Post-translational mechanisms of UDP-glucuronosyltransferase enzyme function

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The UDP-glucuronosyltransferases are an enzyme superfamily that catalyzes the metabolic reaction glucuronidation. This is an elimination reaction that is critical for balancing of steroids and other essential vitamins and nutrients in the body as well as detoxifying chemicals and drugs. Multiple enzymes belonging to the family exist in mammals, fish, birds, insects and invertebrates, differing within and across species. Regulation of UGT activity is complex e.g. enzymes can be present but inactive. Each also has individual genetic and environmental control that can alter activity, causing toxicity and disease. Preliminary data implicates glycosylation in controlling UGT activity and we hypothesize this is fundamental for the enzymes' catalytic reaction. We will investigate the importance of translation, post-translational protein structural changes, namely glycosylation, and endoplasmic reticulum processes (chaperoning, capacity for anchoring) on UGT function. To investigate this we will determine the roles of protein production as well as protein modifications using in vitro cell-free systems. Then using whole cells we will study UGTs protein assembly and processing inside the cell. Preliminary data shows that processing chaperones calnexin and calreticulin are important for UGT protein assembly, because interference with these chaperones alters UGT expression. Also, the absolute capacity inside the cell for UGT protein content and within the ER lumen is unknown, as is how long each UGT exists (protein lifetime) before it is degraded and recycled. We will use fluorescent technologies and expression systems to label UGT enzymes in living cells and determine the absolute capacity [space] for the enzyme within the cell – as amount of protein might be related to how well the protein can perform its detoxification roles. Finally, because the specific protein modification called glycosylation is known to influence UGT function, we will study the effects of two types of glycosylation N-linked, which happens early in protein production and O-linked than tends to happen later and be more structural on the ability of the UGT proteins to metabolize and eliminate essential internal and environmental cehmicals. Understanding the regulation of glucuronidation can identify pathways amenable to manipulation for disease prevention or treatment, aid drugs development and use and improve chemical safety in humans and other species.