Alteration of the Function of the UDP-Glucuronosyltransferase 1A Subfamily by Cytochrome P450 3A4: Different Susceptibility for UGT Isoforms and UGT1A1/7 Variants
Alteration of the Function of the UDP-Glucuronosyltransferase 1A Subfamily by Cytochrome P450 3A4: Different Susceptibility for UGT Isoforms and UGT1A1/7 Variants
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DOI：
10.1124/dmd.113.054833
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发表时间：
2014-02-01
影响因子：
3.9
通讯作者：
Yamada, Hideyuki
中科院分区：
文献类型：
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作者：
Ishii, Yuji;Koba, Hiroki;Yamada, Hideyuki
研究了 UDP-葡萄糖醛酸基转移酶 (UGT)1A 亚型和细胞色素 P450 (CYP)3A4 之间的功能性蛋白质-蛋白质相互作用。为此，在同时表达 UGT 和 CYP3A4 的 Sf-9 细胞中测定了 UGT1A 催化的葡萄糖醛酸化。在 UGT1A6 催化的血清素葡萄糖醛酸化动力学中，CYP3A4 增加了米氏常数 (K-m) 和最大速度 (V-max)。当 CYP3A4 与 UGT1A1 或 1A7 共表达时，伊立替康代谢物 (SN-38) 葡萄糖醛酸化的 V-max 显着增加。 S-50和K-m均为给出0.5V-max的底物浓度，几乎不受CYP3A4同时表达的影响。这项研究还检查了 UGT1A1 和 1A7 等位基因变体的催化特性及其对与 CYP3A4 相互作用的影响。尽管 UGT1A1 催化 4-甲基伞形酮葡萄糖醛酸化的活性在其变体 UGT1A1(star)6 中降低，但 CYP3A4 的共表达将受损的功能恢复到与野生型相当的水平。类似地，CYP3A4的同时表达增加了UGT1A7(star)1（野生型）和（star）2（N129K和R131K）的Vmax，而在UGT1A7（star）3（N129K、R131K和W208R）中没有观察到相同的情况。在涉及不同浓度的 UDP 葡萄糖醛酸 (UDP-GlcUA) 的动力学中，UGT1A7(star)2 和 (star)3 的 UDP-GlcUA 的 Km 显着高于 (star)1。 CYP3A4 增加了 UGT1A7(star)1 和 (star)3 的 Km，而 (star)2 没有表现出任何此类变化。这些结果表明（1）CYP3A4以UGT亚型特异性方式改变UGT1A亚家族的催化功能，（2）UGT1A7（星）3中的非同义突变不仅降低UGT使用UDP-GlcUA的能力，而且降低CYP3A4介导的催化活性增强，而CYP3A4能够恢复UGT1A1（星）6功能。
Functional protein-protein interactions between UDP-glucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells that simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of serotonin, both Michaelis constant (K-m) and maximal velocity (V-max) were increased by CYP3A4. When CYP3A4 was coexpressed with either UGT1A1 or 1A7, the V-max for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. S-50 and K-m both which are the substrate concentration giving 0.5 V-max were little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7 and their effects on the interaction with CYP3A4. Although the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1(star)6, the coexpression of CYP3A4 restored the impaired function to a level comparable with the wild type. Similarly, simultaneous expression of CYP3A4 increased the Vmax of UGT1A7(star)1 (wild type) and (star)2 (N129K and R131K), whereas the same was not observed in UGT1A7(star)3 (N129K, R131K, and W208R). In the kinetics involving different concentrations of UDPglucuronic acid (UDP-GlcUA), the Km for UDP-GlcUA was significantly higher for UGT1A7(star)2 and (star)3 than (star)1. The Km of UGT1A7(star)1 and (star)3 was increased by CYP3A4, whereas (star)2 did not exhibit any such change. These results suggest that (1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoformspecific manner and (2) nonsynonymous mutations in UGT1A7(star)3 reduce not only the ability of UGT to use UDP-GlcUA but also CYP3A4-mediated enhancement of catalytic activity, whereas CYP3A4 is able to restore the UGT1A1(star)6 function.