Mitochondrial and cytosolic maturation of human FeS-dependent molybdenum cofactor synthesis MOCS1A proteins and links between both cofactor pathways

含钼酶催化全球生命循环中的关键反应。它们依赖于Mo辅助因子（Moco）和FeS簇的生物合成，这是Moco合成的第一步所必需的，GTP转化为单磷酸环蝶呤（cPMP），并在Mo酶中起辅助因子的作用。Moco的生物合成缺陷导致一种严重的先天性代谢错误，主要由亚硫酸盐氧化酶活性丧失引起，其特征是迅速进展的神经变性和儿童死亡。我们已经为MOCS1基因突变患者开发了第一种治疗方法，该基因产生编码两种蛋白质的选择性剪接转录物。MOCS1A需要两个[4Fe-4S]簇，属于自由基SAM酶超家族。第二个翻译产物为MOCS1AB蛋白，其MOCS1A结构域无活性，c端MOCS1B结构域具有催化活性。在之前的资助期内，我们能够证明两个MOCS1A剪接变体的细胞质定位，而另外两个MOCS1A剪接变体以及所有MOCS1AB剪接变体定位于线粒体。此外，令人惊讶的是，在导入过程中，我们发现MOCS1AB被蛋白水解裂解，产生一个小的MOCS1B蛋白，该蛋白类似于MOCS1A定位于线粒体基质。MOCS1B的体内活性可以通过SPP内的一个患者病例来证实，其中纯合子移码突变导致MOCS1B单独表达。在接下来的资助期内，我们的目标是解决人类Moco合成和Moco缺乏（MoCD）中未解决的两个关键方面。首先，我们计划研究细胞质和线粒体MOCS1A蛋白的差异，因为这两类蛋白都是在生理条件下表达的，并已被证明在Moco生物合成中发挥作用。因此，我们将根据我们建立的厌氧净化和cPMP体外合成方案，对不同的MOCS1A变体进行生化表征，包括动力学参数的测定。这还将包括MOCS1A （O. Einsle）的结晶，以及从sf9昆虫细胞培养中纯化的细胞质和线粒体MOCS1A蛋白的EPR-（与A. Pierik合作）和Mössbauer-spectroscopy （V. sch<s:1> nemann）。在这个项目的第二部分，我们研究了FeS集群生物发生和Moco合成之间的细胞和稳态联系。我们的目标是通过与不同组的SPP合作，监测细胞培养、小鼠系和患者样本中的Moco酶活性和Moco生物合成，了解铁稳态和fes集群生物发生损伤对Moco生物合成的影响（R. Lill， C. Berndt， A. Steinbicker）。反之亦然，我们将探索亚硫酸盐氧化酶活性的变化如何影响在我们实验室创建的新型亚硫酸盐氧化酶缺陷小鼠系中的fes簇生物发生。

Molybdenum (Mo)-containing enzymes catalyze key reactions in the global cycles of life. They are dependent on the biosynthesis of the Mo cofactor (Moco) as well as FeS clusters, which are required in the first step of Moco synthesis, the conversion of GTP into cyclic pyranopterin monophosphate (cPMP) and function as cofactors in Mo enzymes. A defect in the biosynthesis of Moco leads to a severe form of inborn error in metabolism, mainly caused by the loss of sulfite oxidase activity characterized by rapidly progressing neurodegeneration and childhood death. We have developed a first therapy for patients with mutations in the MOCS1 gene that produces alternatively spliced transcripts encoding for two proteins. MOCS1A requires two [4Fe-4S] clusters and belongs to the superfamily of radical SAM enzymes. The second translation product represents the MOCS1AB protein, with an inactive MOCS1A domain and a C-terminal MOCS1B domain with catalytic activity. During the previous funding period we were able to demonstrate cytosolic localization of two MOCS1A splice variants, while the two other MOCS1A splice variants, as well as all MOCS1AB splice variants localized to mitochondria. Furthermore and surprisingly, we found during the import process that MOCS1AB is proteolytically cleaved resulting in a small MOCS1B protein, which localizes similar to MOCS1A to the mitochondrial matrix. In vivo activity of MOCS1B could be confirmed by a patient case investigated within the SPP, where a homozygous frameshift mutation resulted in separate expression of MOCS1B. In the following funding period we aim to address two key aspects unresolved in human Moco synthesis and Moco deficiency (MoCD). First, we plan to investigate the differences of cytosolic and mitochondrial MOCS1A proteins, given that both classes of proteins are expressed under physiological conditions and have been shown to play a role in Moco biosynthesis. Therefore we will biochemically characterize different MOCS1A variants including the determination of kinetic parameters, based on our established anaerobic purification and cPMP in vitro synthesis protocols. This will also include crystallization of MOCS1A (O. Einsle), as well as EPR- (with A. Pierik) and Mössbauer-spectroscopy (V. Schünemann) of cytosolic and mitochondrial MOCS1A proteins purified from Sf9-insect cell culture. In the second part of this project we investigate the cellular and homeostatic link between FeS cluster biogenesis and Moco synthesis. We aim to understand the impact of impairments of iron homeostasis and FeS-cluster biogenesis on Moco biosynthesis via monitoring Moco-enzyme activities and Moco biosynthesis in cell culture, mouse lines and patient samples in collaboration with different groups of the SPP (R. Lill, C. Berndt, A. Steinbicker). Vice versa, we will probe how alterations in sulfite oxidase activity impact FeS-cluster biogenesis in a novel sulfite oxidase deficient mouse line, which has been created in our lab.