133 research outputs found
UGT pharmacogenomics in drug metabolism and diseases
Glucuronidation, mediated by UDP-glucuronosyltransferase enzymes (UGTs), is a major phase
II biotransformation pathway and, complementary to phase I metabolism and membrane
transport, one of the most important cellular defense mechanism responsible for the inactivation
of therapeutic drugs, other xenobiotics and numerous endogenous molecules. Individual
variability in UGT enzymatic pathways is significant and may have profound pharmacological
and toxicological implications. Several genetic and genomic processes are underlying this
variability and are discussed in the context of drug metabolism and diseases such as cancer
Roles of UGT1A splice variants in cellular defense.
Transcripts of the UGT1A gene, encoding half of human UGT enzymes, undergo alternative
splicing, resulting in active enzymes named isoforms 1 (i1s) and novel truncated isoforms 2
(i2s). Here, we investigated the effects of depleting endogenous i2 on drug response and to
unveil any additional biological role(s) for the truncated novel UGT proteins. We used an
integrated systems biology approach that combines RNA interference with unbiased global
genomic and proteomic screens, and used the HT115 colorectal cancer cells as a model.
Consistent with previous evidence suggesting that i2s negatively regulate i1s through proteinprotein
interactions, i2-depleted cells were less sensitive to drug-induced cell death (IC50 of 0.45
± 0.05 ”M vs. 0.22 ± 0.03 ”M; P=0.006), demonstrating that modulation of i2 levels
meaningfully impacts drug bioavailability and cellular response. We also observed reduced
production of reactive oxygen species by 30 % (P1.2 fold;
P<0.05) of several proteins such as hemoglobin alpha genes and superoxide dismutase 1 that
have network functions associated with antioxidant properties. Interaction proteomics analysis of
endogenous proteins from the cellular model, mainly in human intestine but also in kidney
tissues further uncovered interactions between i2s (but not i1s) and the antioxidant enzymes
catalase and peroxiredoxin 1, which may influence antioxidant potential through sequestration of
these novel partners. Our findings demonstrate for the first time dual roles for i2s in the cellular
defense system as endogenous regulators of drug response as well as in oxidative stress
Regulation of UGT1A1 and HNF1 transcription factor gene expression by DNA methylation in colon cancer cells
<p>Abstract</p> <p>Background</p> <p>UDP-glucuronosyltransferase 1A1 (UGT1A1) is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the <it>UGT1A1 </it>5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4) is found close to a HNF1 response element (HRE), known to be involved in activation of <it>UGT1A1 </it>gene expression, and within an upstream stimulating factor (USF) binding site.</p> <p>Results</p> <p>Gel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in <it>UGT1A1 </it>regulation in colon cancer cells. Based on the differential expression profiles of <it>HNF1A </it>gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the <it>HNF1A </it>promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore <it>HNF1A </it>gene expression, as observed for <it>UGT1A1</it>.</p> <p>Conclusions</p> <p>This study reveals that basal <it>UGT1A1 </it>expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of <it>HNF1A </it>could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.</p
UGT genomic diversity : beyond gene duplication
The human uridine diphospho (UDP)-glucuronosyltransferase (UGT) superfamily comprises enzymes responsible for a major biotransformation phase II pathway: the glucuronidation process. The UGT enzymes are located in the endoplasmic reticulum of almost all tissues, where they catalyze the inactivation of several endogenous and exogenous molecules, including bilirubin, sex steroids, numerous prescribed drugs, and environmental toxins. This metabolic pathway is particularly variable. The influence of inheritable polymorphisms in human UGT-encoding genes has been extensively documented and was shown to be responsible for a fraction of the observed phenotypic variability. Other key genomic processes are likely underlying this diversity; these include copy-number variations, epigenetic factors, and newly discovered splicing mechanisms. This review will discuss novel molecular aspects that may be determinant to UGT phenotypes
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 (38â263% 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
Modulation of the UGT2B7 Enzyme Activity by C-Terminally Truncated Proteins Derived from Alternative Splicing
Immunohistochemical expression of conjugating UGT1A-derived splice proteins in normal and tumoral drug-metabolising tissues in humans
Glucuronidation by UDP-glucuronyltransferase (UGT) enzymes is the prevailing conjugative pathway for the metabolism of both xenobiotics and endogenous compounds. Alterations in this pathway, such as those generated by common genetic polymorphisms, have been shown to significantly impact on the health of individuals, influencing cancer susceptibility, responsiveness to drugs and drug-induced toxicity. Alternative usage of terminal exons leads to UGT1A-derived splice variants, namely the classical and enzymatically active isoforms 1 (i1) and the novel enzymatically inactive isoforms 2 (i2). In vitro functional data from heterologous expression and RNA interference experiments indicate that these i2 isoforms act as negative modulators of glucuronidation, likely by forming inactive complexes with active isoform 1. We used specific antibodies against either active i1 or inactive i2 proteins to examine their distribution in major drug-metabolizing tissues. Data revealed that UGT1A_i1 and inactive UGT1A_i2 are co-produced in the same tissue structures, including liver, kidney, stomach, intestine and colon. Examination of the cellular distribution and semi-quantitative level of expression of UGT1As revealed heterogeneous expression of i1 and i2 proteins, with increased expression of i2 in liver tumours and decreased levels of i1 and i2 in colon cancer specimens, compared to normal tissues. These differences in expression may be relevant to human colon and liver cancer tumorigenesis. Our data clearly demonstrate the similar immunolocalization of active and inactive UGT1A isoforms in most UGT1A-expressing cell types of major tissues involved in drug metabolism. These expression patterns are consistent with a dominant-negative function for the i2 encoded by the UGT1A gene
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 5â and/or 3â 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
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