The oligomerization of UGTs [UDP (uridine diphosphate)-glucuronosyltransferases] modulates their enzyme activities. Recent findings also indicate that glucuronidation is negatively regulated by the formation of inactive oligomeric complexes between UGT1A enzymes [i1 (isoform 1)] and an enzymatically inactive alternatively spliced i2 (isoform 2). In the present paper, we assessed whether deletion of the UGT-interacting domains previously reported to be critical for enzyme function might be involved in i1–i2 interactions. The bilirubin-conjugating UGT1A1 was used as a prototype. We also explored whether intermolecular disulfide bonds are involved in i1–i2 interactions and the potential role of selected cysteine residues. Co-immunoprecipitation assays showed that UGT1A1 lacking the SP (signal peptide) alone or also lacking the transmembrane domain (absent from i2) did not self-interact, but still interacted with i2. The deletion of other N- or C-terminal domains did not compromise i1–i2 complex formation. Under non-reducing conditions, we also observed formation of HMWCs (high-molecular-mass complexes) for cells overexpressing i1 and i2. The presence of UGTs in these complexes was confirmed by MS. Mutation of individual cysteine residues throughout UGT1A1 did not compromise i1–i1 or i1–i2 complex formation. These findings are compatible with the hypothesis that the interaction between i1 and i2 proteins (either transient or stable) involves binding of more than one domain that probably differs from those involved in i1–i1 interactions.
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Research Article|
January 24 2013
Protein–protein interactions between the bilirubin-conjugating UDPglucuronosyltransferase UGT1A1 and its shorter isoform 2 regulatory partner derived from alternative splicing
Mélanie Rouleau;
Mélanie Rouleau
1Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHUQ) Research Center, 2705 Boul. Laurier, Québec, Canada, G1V 4G2, and Faculty of Pharmacy, Laval University, Pavillon Ferdinand-Vandry, Université Laval, Québec, Canada, G1V 0A6
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Pierre Collin;
Pierre Collin
1Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHUQ) Research Center, 2705 Boul. Laurier, Québec, Canada, G1V 4G2, and Faculty of Pharmacy, Laval University, Pavillon Ferdinand-Vandry, Université Laval, Québec, Canada, G1V 0A6
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Judith Bellemare;
Judith Bellemare
1Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHUQ) Research Center, 2705 Boul. Laurier, Québec, Canada, G1V 4G2, and Faculty of Pharmacy, Laval University, Pavillon Ferdinand-Vandry, Université Laval, Québec, Canada, G1V 0A6
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Mario Harvey;
Mario Harvey
1Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHUQ) Research Center, 2705 Boul. Laurier, Québec, Canada, G1V 4G2, and Faculty of Pharmacy, Laval University, Pavillon Ferdinand-Vandry, Université Laval, Québec, Canada, G1V 0A6
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Chantal Guillemette
Chantal Guillemette
1
1Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHUQ) Research Center, 2705 Boul. Laurier, Québec, Canada, G1V 4G2, and Faculty of Pharmacy, Laval University, Pavillon Ferdinand-Vandry, Université Laval, Québec, Canada, G1V 0A6
1To whom correspondence should be addressed (email chantal.guillemette@crchul.ulaval.ca).
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Publisher: Portland Press Ltd
Received:
October 16 2012
Revision Received:
November 12 2012
Accepted:
November 14 2012
Accepted Manuscript online:
November 14 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 450 (1): 107–114.
Article history
Received:
October 16 2012
Revision Received:
November 12 2012
Accepted:
November 14 2012
Accepted Manuscript online:
November 14 2012
Citation
Mélanie Rouleau, Pierre Collin, Judith Bellemare, Mario Harvey, Chantal Guillemette; Protein–protein interactions between the bilirubin-conjugating UDPglucuronosyltransferase UGT1A1 and its shorter isoform 2 regulatory partner derived from alternative splicing. Biochem J 15 February 2013; 450 (1): 107–114. doi: https://doi.org/10.1042/BJ20121594
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