Connecting sulfur transfer pathways for molybdenum cofactor biosynthesis and tRNA thiolation in humans

硫是所有生物体的必需元素。硫向tRNA中的钼辅因子和核苷的运输和递送是一个高度调节的过程，其在涉及几种硫转移蛋白的复杂途径中进行。在连接人类的两种途径方面仍然存在许多悬而未决的问题，我们希望在这个项目中更详细地研究。在人类中，tRNAsLys、Gln、Glu中mcm 5s 2U 34的胞质硫修饰需要蛋白质NFS 1、TUM 1、MOCS 3、URM 1、CTU 1和CTU 2。相比之下，线粒体tRNALys、Gln、Glu中用于tm 5s 2U 34修饰的硫插入涉及蛋白质NFS 1、TUM 1和MTU 1。此外，对于Moco的二硫杂环戊烯基团的合成，涉及蛋白质MOCS 2A、MOCS 3、TUM 1和NFS 1，这是在人的胞质溶胶中发生的反应。总之，Moco生物合成和tRNA巯基化在胞质溶胶中连接，并且共享由NFS 1、MOCS 3和可能的TUM 1组成的相同硫递送途径蛋白。要剖析复杂的硫转移途径的系统中的线粒体和胞质tRNA巯基化和Moco的生物合成，一个目标是分析的作用，两个新发现的TUM 1-异构体的tm 5s 2U形成在线粒体和形成的mcm 5s 2U 34和Moco在胞质溶胶。我们的目标是详细剖析硫转移途径的特异性，并分析是否存在不同途径的硫中继系统的胞质tRNA硫醇化和莫科生物合成。此外，分析酵母和人MOCS 3之间是否存在差异是有意义的。使用构建的CRISPR/Cas9敲除细胞系（MOCS 3、TUM 1）以及与酵母Uba 4蛋白、不同的TUM 1同种型或MOCS 3片段和变体的功能互补将详细揭示每种蛋白的细胞作用。总之，我们的研究将详细剖析tRNA巯基化的作用及其与人类细胞中每个隔室中Moco生物合成的联系。

Sulfur is an essential element for all living organisms. The trafficking and delivery of sulfur to the molybdenum cofactor and nucleosides in tRNA is a highly regulated process which proceeds in a complex pathway involving several sulfur transferring proteins. Still numerous open questions remain in connecting both pathways in humans, which we want to study in this project in more detail. In humans, the cytosolic sulfur modification of mcm5s2U34 in tRNAsLys, Gln, Glu requires the proteins NFS1, TUM1, MOCS3, URM1, CTU1 and CTU2. In contrast, the sulfur insertion for tm5s2U34 modifications in mitochondrial tRNALys, Gln, Glu involves the proteins NFS1, TUM1 and MTU1. Further, for the synthesis of the dithiolene group of Moco, the proteins MOCS2A, MOCS3, TUM1 and NFS1 are involved, a reaction which occurs in the cytosol of humans. Conclusively, Moco biosynthesis and tRNA thiolation are connected in the cytosol and share the same sulfur delivery pathway proteins composed of NFS1, MOCS3 and possibly TUM1. To dissect the complex sulfur transfer pathways in the systems of mitochondrial and cytosolic tRNA thiolation and Moco biosynthesis, one goal is to analyze the role of two newly identified TUM1-isoforms for tm5s2U formation in mitochondria and the formation of mcm5s2U34 and Moco in the cytosol. The goal is to dissect the specificity of the sulfur transfer pathways in detail and to analyze whether different routes of sulfur relay systems exist for cytosolic tRNA thiolation and Moco biosynthesis. Further it is of interest to analyze whether differences between yeast and human MOCS3 exist. The usage of constructed CRISPR/Cas9 knock out cell lines (MOCS3, TUM1) and the functional complementation with the yeast Uba4 protein, the different TUM1 isoforms or MOCS3 fragments and variants will reveal the cellular role of each protein in detail. In total, our studies will dissect the role of tRNA thiolation and its connection to Moco biosynthesis in human cells in each compartment in detail.