Impact of UGT2B7 gene deletion genotype on human liver proteome

Abstract

Background and objectivesUDP‐Glucuronosyltransferase 2B17 (UGT2B17) is a highly variable enzyme, which plays an important role in metabolism of endogenous steroids (e.g., testosterone), drugs or active metabolites (e.g., vorinostat, 17‐dihydroexemestane), and environmental chemicals (e.g., nicotine nitrosamine ketone, NNK). UGT2B17 is the most commonly deleted gene and we have previously demonstrated that age, sex, copy number variation, and certain single nucleotide polymorphisms can affect its abundance and activity (1). The aim of this study was to identify steroid and drug metabolism pathways that are impacted by UGT2B17 deletion using label‐free untargeted proteomics approach.MethodsThe untargeted proteomics study was performed on the liver S9 samples isolated from liver tissue of subjects with UGT2B17 gene deletion (n=3) and UGT2B17 high expression (n=3) (1). The proteins were reduced, alkylated, and digested with trypsin using an optimized protocol (1). Proteomics data were acquired in high resolution mode in QE‐HF (Thermo Scientific) coupled with Easy nano‐LC 1200. The tryptic digest was injected onto a nano‐column for peptide separation and the peptides were detected using data‐dependent acquisition (DDA) method. The survey scans of peptide precursors were performed from 375 to 1500 m/z at 120 K resolution with a 3e6 ion count target and a maximum injection time of 50 ms. The untargeted proteomic data was analyzed using MaxQuant with 1% FDR followed by data mining using Perseus and STRING analysis for pathway mapping.Results and conclusionA total of 1002 liver proteins were quantified based on the signature peptides. Out of 1002 proteins, 14 and 39 proteins were exclusively detected and quantified in UGT2B17 deletion and high expression groups, respectively (Fig. 1A), whereas 949 proteins were quantified in both the groups. The following pathways were upregulated in UGT2B17 deletion group: oxidation reduction process, drug metabolic process, steroid and fatty acid metabolic process. Notable proteins that were differentially expressed in the gene deletion groups were: a) AKR1D1, AKR1C4, AKR7DA3, AKR1A1, DHCR7, ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A1 (upregulated) and b) CYP3A5, CYP1A2, LDHA, NNMT, Kynureninase, and ACACB (downregulated). A deeper molecular pathway analysis reveals that the enzymes associated with steroidal and bile acid homeostasis (e.g., aldo‐keto reductases (AKRs) and aldehyde dehydrogenases) were significantly elevated in the UGT2B17 deletion group. Interestingly, AKR1D1 acts as the sole Δ4‐3‐ketosteroid‐5β‐reductase, that metabolizes testosterone to inactive 5β‐dihydrotestosterone (2). In contrast, this pathway is a minor contributor to testosterone metabolism in the UGT2B17 high expressers. Similarly, AKR1C4 and UGT2B7, which are responsible for sequential conversion of 5β‐ dihydrotestosterone to 3α‐5β‐tetrahydrotestosterone (THT) to THT‐glucuronide (Fig. 1B), are upregulated in the UGT2B17 gene deletion groups. These novel findings of testosterone homeostasis in the UGT2B17 gene individuals can provide deeper insights into the mechanisms of UGT2B17 gene deletion‐associated pathophysiological conditions (osteoporosis, obesity, insulin sensitivity, and cancers).A) Number of hepatic proteins detected in UGT2B17 gene deletion (red) and high expressers (blue) individuals. B) Proposed compensatory mechanism of testosterone hepatic metabolism in UGT2B17 gene deletion individuals.Figure 1