白之前还是之后发挥作用。对BOL蛋白功能的全面机制了解将对BOLA3相关线粒体疾病MMDS2的分子观点具有重要意义。