Evaluation of UGT Protein Interactions in Human Hepatocytes: Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on Propofol Glucuronidation

Abstract

Previous experiments performed in recombinant systems that over‐express either a single or multiple UDP‐glucuronosyltranseferase (UGT) isoforms have suggested that protein‐protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein‐protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In the current study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation due to disrupted protein interactions. Selective siRNA down regulation of UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 probe substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. To our knowledge, this represents the first piece of evidence that UGT protein‐protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates such as propofol.