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Examination of Human UDP-glucose Dehydrogenase Structure and Function Reveals Regulatory Approaches
UDP-glucose dehydrogenase (UGDH) is an essential enzyme that catalyzes the two step NAD+-dependent conversion of UDP-glucose to UDP-glucuronate. Structural studies of human UGDH revealed that it is a trimer of dimers assembled into a disc-like structure. In the absence of substrate and cofactor, hUGDH purifies as equimolar amounts of homohexameric and homodimeric species, but upon addition of substrate and cofactor, UGDH is transformed into primarily the hexameric species. Hexamer formation was previously shown to be critical for attaining maximal activity and cellular function. We have now shown that dissociation at the dimer-dimer interfaces is equally as important and that quaternary assembly directly impacts enzymatic activity. Additionally, we identified novel missense UGDH DNA mutations in patients presenting epileptic encephalopathy, in which the resulting UGDH point mutations were found to significantly destabilize UGDH, prevent hexamer formation, reduce catalytic activity, and alter downstream UDP-glucuronate pathways. These are the first reported neurological disease causing mutations identified in UGDH.^ UDP-glucuronate, the product of UGDH catalysis, is a critical metabolite utilized in three key pathways: hyaluronan (HA) synthesis, proteoglycan and other glycosaminoglycan production, and phase II detoxification via UDP-glucuronosyltransferases (UGTs) which transfer the glucuronide to xenobiotics and other lipophilic compounds for secretion. The partitioning into the aforementioned pathways and local production of UDP-glucuronate can have significant impacts on a variety of diseases including prostate cancer progression. Therefore, we examined the UGDH interactome in a prostate cancer cell line through use of site-specific photocrosslinking in permeabilized cells, UGDH immunoprecipitation, and LC-MS/MS analysis to identify novel intracellular regulators of UGDH. Identification of cellular regulators of UGDH function will provide the essential information to modulate UGDH function, subcellular localization, and/or local UDP-glucuronate pools. Another strategy we employed to target UGDH activity and function was high throughput screening and peptide phage display for inhibitors and activators of UGDH. We have identified two lead small molecule inhibitors and several 10-mer peptides which can be optimized to depress inhibition doses and generate a more therapeutically and pharmacogenetically relevant set of UGDH modulators. ^
Grady, George D, "Examination of Human UDP-glucose Dehydrogenase Structure and Function Reveals Regulatory Approaches" (2018). ETD collection for University of Nebraska - Lincoln. AAI10843348.