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

含钼（Mo）的酶催化全球生命周期中的关键反应。它们依赖于Mo辅因子（Moco）以及FeS簇的生物合成，这是Moco合成的第一步所需的，GTP转化为环状吡喃蝶呤单磷酸（cPMP）并在Mo酶中作为辅因子发挥作用。Moco生物合成的缺陷导致代谢中严重的先天性缺陷，主要由亚硫酸盐氧化酶活性丧失引起，其特征在于快速进展的神经变性和儿童死亡。我们已经开发了第一种治疗方法，用于MOCS 1基因突变的患者，该基因产生编码两种蛋白质的选择性剪接转录本。MOCS 1A需要两个[4Fe-4S]簇，属于自由基SAM酶超家族。第二个翻译产物代表MOCS 1AB蛋白，具有无活性的MOCS 1A结构域和具有催化活性的C-末端MOCS 1B结构域。在之前的资助期间，我们能够证明两个MOCS 1A剪接变体的胞质定位，而另外两个MOCS 1A剪接变体以及所有MOCS 1AB剪接变体定位于线粒体。此外，令人惊讶的是，我们发现在输入过程中，MOCS 1AB被蛋白水解切割，产生小的MOCS 1B蛋白，其与MOCS 1A类似地定位于线粒体基质。MOCS 1B的体内活性可以通过SPP内研究的患者病例来证实，其中纯合移码突变导致MOCS 1B的单独表达。 在接下来的资助期内，我们的目标是解决人类Moco合成和Moco缺乏症（MoCD）中尚未解决的两个关键问题。首先，我们计划研究细胞溶质和线粒体MOCS 1A蛋白的差异，因为这两类蛋白在生理条件下表达，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纯化和cPMP体外合成方案，对不同的MOCS 1A变体进行生物化学表征，包括动力学参数的测定。这也将包括MOCS 1A（O. Einsle），以及EPR-（与A. Pierik）和穆斯堡尔谱（V. Schünemann）从Sf 9昆虫细胞培养物中纯化的胞质和线粒体MOCS 1A蛋白。在这个项目的第二部分，我们调查的细胞和稳态之间的联系FeS簇的生物合成和莫科合成。我们的目的是了解铁稳态和FeS簇生物合成的损伤对Moco生物合成的影响，通过监测Moco酶活性和Moco生物合成在细胞培养物中，小鼠品系和患者样品与SPP（R.利尔角Berndt，A. Steinbicker）。反之亦然，我们将探讨亚硫酸氧化酶活性的改变如何影响FeS-簇在一个新的亚硫酸氧化酶缺陷小鼠系，这已经在我们的实验室创建的生物合成。

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.