Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential

Abstract

Phase II metabolism is prominently governed by UDPglucuronosyltransferases (UGTs) in humans. These enzymes regulate the bioactivity of many drugs and endogenous small molecules in many organs, including the liver, a major site of regulation by the glucuronidation pathway. This study determined the expression of hepatic UGTs by targeted proteomics in 48 liver samples and by measuring the glucuronidation activity using probe substrates. It demonstrates the sensitivity and accuracy of nano-ultra-performance liquid chromatography with tandem mass spectrometry to establish the complex expression profiles of 14 hepatic UGTs in a single analysis. UGT2B7 is the most abundant UGT in our collection of livers, expressed at 69 pmol/mg microsomal proteins, whereas UGT1A1, UGT1A4, UGT2B4, and UGT2B15 are similarly abundant, averaging 30-34 pmol/mg proteins. The average relative abundance of these five UGTs represents 81% of the measured hepatic UGTs. Our data further highlight the strong relationships in the expression of several UGTs. Most notably, UGT1A4 correlates with most measured UGTs, and the expression levels of UGT2B4/UGT2B7 displayed the strongest correlation. However, significant interindividual variability is observed for all UGTs, both at the level of enzyme concentrations and activity (coefficient of variation: 45%-184%). The reliability of targeted proteomics quantification is supported by the high correlation between UGT concentration and activity. Collectively, these findings expand our understanding of hepatic UGT profiles by establishing absolute hepatic concentrations of 14 UGTs and further suggest coregulated expression between most abundant hepatic UGTs. Data support the value of multiplexed targeted quantitative proteomics to accurately assess specific UGT concentrations in liver samples and hepatic glucuronidation potential. IntroductionThe liver is a key organ for the metabolism of drugs and endogenous compounds such as hormones and bile acids. Through the expression of a complex cocktail of enzymes such as uridine 59-diphosphoglucuronosyltransferases (UGTs), the biochemical properties and bioactivity of these compounds are highly regulated by the liver. Glucuronidation contributes 35% of the phase II drug metabolic pathways and is involved in the clearance of 55% of the 200 most prescribed drugs (Guillemette et al., 2014). The liver expresses a diversified array of UGTs, with 12 of the 16 UGT1A and UGT2B proteins (Guillemette et al., 2014).The expression profile of UGTs was first described using various techniques based on mRNA and immunochemical quantification (Court et al., 2012). However, significant quantitative inaccuracy arises from the lack of correlation between mRNA and protein expression levels and the high sequence similarity among UGTs (Margaillan et al., 2015). A number of research groups including ours have developed mass spectrometry-based methods to quantify UGTs in human tissues (Table 1) (Harbourt et al., 2012;Ohtsuki et al., 2012;Fallon et al., 2013b;Achour et al., 2014; Gr...

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…However, spurious correlations in relation to the activities of UGTs 1A4, 2B7, and 2B15 were also uncovered, which were not as strong as true correlations and could be explained by natural correlation of expression between UGT1A4/2B4 and UGT2B7/2B15, shown to be strong and significant in both SIL-based and QconCAT-based datasets and corroborated by recent literature (Achour et al, 2014c, Margaillan et al, 2015b. A possible ramification of such apparent relationships can be compromised reaction phenotyping of substrates of such enzymes if phenotyping is carried out using correlation of activity with enzyme content in an in vitro system, such as human liver microsomes.…”

“…Quantitative Proteomics and Catalytic Activity of UGTs 1109 at ASPET Journals on September 13, 2017 dmd.aspetjournals.org sample set (n = 60), with levels of variability ranging between 27 and 67%, in line with previous studies on UGT protein expression and activity that used similar methodologies (Sato et al, 2012;Margaillan et al, 2015b). In contrast, higher interindividual variability was recorded with the QconCAT abundance dataset (43%-101%, n = 23 or 24).…”

“…The cases of UGTs 1A6, 1A9, and 2B15 showed a significant but lower level of correlation, possibly due to less specific substrates or less efficient proteomic methods. The same substrates (propofol and zidovudine) were previously used to assess correlation of activity rates with UGTs 1A9 and 2B7 protein contents in human liver and kidney microsomal samples and demonstrated strong correlation in both cases (Margaillan et al, 2015b;Knights et al, 2016), pointing to the need for further optimization of the proteomic workflows in the cases where strong correlations were not demonstrated. In contrast, the QconCAT-based dataset showed overall poor correlation with catalytic activity, with moderate correlations observed only for UGTs 1A1, 1A3, and 2B7.…”

Quantitative profiling of the UGT transcriptome in human drug-metabolizing tissues

“…17 Still, expression data are consistent with lower expression of UGTs in kidney and colon tissues as previously reported using other approaches, at the mRNA and protein levels. [18][19][20][21][22] Our preliminary observations also suggest a shift in the transcript profiles in kidney and intestine/colon tumors that requires additional investigations using a larger set of well-characterized tumor samples.…”

Stereoselective Glucuronidation of Bupropion Metabolites In Vitro and In Vivo

“…UGT2B7 and UGT1A9, both being prominently expressed in the liver (Margaillan et al, 2015b), are likely the dominant isoforms responsible for hepatic glucuronidation of bupropion metabolites. These isoforms may also play an important role in the extrahepatic glucuronidation of bupropion, particularly in the kidney where UGT1A9 is the predominant isoform expressed (Margaillan et al, 2015a) and in the gut where UGT2B7 and UGT1A9 are present (Harbourt et al, 2012;Fallon et al, 2013).…”