Modulation of the human glucuronosyltransferase UGT1A pathway by splice isoform polypeptides is mediated through protein-protein interactions.
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K(m) (app)) for all substrates tested and the K(m) for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
Bellemare
Judith
J
From the Pharmacogenomics Laboratory, Centre Hospitalier de l'Université Laval Research Center and Faculty of Pharmacy, Laval University, Québec, Québec G1V 4G2, Canada.
Rouleau
Mélanie
M
From the Pharmacogenomics Laboratory, Centre Hospitalier de l'Université Laval Research Center and Faculty of Pharmacy, Laval University, Québec, Québec G1V 4G2, Canada.
Harvey
Mario
M
From the Pharmacogenomics Laboratory, Centre Hospitalier de l'Université Laval Research Center and Faculty of Pharmacy, Laval University, Québec, Québec G1V 4G2, Canada.
Guillemette
Chantal
C
From the Pharmacogenomics Laboratory, Centre Hospitalier de l'Université Laval Research Center and Faculty of Pharmacy, Laval University, Québec, Québec G1V 4G2, Canada. Electronic address: Chantal.Guillemette@crchul.ulaval.ca.
D017398:Alternative Splicing; D015153:Blotting, Western; D002460:Cell Line; D004440:Ecdysone / Q000494:pharmacology; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization; D015398:Signal Transduction; D013379:Substrate Specificity; D014162:Transfection; D000099148:UGT1A1 Enzyme
D004440:Ecdysone; D020719:Glucuronides; D014453:Glucuronosyltransferase; D007527:Isoenzymes; D010455:Peptides; D000099148:UGT1A1 Enzyme; C454320:UDP-glucuronosyltransferase, UGT1A8; C524630:UGT1A7 protein, human