Nuclear de novo thymidylate biosynthesis complex formation and computational modeling

Abstract

Folate-mediated one-carbon metabolism (FOCM) consists of a network of metabolic pathways connected through the common use of folate derivatives that carry and donate one-carbon (1C) units for de novo biosynthesis of purines and thymidylate (dTMP or deoxythymidine monophosphate), and homocysteine remethylation. Impairments in FOCM are associated with common pathologies, including neural tube defects (NTDs), neurodegenerations, and cancer, but the causal mechanisms are not established. This dissertation research investigated the nutritional, biochemical and genetic factors that regulate FOCM, with a focus on de novo thymidylate biosynthesis pathway. Regulation of the partitioning of folate cofactors among FOCM pathways is essential to address metabolic needs that fluctuate through cell cycle progression. The first objective aimed to summarize the evidence for temporal regulation of expression, activity and cellular localization of enzymes and pathways in the FOCM network in mammalian cells through the cell cycle. The second objective sought to investigate the structure of the nuclear de novo thymidylate synthesis multienzyme complex and the nature of the protein-protein interactions. Multiple SUMO1 modification sites were identified on enzymes of the de novo dTMP biosynthesis pathway by mass spectrometry (MS) analysis. Further experiments are needed characterize the protein-protein interactions within the complex. Computational modeling is useful to access the complexity of FOCM network and guide the design of biological experiments. The third objective aimed to extend the current FOCM model to include the nuclear compartment. The model confirms that accounting for the kinetic effects of nuclear multienzyme complex formation and substrate channeling is essential for the functioning of de novo dTMP synthesis. The fourth objective sought to investigate the unique requirement for glycine for C2C12 myoblasts proliferation. Glycine contributed 1C units to FOCM and de novo thymidylate biosynthesis. However, formate, hypoxanthine or thymidine supplementation failed to rescue the growth of C2C12 myoblasts under glycine depletion, suggesting that generating 1C units is not the primary mechanism by which glycine supports C2C12 myoblasts proliferation.