Structural characterization of human uridine monophosphate synthase (UMPS) and its spatio‐temporal localization in cells

Abstract

In humans, uridine monophosphate synthase (UMPS) catalyzes the last two reactions in the pyrimidine de novo biosynthetic pathway, with N‐terminal OPRT domain and C‐terminal OMPDC domain. OMPDC has been reported to dimerize in its active state, whereas the oligomerization and structural characterization of OPRT and UMPS are not known. To understand the regulation of proteins in this pathway, we are investigating the structure of UMPS and its spatio‐temporal localization in the cell. Wild type and mutant UMPS were expressed in BL21 E.coli and purified with affinity chromatography and size exclusion chromatography for structural and biochemical studies. Our results indicate that wild type UMPS equilibrates in two different oligomeric states. In cells, UMPS is known to be a cytosolic protein, however, its localization has not been observed when the pyrimidine nucleotide demand increases. We predict that UMPS gather around the mitochondria to increase flux of intermediates under nucleotide depleted conditions. We transiently transfected FLAG‐tagged UMPS into HeLa cells and observed its localization with the mitochondria, which will be further confirmed with super resolution microscopy. UMPS has become an attractive drug target for cancer, autoimmune disease and antiviral therapeutics among others, therefore, structural characterization and cellular localization of UMPS is crucial to determine the regulatory mechanism of the pathway.Support or Funding InformationSUNY at Stony BrookThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.