The goal of the Section on Genetic Disorders of Drug Metabolism is to understand the molecular genetics of the human UDP-glucuronosyltransferase (UGT1) enzyme system, which is critical for removing from the body bilirubin and other endogenous metabolites as well as a large and indeterminate number of xenochemicals. To reach that goal, our studies focus on the characterization of the multigenic UGT1 locus, which includes understanding the regulation of certain isozymes; understanding the tissue distribution and biochemical properties of the encoded isozymes; understanding the phosphorylation requirement for UGTs and their participation in signaling events; and purifying isozymes as models for structural analysis.

Over the last year, our characterization of UGT1-encoded isozymes indicated that the five isozymes distributed to the gastrointestinal (GI) tract are located in the mucosal surface of each tissue, presumably in a strategic location to metabolize at the site of absorption the many potentially toxic chemicals ingested in the diet and in contaminants of the diet. We tested 38 chemicals from four different categories. Separately, our in vitro studies with the individually expressed recombinant isozymes showed that each isozyme metabolizes the same chemical with different pH optima and with different substrate concentration effects. A summary of the data shows, in effect, that the isozymes are not redundant. The unique optimal assay conditions for an isozyme suggest, perhaps, differential adaptations to the properties of the GI tract.

It is well known that the detoxification of many endogenous and ingested polyphenols and related chemicals occurs by their conversion to glucuronides by a family of endoplasmic reticulum-bound UGT isozymes. In the last year, we discovered that these enzymes require phosphorylation that is regulated by a PKCsignaling system. Our phosphorylation studies were prompted by the transient inhibition of UGT in LS180 cells by, respectively, the general kinase and specific PKC inhibitors curcumin and calphostin-C. Phosphorylation was confirmed by mutation studies and by radiolabeling of UGTs with [33P] ortho phosphate in LS180 cells as shown in Figure 9.

Figure 9

In addition, we studied the effect of glucuronidation on the uptake of free drug and therapeutic efficacy. Given that the promising immunosuppressant mycophenolic acid (MPA) has been introduced into tissue transplantation protocols, we studied MPA’s metabolism in greater detail to understand rapid glucuronidation and its high dosage requirement. We demonstrated that curcumin inhibits MPA’s metabolism in both cell culture and the mouse duodenum. Further, our in vivo studies show that the coadministration of curcumin with MPA can transiently down-regulate glucuronidation of MPA to increase its immunosuppressant effects by two- to three-fold.

In an attempt to determine the tertiary structure of a UGT isozyme, we used computer modeling to uncover the predicted binding sites in the UGT1A10 isozyme. We will use both forward and back mutations to establish the role, if any, of lysine residues 404 and 314 in binding of the UGT universal and common substrate, UDP-glucuronic acid.