37 research outputs found
The relative protein abundance of UGT1A alternative splice variants as a key determinant of glucuronidation activity in vitro
Alternative splicing (AS) is one of the most significant components of the functional complexity of human UDP-glucuronosyltransferase enzymes (UGTs), particularly for the UGT1A gene, which represents one of the best examples of a drug-metabolizing gene regulated by AS. Shorter UGT1A isoforms [isoform 2 (i2)] are deficient in glucuronic acid transferase activity but function as negative regulators of enzyme activity through protein-protein interaction. Their abundance, relative to active UGT1A enzymes, is expected to be a determinant of the global transferase activity of cells and tissues. Here we tested whether i2-mediated inhibition increases with greater abundance of the i2 protein relative to the isoform 1 (i1) enzyme, using the extrahepatic UGT1A7 as a model and a series of 23 human embryonic kidney 293 clonal cell lines expressing variable contents of i1 and i2 proteins. Upon normalization for i1, a significant reduction of 7-ethyl-10-hydroxycamptothecin glucuronide formation was observed for i1+i2 clones (mean of 53%) compared with the reference i1 cell line. In these clones, the i2 protein content varied greatly (38–263% relative to i1) and revealed two groups: 17 clones with i2 < i1 (60% ± 3%) and 6 clones with i2 = i1 (153% ± 24%). The inhibition induced by i2 was more substantial for clones displaying i2 = i1 (74.5%; P = 0.001) compared with those with i2 < i1 (45.5%). Coimmunoprecipitation supports a more substantial i1-i2 complex formation when i2 exceeds i1. We conclude that the relative abundance of regulatory i2 proteins has the potential to drastically alter the local drug metabolism in the cells, particularly when i2 surpasses the protein content of i1
A rare UGT2B7 variant creates a novel N-glycosylation site at codon 121 with impaired enzyme activity
UDP-glucuronosyltransferase (UGT) superfamily are glycoproteins resident of the endoplasmic
reticulum membranes that undergo post-translational modifications (PTM). UGT2B7 is of
particular interest because of its action on a wide variety of drugs. Most studies currently survey
common variants and are only examining a small fraction of the genetic diversity. However, rare
variants (frequency <1%) might have significant effect as they are predicted to greatly
outnumber common variants in the human genome. Here, we discovered a rare single nucleotide
UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino
acid substitution at codon 121. This low-frequency variation, found in two individuals of a
population of 305 healthy volunteers, leads to the translation of an asparagine (Asn) instead of an
aspartic acid (Asp) (UGT2B7 p.D121N). This amino acid change was predicted to create a
putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H
treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced
UGT2B7 with tunicamycin from HEK293 cells. The presence of an additional N-linked glycan
on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant
decrease, respectively by 49 and 40%, in the formation of zidovudine and mycophenolic acid
glucuronides. A systematic survey of the dbSNP database uncovered 32 rare and naturally
occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in
the other UGT2B enzymes. Collectively, these variants have the potential to increase the
proportion of variance explained in the UGT pathway due to changes in PTM such as N-linked
glycosylation with consequences on drug metabolism
Posttranscriptional regulation of UGT2B10 hepatic expression and activity by alternative splicing
The detoxification enzyme UDP-glucuronosyltransferase UGT2B10 is specialized in the
N-linked glucuronidation of many drugs and xenobiotics. Preferred substrates possess
tertiary aliphatic amines and heterocyclic amines such as tobacco carcinogens and
several anti-depressants and anti-psychotics. We hypothesized that alternative splicing
(AS) constitutes a mean to regulate steady state levels of UGT2B10 and enzyme
activity. We established the transcriptome of UGT2B10 in normal and tumoral tissues of
multiple individuals. Highest expression was in the liver, where ten AS transcripts
represented 50% of the UGT2B10 transcriptome in 50 normal livers and 44
hepatocellular carcinomas. One abundant class of transcripts involves a novel exonic
sequence and leads to two alternative (alt.) variants with novel in-frame C-termini of 10
or 65 amino acids. Their hepatic expression was highly variable among individuals,
correlated with canonical transcript levels, and was 3.5 fold higher in tumors. Evidence
for their translation in liver tissues was acquired by mass spectrometry. In cell models,
they co-localized with the enzyme and influenced the conjugation of amitriptyline and
levomedetomidine by repressing or activating the enzyme (40-70%; P<0.01), in a cell
context-specific manner. A high turnover rate for the alt. proteins, regulated by the
proteasome, was observed in contrast to the more stable UGT2B10 enzyme. Moreover,
a drug-induced remodelling of UGT2B10 splicing was demonstrated in the HepaRG
hepatic cell model, which favored alt. variants expression over the canonical transcript.
Our findings support a significant contribution of AS in the regulation of UGT2B10
expression in the liver with an impact on enzyme activity
Quantitative profiling of the UGT transcriptome in human drug metabolizing tissues
Alternative splicing as a mean to control gene expression and diversify function is
suspected to considerably influence drug response and clearance. We report the
quantitative expression profiles of the human UGT genes including alternatively spliced
variants not previously annotated established by deep RNA-sequencing in tissues of
pharmacological importance. We reveal a comprehensive quantification of the
alternative UGT transcriptome that differ across tissues and among individuals.
Alternative transcripts that comprise novel in-frame sequences associated or not with
truncations of the 5’ and/or 3’ termini, significantly contribute to the total expression
levels of each UGT1 and UGT2 gene averaging 21% in normal tissues, with expression
of UGT2 variants surpassing those of UGT1. Quantitative data expose preferential
tissue expression patterns and remodelling in favour of alternative variants upon
tumorigenesis. These complex alternative splicing programs have the strong potential to
contribute to interindividual variability in drug metabolism in addition to diversify the UGT
proteome
Unravelling the transcriptomic landscape of the major phase II UDP-glucuronosyltransferase drug metabolizing pathway using targeted RNA sequencing
A comprehensive view of the human UDP-glucuronosyltransferase (UGT) transcriptome is a
prerequisite to the establishment of an individual’s UGT metabolic glucuronidation signature. Here,
we uncover the transcriptome landscape of the ten human UGT loci genes in normal and tumoral
metabolic tissues by targeted RNA next generation sequencing. Alignment on the human hg19
reference genome identifies 234 novel exon-exon junctions. We recover all previously known
UGT1 and UGT2 enzyme-coding transcripts and identify over 130 structurally and functionally
diverse novel UGT variants. We further expose a revised genomic structure of UGT loci and
provide a comprehensive repertoire of transcripts for each UGT gene. Data also uncover a
remodelling of the UGT transcriptome occurring in a tissue- and tumor-specific manner. The
complex alternative splicing program regulating UGT expression and protein functions is likely
critical in determining detoxification capacity of an organ and stress-related responses, with
significant impact on drug responses and diseases. Keywords: Alternative splicing, transcriptome, glucuronidation, RNA sequencing, drug
metabolism, glucuronosyltransferase (UGT
UGT1A1 and UGT1A9 functional variants, meat intake, and colon cancer, among Caucasians and African-Americans
Glucuronidation by the UDP-glucuronosyltransferase enzymes (UGTs) is one of the primary detoxification pathways of dietary heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs). In a population-based case-control study of 537 cases and 866 controls, we investigated whether colon cancer was associated with genetic variations in UGT1A1 and UGT1A9 genes and we determined if those variations modify the association between colon cancer and dietary HCA and PAH exposure. We measured functional UGT1A1 polymorphisms at positions −53 (*28; A(TA)6TAA to A(TA)7TAA), −3156 (G>A), −3279 (T>G) and the UGT1A9-275(T>A) polymorphism, and found no association with colon cancer overall. However, when stratified by race, the UGT1A1-3279 GG/TG intermediate/low activity genotypes were associated with an increased risk of colon cancer (odds ratio (OR) = 1.5, 95% confidence interval (CI)=1.1–2.0) in Caucasians. This finding is also supported by haplotype analyses where the UGT1A1-3279G-allele-bearing haplotype is overrepresented in case group. Overall, UGT1A1-53 and -3156 genotypes modified the association between dietary benzo(a)pyrene (BaP) and colon cancer (P for interaction=0.02 and 0.03, respectively). The strongest association was observed for those with <7.7 ng/day BaP exposure and the low activity genotypes, for both UGT1A1*28/*28 (OR=1.8, 95% CI=1.1–2.9) and −3156AA (OR=1.7, 95% CI=1.0–3.0), compared to ≥7.7 ng/day and combined high/intermediate genotypes. These data support a hypothesis that UGTs modify the association between meat-derived PAH exposure and colon cancer by their role in the elimination of dietary carcinogens
Quantitative Profiling of Human Renal UDP-glucuronosyltransferases and Glucuronidation Activity: A Comparison of Normal and Tumoral Kidney Tissues
Renal metabolism by UDP-glucuronosyltransferase (UGT) enzymes is central to the clearance of many drugs. However, significant discrepancies about the relative abundance and activity of individual UGT enzymes in the normal kidney prevail among reports, whereas glucuronidation in tumoral kidney has not been examined. In this study, we performed an extensive profiling of glucuronidation metabolism in normal (n = 12) and tumor (n = 14) kidneys using targeted mass spectrometry quantification of human UGTs. We then correlated UGT protein concentrations with mRNA levels assessed by quantitative polymerase chain reaction and with conjugation activity for the major renal UGTs. Beyond the wide interindividual variability in expression levels observed among kidney samples, UGT1A9, UGT2B7, and UGT1A6 are the most abundant renal UGTs in both normal and tumoral tissues based on protein quantification. In normal kidney tissues, only UGT1A9 protein levels correlated with mRNA levels, whereas UGT1A6, UGT1A9, and UGT2B7 quantification correlated significantly with their mRNA levels in tumor kidneys. Data support that posttranscriptional regulation of UGT2B7 and UGT1A6 expression is modulating glucuronidation in the kidney. Importantly, our study reveals a significant decreased glucuronidation capacity of neoplastic kidneys versus normal kidneys that is paralleled by drastically reduced UGT1A9 and UGT2B7 mRNA and protein expression. UGT2B7 activity is the most repressed in tumors relative to normal tissues, with a 96-fold decrease in zidovudine metabolism, whereas propofol and sorafenib glucuronidation is decreased by 7.6- and 5.2-fold, respectively. Findings demonstrate that renal drug metabolism is predominantly mediated by UGT1A9 and UGT2B7 and is greatly reduced in kidney tumors
Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential
Phase II metabolism is prominently governed by UDP-glucuronosyltransferases (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
The Glycosyltransferase Pathway: An Integrated Analysis of the Cell Metabolome
Nucleotide sugar-dependent glycosyltransferases (UGTs) are critical to the homeostasis of endogenous metabolites and the detoxification of xenobiotics. Their impact on the cell metabolome remains unknown. Cellular metabolic changes resulting from human UGT expression were profiled by untargeted metabolomics. The abundant UGT1A1 and UGT2B7 were studied as UGT prototypes along with their alternative (alt.) splicing-derived isoforms displaying structural differences. Nineteen biochemical routes were modified, beyond known UGT substrates. Significant variations in glycolysis and pyrimidine pathways, and precursors of the co-substrate UDP-glucuronic acid were observed. Bioactive lipids such as arachidonic acid and endocannabinoids were highly enriched by up to 13.3-fold (p < 0.01) in cells expressing the canonical enzymes. Alt. UGT2B7 induced drastic and unique metabolic perturbations, including higher glucose (18-fold) levels and tricarboxylic acid cycle (TCA) cycle metabolites and abrogated the effects of the UGT2B7 canonical enzyme when co-expressed. UGT1A1 proteins promoted the accumulation of branched-chain amino acids (BCAA) and TCA metabolites upstream of the mitochondrial oxoglutarate dehydrogenase complex (OGDC). Alt. UGT1A1 exacerbated these changes, likely through its interaction with the OGDC component oxoglutarate dehydrogenase-like (OGDHL). This study expands the breadth of biochemical pathways associated with UGT expression and establishes extensive connectivity between UGT enzymes, alt. proteins and other metabolic processes