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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.


Detection of the FRET phenomenon using the acceptor photobleaching method in UGT1A-CFP and UGT1A-YFP co-infected cells.Sf9 cells were co-infected with recombinant CFP and YFP Baculovirus (negative control, A) and no increase in CFP fluorescence was observed in the circle zones bleached by the 515 nm laser line. In CFP-linker-YFP (positive control, B) UGT1A1*1-CFP + UGT1A1*1-YFP (C) and UGT1A1*9-CFP + UGT1A1*9-YFP (D)-infected cells, an increase in CFP fluorescence was observed in the circle zones bleached by the 515 nm laser line indicating oligomerization between UGT1A1 or UGT1A9 allozymes.
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f2: Detection of the FRET phenomenon using the acceptor photobleaching method in UGT1A-CFP and UGT1A-YFP co-infected cells.Sf9 cells were co-infected with recombinant CFP and YFP Baculovirus (negative control, A) and no increase in CFP fluorescence was observed in the circle zones bleached by the 515 nm laser line. In CFP-linker-YFP (positive control, B) UGT1A1*1-CFP + UGT1A1*1-YFP (C) and UGT1A1*9-CFP + UGT1A1*9-YFP (D)-infected cells, an increase in CFP fluorescence was observed in the circle zones bleached by the 515 nm laser line indicating oligomerization between UGT1A1 or UGT1A9 allozymes.

Mentions: As shown in Fig. 2, compared to the negative control (Fig. 2A), the fluorescence intensity of CFP channel showed a significant increase in the number of CFP-linker-YFP (Fig. 2B) infected cells and UGT1A1*1-CFP/UGT1A1*1-YFP (Fig. 2C) and UGT1A9*1-CFP/UGT1A9*1-YFP (Fig. 2D) co-infected cells. The significant FRET phenomenon indicated that two proteins reside in close proximity to each other within the ER membrane; confirming oligomerization among UGT1A1 or UGT1A9 allozymes. FRET efficiencies and donor-acceptor r distances were calculated to visually exhibit the interactions of the two protein isoforms (Table 1). FRET is a powerful technique that can be applied to measure distances between 1 and 10 nm, which is small enough to characterize the proximity of interacting molecules262728. The donor-acceptor r distance correlated to each pair of UGT1A1 or UGT1A9 allozymes revealed a dimeric distance29. These results suggested that, all UGT1A1 and UGT1A9 allozymes used in this study were capable of oligomerization. It has been reported that FRET donor-acceptor distance is positively associated with tagged domain-domain distance30, in other words, FRET donor-acceptor distance reflects the interaction ability between target proteins. Interestingly, the donor-acceptor distance of two non-functional UGT1A1*1b was slightly smaller than wild-type UGT1A1; conversely, UGT1A1*1/UGT1A1*1b dimer showed weaker interaction than UGT1A1*1/1A1*1 dimer. This suggests that the alternative splice is associated with interaction ability of UGT1A1. In UGT1A9 FRET analysis, UGT1A9*1/1A9*1 exhibited shorter donor-acceptor r distance and stronger interaction than the other three self-interaction dimers. UGT1A9*1/1A9*3 and UGT1A9*1/1A9*5 showed stronger interactions than that of UGT1A9*3/1A9*3 and UGT1A9*5/1A9*5. This indicates that the interaction ability is somewhat decreased among mutants and the loss of interaction can be partially recovered by interacting with wild-type protein. UGT1A9*2/1A9*3 and UGT1A9*2/1A9*5 exhibited similar interaction ability to UGT1A9*3/1A9*3 and UGT1A9*5/1A9*5. Moreover, the interaction between UGT1A9*1 and UGT1A9*2 was weakened. However, the interaction between UGT1A9*3 and UGT1A9*5 was stronger than that of UGT1A9*5/1A9*5. It seemed that all of the mutants in UGT1A1 and UGT1A9 showed different spatial structure with wild-type protein, and changed the interaction ability of UGTs. The diversity of spatial structure might be associated with substrate specificity and enzymatic activity.


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)

Detection of the FRET phenomenon using the acceptor photobleaching method in UGT1A-CFP and UGT1A-YFP co-infected cells.Sf9 cells were co-infected with recombinant CFP and YFP Baculovirus (negative control, A) and no increase in CFP fluorescence was observed in the circle zones bleached by the 515 nm laser line. In CFP-linker-YFP (positive control, B) UGT1A1*1-CFP + UGT1A1*1-YFP (C) and UGT1A1*9-CFP + UGT1A1*9-YFP (D)-infected cells, an increase in CFP fluorescence was observed in the circle zones bleached by the 515 nm laser line indicating oligomerization between UGT1A1 or UGT1A9 allozymes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Detection of the FRET phenomenon using the acceptor photobleaching method in UGT1A-CFP and UGT1A-YFP co-infected cells.Sf9 cells were co-infected with recombinant CFP and YFP Baculovirus (negative control, A) and no increase in CFP fluorescence was observed in the circle zones bleached by the 515 nm laser line. In CFP-linker-YFP (positive control, B) UGT1A1*1-CFP + UGT1A1*1-YFP (C) and UGT1A1*9-CFP + UGT1A1*9-YFP (D)-infected cells, an increase in CFP fluorescence was observed in the circle zones bleached by the 515 nm laser line indicating oligomerization between UGT1A1 or UGT1A9 allozymes.
Mentions: As shown in Fig. 2, compared to the negative control (Fig. 2A), the fluorescence intensity of CFP channel showed a significant increase in the number of CFP-linker-YFP (Fig. 2B) infected cells and UGT1A1*1-CFP/UGT1A1*1-YFP (Fig. 2C) and UGT1A9*1-CFP/UGT1A9*1-YFP (Fig. 2D) co-infected cells. The significant FRET phenomenon indicated that two proteins reside in close proximity to each other within the ER membrane; confirming oligomerization among UGT1A1 or UGT1A9 allozymes. FRET efficiencies and donor-acceptor r distances were calculated to visually exhibit the interactions of the two protein isoforms (Table 1). FRET is a powerful technique that can be applied to measure distances between 1 and 10 nm, which is small enough to characterize the proximity of interacting molecules262728. The donor-acceptor r distance correlated to each pair of UGT1A1 or UGT1A9 allozymes revealed a dimeric distance29. These results suggested that, all UGT1A1 and UGT1A9 allozymes used in this study were capable of oligomerization. It has been reported that FRET donor-acceptor distance is positively associated with tagged domain-domain distance30, in other words, FRET donor-acceptor distance reflects the interaction ability between target proteins. Interestingly, the donor-acceptor distance of two non-functional UGT1A1*1b was slightly smaller than wild-type UGT1A1; conversely, UGT1A1*1/UGT1A1*1b dimer showed weaker interaction than UGT1A1*1/1A1*1 dimer. This suggests that the alternative splice is associated with interaction ability of UGT1A1. In UGT1A9 FRET analysis, UGT1A9*1/1A9*1 exhibited shorter donor-acceptor r distance and stronger interaction than the other three self-interaction dimers. UGT1A9*1/1A9*3 and UGT1A9*1/1A9*5 showed stronger interactions than that of UGT1A9*3/1A9*3 and UGT1A9*5/1A9*5. This indicates that the interaction ability is somewhat decreased among mutants and the loss of interaction can be partially recovered by interacting with wild-type protein. UGT1A9*2/1A9*3 and UGT1A9*2/1A9*5 exhibited similar interaction ability to UGT1A9*3/1A9*3 and UGT1A9*5/1A9*5. Moreover, the interaction between UGT1A9*1 and UGT1A9*2 was weakened. However, the interaction between UGT1A9*3 and UGT1A9*5 was stronger than that of UGT1A9*5/1A9*5. It seemed that all of the mutants in UGT1A1 and UGT1A9 showed different spatial structure with wild-type protein, and changed the interaction ability of UGTs. The diversity of spatial structure might be associated with substrate specificity and enzymatic activity.

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.