Role of the proteasome complex in degradation of human CYP2E1 in transfected HepG2 cells.
Role of the proteasome complex in degradation of human CYP2E1 in transfected HepG2 cells.
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蛋白酶体复合物在转染的 HepG2 细胞中人 CYP2E1 降解中的作用。
DOI：
10.1006/bbrc.1996.1418
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Cederbaum,AI
中科院分区：
文献类型：
--
作者：
Yang,MX;Cederbaum,AI
本研究的目的是表征人CYP 2 E1营业额和检查的蛋白酶体蛋白水解途径中的快速降解CYP 2 E1在转染的HepG 2细胞系表达人CYP 2 E1的可能作用。从MVh 2 E1 -9细胞中分离的微粒体催化表达的CYP 2 E1的缓慢降解，通过加入4-甲基吡唑（一种稳定CYP 2 E1的配体）可防止这种降解。向微粒体中加入HepG 2细胞的胞质组分可使CYP 2 E1快速降解。这种快速降解需要MgATP，并通过4-甲基吡唑完全防止。用[35 S]-甲硫氨酸标记CYP 2 E1和用抗人CYP 2 E1 IgG免疫沉淀后的脉冲追踪实验表明CYP 2 E1的双相转换，半衰期为2.5和6小时。加入Czb-Ile-Glu（OtBu）-Ala-Leucinal（PSI）作为细胞穿透蛋白酶体抑制剂，浓度范围为5 - 80 μM，可防止CYP 2 E1降解。PSI还增加了CYP 2 E1的稳态蓄积，与其抑制CYP 2 E1转换一致。这些结果表明，蛋白酶体复合物在转染的HepG 2细胞中的人CYP 2 E1的降解中起主要作用。
The aim of the present study was to characterize human CYP2E1 turnover and examine the possible role of the proteasome proteolytic pathway in the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Microsomes isolated from MVh2E1-9 cells catalyzed a slow degradation of the expressed CYP2E1, which was prevented by the addition of 4-methylpyrazole, a ligand which stabilizes CYP2E1. The addition of the cytosolic fraction of the HepG2 cells to the microsomes produced rapid degradation of CYP2E1. This rapid degradation required MgATP and was completely prevented by 4-methylpyrazole. Pulse-chase experiments after labeling CYP2E1 with [35S]-methionine and immunoprecipitation with anti-human CYP2E1 IgG indicated a biphasic turnover of CYP2E1 with half-lives of 2.5 and 6 hours. The addition of Czb-Ile-Glu(OtBu)-Ala-Leucinal(PSI) as a cell penetrating proteasome inhibitor, at concentrations ranging from 5 to 80 μM resulted in protection against the degradation of CYP2E1. PSI also increased the steady state accumulation of CYP2E1, consistent with its inhibition of CYP2E1 turnover. These results suggest that the proteasome complex plays a major role in the degradation of human CYP2E1 in the transfected HepG2 cells.