Role of redox-active thiols in the biogenesis of cytosolic and nuclear iron-sulfur proteins

铁硫蛋白（Fe/S）分布于线粒体、细胞质和细胞核中，在代谢、呼吸、蛋白质翻译、DNA合成和修复以及抗病毒防御中起着重要作用。它们的Fe/S簇在线粒体和胞质溶胶中通过复杂的机制组装并插入脱辅基蛋白中。细胞溶质Fe/S蛋白组装（CIA）机制包括十种已知的蛋白质，其在三个主要步骤中协助成熟过程。首先，在支架复合物（Cfd 1-Nbp 35）上合成[4Fe-4S]簇，其需要线粒体作为硫供体和用于还原的电子转移链。其次，Fe/S簇通过Nar 1和CIA靶向复合物以靶特异性方式转移到脱辅基蛋白。第三，Yae 1-Lto 1衔接子复合物将Fe/S蛋白Rli 1募集至CIA靶向复合物以进行靶特异性成熟。Cia 2是CIA靶向复合物的重要组成部分，含有最具反应性的细胞巯基（酵母中的Cys 161），是该项目的中心。在正在进行的资助期间，我们将Cia 2表征为一般的Fe/S蛋白生物发生因子，并在光谱上发现Cys 161在两个Cia 2单体之间协调[4Fe-4S]簇。基于这些（和其他）发现，我们现在将研究五个主题。首先，我们将在体外重建Cia 2 [4Fe-4S]簇的组装和位错途径，以阐明其他CIA因子参与其中。其次，我们将尝试积累Fe/S簇与Cia 2结合的体内证据，例如，通过检测其依赖于簇的二聚化。第三，我们将研究高反应性Cys 161是否经历任何其他翻译后修饰，如二硫键形成或谷胱甘肽化，特别是在低巯基还原酶活性的条件下，这是与胞质Fe/S蛋白生物合成缺陷。第四，我们将测试细菌Cia 2同系物是否也能够结合[4Fe-4S]簇。第五，我们将研究Cia 2如何在Fe/S簇转移和插入中建立其靶特异性。我们将利用我们最近的研究结果对两个人类亚型CIA 2A和CIA 2B，结合到不同的，短区域的抗病毒自由基SAM Fe/S蛋白蝰蛇蛋白。总的来说，我们的研究将有助于更好地了解Fe/S蛋白生物合成中最具反应性的细胞硫醇之一的机制作用。

Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol and nucleus, and fulfill essential tasks in metabolism, respiration, protein translation, DNA synthesis and repair as well as in antiviral defense. Their Fe/S clusters are assembled and inserted into apoproteins by complex machinery in both mitochondria and cytosol. The cytosolic Fe/S protein assembly (CIA) machinery comprises ten known proteins which assist the maturation process in three major steps. First, a [4Fe-4S] cluster is synthesized on a scaffold complex (Cfd1-Nbp35) which requires mitochondria as a sulfur donor and an electron transfer chain for reduction. Second, the Fe/S cluster is transferred to apoproteins by Nar1 and the CIA targeting complex in a target-specific manner. Third, the Yae1-Lto1 adapter complex recruits the Fe/S protein Rli1 to the CIA targeting complex for target-specific maturation. Cia2 is a crucial component of the CIA targeting complex, and contains one of the most reactive cellular thiol groups (Cys161 in yeast) that is in the center of this project. In the ongoing funding period, we have characterized Cia2 as a general Fe/S protein biogenesis factor and spectroscopically found that Cys161 coordinates a [4Fe-4S] cluster between two Cia2 monomers. Based on these (and other) findings, we now will investigate five topics. First, we will in vitro reconstitute the assembly and dislocation pathway of the Cia2 [4Fe-4S] cluster to elucidate which other CIA factors are involved. Second, we will try to accumulate in vivo evidence for Fe/S cluster binding to Cia2, e.g., by detecting its cluster-dependent dimerization. Third, we will study whether the hyper-reactive Cys161 undergoes any other post-translational modification such as disulfide bond formation or glutathionylation, especially under conditions of low thiol reductase activity which is associated with a cytosolic Fe/S protein biogenesis defect. Forth, we will test, if bacterial Cia2 homologs are also able to bind a [4Fe-4S] cluster. Fifth, we will investigate how Cia2 establishes its target specificity in Fe/S cluster transfer and insertion. We will exploit our recent findings on the two human isoforms CIA2A and CIA2B that bind to distinct, short regions of the antiviral radical-SAM Fe/S protein viperin. Collectively, our studies will contribute to a better molecular understanding of the mechanistic roles of one of the most reactive cellular thiols in Fe/S protein biogenesis.