Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells.
Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells.
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DOI：
10.1016/j.taap.2003.12.019
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发表时间：
2004-09
影响因子：
3.8
通讯作者：
J. Huan;J. Streicher;L. Bleyle;D. Koop
中科院分区：
文献类型：
--
作者：
J. Huan;J. Streicher;L. Bleyle;D. Koop
研究了四环素诱导的HeLa细胞株中乙醇诱导的细胞色素P450 2E1 （CYP2E1）和苯巴比妥诱导的细胞色素P450 2B1 （CYP2B1）的降解。采用稳态脉冲追踪分析确定CYP2E1的半衰期为3.8 h，而CYP2B1的半衰期在同一细胞系中是其2.3倍。相比之下，在Tc-HeLa细胞中组成表达的NADPH细胞色素P450还原酶的半衰期约为30小时。Lactacystin和其他选择性蛋白酶体抑制剂包括n -benzyloxycarbonyl-leucyl-leucyl- leucyl-leucyl- leucyl-leucyl-l - normvalinal （MG115）和n -benzyloxycarbonyl-l-leucyl-l-leucyl-l- normvalinal （MG115）显著抑制CYP2E1和CYP2B1的降解。calpain抑制剂可轻微抑制CYP2E1的周转，而CYP2B1的周转不受影响。溶酶体蛋白水解抑制剂对两种蛋白的降解均无影响。用brefeldin A处理细胞没有改变P450的降解，这表明降解发生在内质网（ER）。即使存在蛋白酶体抑制剂，也未观察到高分子量泛素偶联物。两个假定的泛素化位点（赖氨酸317和324）的突变没有改变CYP2E1的降解。在含有耐热泛素激活酶（E1）的中国仓鼠突变细胞系E36ts20中，研究了泛素化在CYP2E1降解中的作用。与对照E36细胞相比，ts20细胞在非允许温度下CYP2E1的周转无显著差异。此外，hsp90抑制剂格尔达霉素、herbyycin和radicicol的加入对CYP2E1的周转没有影响，这将CYP2E1的降解与其他蛋白酶体依赖性降解的底物区分开来。
The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-l-leucyl-l-leucyl-l-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.