title: Protein-protein interactions between the bilirubin-conjugating UDP-glucuronosyltransferase UGT1A1 and its shorter isoform 2 regulatory partner derived from alternative splicing.
abstract: 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.
authors.affiliation: Pharmacogenomics 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.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Rouleau
authors.forename: Pierre
authors.initials: P
authors.lastname: Collin
authors.forename: Judith
authors.initials: J
authors.lastname: Bellemare
authors.forename: Mario
authors.initials: M
authors.lastname: Harvey
authors.forename: Chantal
authors.initials: C
authors.lastname: Guillemette
mesh_terms: D017398:Alternative Splicing; D001663:Bilirubin / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000099148:UGT1A1 Enzyme
chemical_list: D001663:Bilirubin; D014453:Glucuronosyltransferase; D020033:Protein Isoforms; D000099148:UGT1A1 Enzyme