UGT PROTEIN NETWORK AND THE CONTROL OF CELL METABOLISM.

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Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), is crucial in higher eukaryotes for protection against imbalances in endobiotics (e.g., hormones, neurotransmitters) and harmful xenobiotics (e.g., pollutants, drugs). In humans, nine functional UGTs enzymes are encoded by a single gene, UGT1A. As a major breakthrough in the field, we discovered a novel post-transcriptional regulatory mechanism of cellular glucuronidation involving nine other shorter UGT1A proteins (named i2s) produced by alternative splicing. These i2s are enzymatically inactive but act as negative modulators of cellular metabolism by inhibiting the i1 enzymes in the endoplasmic reticulum (ER) through protein-protein interaction. In addition, i2s localize outside the ER, suggesting potential additional roles and interactions with new partner proteins. We hypothesized that UGT1As comprise a highly organized protein network that is shaped by proteins likely involved in modulating UGT1A-mediated activities (as initially observed for i2s) and possibly through interaction with novel partners that would reveal additional cellular functions for this family of proteins. Aim 1: To study protein-protein interactions between i1 enzymes and i2 regulators in the ER and define the interaction domains involved. Aim 2: To identify novel interacting partners (ER-resident and non-resident) to provide new insights into UGT1A-associated proteins involved in modulating glucuronidation activity and explore putative new functions for UGTs. We will use complementary approaches including mass spectrometry and the models and tools established in the last grant period. The findings may provide novel approaches to regulate or modulate the glucuronidation pathway. Identifying and understanding the role of protein-protein interactions is fundamental to understanding glucuronidation at the systems level and developing novel therapeutics. Our group has discover UGT1A splice variants and reveal their regulatory function for the first time; we thus have the unique expertise and tools to achieve the goals set forth in this proposal. Highly qualified students will continue to be trained throughout this research program.