TY - JOUR
T1 - The human UGT2B7 nanodisc
AU - Cook, Ian
AU - Asenjo, Anna B.
AU - Sosa, Hernando
AU - Leyh, Thomas S.
N1 - Publisher Copyright:
© 2020 by The American Society for Pharmacology and Experimental Therapeutics.
PY - 2020
Y1 - 2020
N2 - The 20 uridine diphosphate glycosyl-transferases (UGTs) encoded in the human genome form an essential homeostatic network of overlapping catalytic functions that surveil and regulate the activity and clearance of scores of small molecule metabolites. Biochemical and biophysical UGT studies have been hampered by the inability to purify these membrane-bound proteins. Here, using cell-free expression and nanodisc technology, we assemble and purify to homogeneity the first UGT nanodisc-the human UGT2B7 nanodisc. The complex is readily isolated in milligram quantities. It is stable and its initial-rate parameters are identical within error to those associated with UGT2B7 in microsomal preparations (i.e., Supersomes). The high purity of the nanodisc preparation simplifies UGT assays, which allows complexities traditionally associated with microsomal assays (latency and the albumin effect) to be circumvented. Each nanodisc is shown to harbor a single UGT2B7 monomer. The methods described herein should be widely applicable to UGTs, and these findings are expected to set the stage for experimentalists to more freely explore the structure, function, and biology of this important area of phase II metabolism.
AB - The 20 uridine diphosphate glycosyl-transferases (UGTs) encoded in the human genome form an essential homeostatic network of overlapping catalytic functions that surveil and regulate the activity and clearance of scores of small molecule metabolites. Biochemical and biophysical UGT studies have been hampered by the inability to purify these membrane-bound proteins. Here, using cell-free expression and nanodisc technology, we assemble and purify to homogeneity the first UGT nanodisc-the human UGT2B7 nanodisc. The complex is readily isolated in milligram quantities. It is stable and its initial-rate parameters are identical within error to those associated with UGT2B7 in microsomal preparations (i.e., Supersomes). The high purity of the nanodisc preparation simplifies UGT assays, which allows complexities traditionally associated with microsomal assays (latency and the albumin effect) to be circumvented. Each nanodisc is shown to harbor a single UGT2B7 monomer. The methods described herein should be widely applicable to UGTs, and these findings are expected to set the stage for experimentalists to more freely explore the structure, function, and biology of this important area of phase II metabolism.
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U2 - 10.1124/DMD.119.089946
DO - 10.1124/DMD.119.089946
M3 - Article
C2 - 31892527
AN - SCOPUS:85096716073
SN - 0090-9556
VL - 48
SP - 198
EP - 204
JO - Drug Metabolism and Disposition
JF - Drug Metabolism and Disposition
IS - 3
ER -