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Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Liu YQ, Yuan LM, Gao ZZ, Xiao YS, Sun HY, Yu LS, Zeng S - Sci Rep (2016)

Bottom Line: Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans.Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity.SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances.

View Article: PubMed Central - PubMed

Affiliation: Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.

No MeSH data available.


Analysis of the dimerization of UGT1As by Co-IP.The HA-tagged and CFP-tagged proteins were directly mixed and no CFP-tagged protein bands were observed after Co-IP (A,B). The HA- and CFP-tagged proteins were co-expressed in host cells followed by anti-HA beads immunoprecipitation and western blotting detection, wherein two bands (56 kDa and 81 kDa) were observed (C,D).
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f3: Analysis of the dimerization of UGT1As by Co-IP.The HA-tagged and CFP-tagged proteins were directly mixed and no CFP-tagged protein bands were observed after Co-IP (A,B). The HA- and CFP-tagged proteins were co-expressed in host cells followed by anti-HA beads immunoprecipitation and western blotting detection, wherein two bands (56 kDa and 81 kDa) were observed (C,D).

Mentions: The HA- and CFP-tagged fusion proteins were expressed in Sf9 cells individually. Solubilized proteins were mixed with anti-HA beads, and resuspended protein was detected by western blotting using anti-UGT1A antibody (Fig. S2). As a result, only HA-tagged proteins (56 kDa) were pulled down and revealed bands in western blotting, suggesting that CFP-tagged (81 kDa) proteins were not immunoprecipitated with beads. The HA-tagged and CFP-tagged proteins were directly mixed, and no CFP-tagged protein bands were observed after Co-IP as well (Fig. 3A,B). Furthermore, the HA- and CFP-tagged proteins were co-expressed in host cells followed by anti-HA beads immunoprecipitation and western blotting detection. Interestingly, two bands (56 kDa and 81 kDa) were observed, which suggested that the HA-tagged protein immunoprecipitated with CFP-tagged protein (Fig. 3C,D). As no signals were detected in directly mixed samples, intracellular environment was therefore necessary for the interaction between two forms of UGT1A. Co-IP analysis strengthened the results of FRET analysis and supported direct evidence for dimerization among UGT1A1 or UGT1A9 allozymes.


Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Liu YQ, Yuan LM, Gao ZZ, Xiao YS, Sun HY, Yu LS, Zeng S - Sci Rep (2016)

Analysis of the dimerization of UGT1As by Co-IP.The HA-tagged and CFP-tagged proteins were directly mixed and no CFP-tagged protein bands were observed after Co-IP (A,B). The HA- and CFP-tagged proteins were co-expressed in host cells followed by anti-HA beads immunoprecipitation and western blotting detection, wherein two bands (56 kDa and 81 kDa) were observed (C,D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837415&req=5

f3: Analysis of the dimerization of UGT1As by Co-IP.The HA-tagged and CFP-tagged proteins were directly mixed and no CFP-tagged protein bands were observed after Co-IP (A,B). The HA- and CFP-tagged proteins were co-expressed in host cells followed by anti-HA beads immunoprecipitation and western blotting detection, wherein two bands (56 kDa and 81 kDa) were observed (C,D).
Mentions: The HA- and CFP-tagged fusion proteins were expressed in Sf9 cells individually. Solubilized proteins were mixed with anti-HA beads, and resuspended protein was detected by western blotting using anti-UGT1A antibody (Fig. S2). As a result, only HA-tagged proteins (56 kDa) were pulled down and revealed bands in western blotting, suggesting that CFP-tagged (81 kDa) proteins were not immunoprecipitated with beads. The HA-tagged and CFP-tagged proteins were directly mixed, and no CFP-tagged protein bands were observed after Co-IP as well (Fig. 3A,B). Furthermore, the HA- and CFP-tagged proteins were co-expressed in host cells followed by anti-HA beads immunoprecipitation and western blotting detection. Interestingly, two bands (56 kDa and 81 kDa) were observed, which suggested that the HA-tagged protein immunoprecipitated with CFP-tagged protein (Fig. 3C,D). As no signals were detected in directly mixed samples, intracellular environment was therefore necessary for the interaction between two forms of UGT1A. Co-IP analysis strengthened the results of FRET analysis and supported direct evidence for dimerization among UGT1A1 or UGT1A9 allozymes.

Bottom Line: Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans.Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity.SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances.

View Article: PubMed Central - PubMed

Affiliation: Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.

No MeSH data available.