Abstracts 2025

DEFINING THE INTERACTOME OF UDP-GLUCOSE DEHYDROGENASE (UGDH) Emily Allego^1* , Brenna Zimmer¹ , Linlin Ma¹ , George Grady¹ , Pengda Liu² , Joseph Barycki¹ , Melanie Simpson^{1 1}North Carolina State University, Raleigh, USA. ²University of North Carolina, Chapel Hill, USA UDP-glucose dehydrogenase (UGDH) is an essential cytosolic enzyme that catalyzes the NAD+ dependent conversion of UDP-glucose to UDP-glucuronate. UDP-glucuronate cannot be produced by any other human protein except UGDH, and this key metabolite is utilized in three downstream cellular pathways that are compartmentalized. Specifically, the glucuronidation of androgens and xenobiotics occurs in the endoplasmic reticulum, hyaluronan is produced at the plasma membrane, and proteoglycan synthesis is in the Golgi. The partitioning of UDP-glucuronate between these energy demanding pathways necessitates tight regulation of UDP-glucuronate pools. Here, we sought to determine how UGDH regulation through novel protein-protein interactions could impact the fate of UDP-glucuronate using complementary targeted and unbiased approaches. A kinase screen of the UGDH primary sequence revealed several canonical phosphorylation consensus sequences. We pursued a functional screen of the relevant kinase families, and identified and validated one kinase that phosphorylated UGDH at a specific consensus site. We generated phosphodeficient and phosphomimetic point mutants of UGDH and stably expressed them in LNCaP cells, a human prostate tumor cell line, and found significant shifts in the downstream fates of UDP-glucuronate. To identify additional protein-protein interactions that could regulate UGDH, we next created recombinant UGDH point mutants to incorporate an unnatural amino acid at various positions. We synthesized the protein with the photocrosslinker, benzoylphenylalanine (Bpa), which allowed us to photocrosslink UGDH to putative intracellular interacting partners in LNCaP cells. Candidate regulatory proteins that co-immunoprecipitated with UGDH were analyzed by liquid chromatography followed by mass spectrometry. Approximately thirty proteins were found in each UGDH Bpa crosslinked sample. To discriminate and validate which interactions were reliable, we then performed biotin proximity labeling, an in-cell technique to probe the UGDH interactome. We generated a fusion protein of UGDH with BirA, a bacterial biotin protein ligase, that promiscuously biotinylates proteins within a several nanometers of UGDH. Biotin-labeled proteins were isolated from cell lysates through a streptavidin pulldown from LNCaP cells transiently transfected to express BirA-UGDH. Proteomic analysis resulted in the detection of approximately forty proteins from each sample. Comparison of the two datasets allowed us to streamline the validation of genuine UGDH-interacting proteins and determine which interactions impact UGDH function and/or the partitioning of UDP-glucuronate. Novel protein-protein interactions offer new targets for anti-cancer therapeutic development.