UDP-glucose dehydrogenase (UGDH) is a unique, essential enzyme with the central role of providing UDP- glucuronate, a rate-limiting precursor for plasma membrane hyaluronan synthesis, Golgi proteoglycan production, and ER-localized modification of hormones for elimination. Our laboratory has shown that insufficiency of UGDH contributes to loss of control of intracellular steroid levels, and dysregulated tumor cell growth rate in prostate cancer. It is not known how the cytosolic UDP-glucuronate is partitioned to its respective fates in the high levels needed for specifically timed product formation, nor how UGDH activity is controlled to limit competition with other pathways. Our hypothesis for this proposal is that hexameric and dimeric units of UGDH sense metabolic status of the cell and respond with increased or decreased enzymatic activity. Information for molecular sensing is conveyed partly through differential protein-protein interactions that occur upon exposure of the dimer-dimer interface. We will test this with two aims.
Aim 1 : Determine the functional outcome of specific UGDH interactions with components of the androgen elimination pathway. We will directly measure interactions of UGDH with hyaluronan synthase, the Golgi UDP-xylose transporter, and the ER UDP-glucuronate transporter, as the three proteins that mediate the demands for UDP-glucuronate flux. We will quantify UDP-glucuronate, steroid-glucuronide, notch glycosylation and hyaluronan production, which will respectively report overall UGDH activity, and functional distribution to the ER for androgen elimination, the Golgi for proteoglyca secretion, or the plasma membrane for hyaluronan synthesis.
Aim 2 : Characterize and validate the UGDH interactome using an unbiased approach. We will use mass spectrometry to identify proteins that differentially co-fractionate by size exclusion chromatography with our well- characterized hexameric and dimeric point mutants. As a complementary approach, we will identify proteins that cross-link with hexameric versus dimeric UGDH point mutants that incorporate a photo-activatable crosslinker as a non-natural amino acid. Validated interactions and/or post-translational modifications will be used to design strategies for selective partitionin of UDP-glucuronate to favor hormone elimination in future preclinical studies of prostate cancer.
The goal of this proposal is to identify cellular proteins that modulate the activity of UDP-glucose dehydrogenase (UGDH), and determine how they orchestrate the flux of UGDH products through three critical pathways. UGDH is an enzyme that has a pivotal role in general detoxification and hormone activity in the prostate so selectively targeting its activity to steroid removal may offer a new strategy to improve response to androgen deprivation therapy in prostate cancer patients.
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| Zimmer, Brenna M; Howell, Michelle E; Wei, Qin et al. (2016) Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential. Horm Cancer 7:260-71 |
| Grady, George; Thelen, Ashley; Albers, Jaleen et al. (2016) Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking. Biochemistry 55:3157-64 |
