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

• Applicant: Professor Dr. Günter Schwarz
• Subject Area: Biochemistry
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 311772630

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.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1927: Iron-Sulfur for Life