Evaluation of UGT protein interactions in human hepatocytes: Effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes

Abstract

Previous experiments performed in recombinant systems 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 this 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 of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. 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.

Reduction of p53 by Knockdown of the UGT1 Locus in Colon Epithelial Cells Causes an Increase in Tumorigenesis

“… 46 The UGTs have also been shown to dimerize with other ER-based proteins, such as the cytochrome P450s. 47 , 48 , 49 , 50 Because knockout of the UGT1A proteins during stress in colon tissue leads to decreases in p53 abundance, the possibility exists that the UGTs may interact through protein–protein interactions and play an important role in controlling activation of ER-localized synoviolin, which controls the levels of phosphorylated cytoplasmic p53. Our working model of this study is illustrated in Figure 8 .…”

Extensive Protein-Protein Interactions Involving UDP-glucuronosyltransferase (UGT) 2B7 in Human Liver Microsomes

“…However, these interactions had not been determined under physiological conditions until recently, when a functional interaction between human UGTs in human hepatocytes was demonstrated. 27) Nevertheless, physical interactions between UGTs under physiological conditions, such as in human hepatocytes and liver microsomes, have not been proved yet. In our large-scale analysis of protein-protein interactions by shotgun analysis, the presence of various UGT-UGT and UGT-CYP interactions in human liver microsomes was revealed.…”

Quantitative analysis of the UDP‐glucuronosyltransferase transcriptome in human tissues

“…32,33,38 There was also large interperson variability in the composition of UGT pools in different tissues (Figure 4), as previously reported in the liver. 33,38 The UGT proteins exist as homo-and heterodimers, 55,56 and oligomerization between different UGTs can increase or decrease their enzymatic activity depending on the different UGT partners or substrates. 57,58 Thus, the variable composition of UGT pools will likely change the landscape of UGT oligomers and their activity for some substrates, thereby contributing to interperson variability in UGT activity.…”