Goasduff T, Cederbaum A I
Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Arch Biochem Biophys. 2000 Jul 15;379(2):321-30. doi: 10.1006/abbi.2000.1870.
One major mode of regulation of cytochrome P450 2E1 (CYP2E1) is at the posttranscriptional level, since many low-molecular-weight compounds stabilize the enzyme against proteolysis by the proteasome complex. In an in vitro system containing human liver microsomes, degradation of CYP2E1 in the microsomes required addition of the human liver cytosol fraction in a reaction sensitive to inhibitors of the proteasome complex. It is not clear how CYP2E1 in the microsomal membrane becomes accessible to the cytosolic proteasome. Since molecular chaperones play a role in protein folding and degradation, the possible role of heat shock proteins in CYP2E1 degradation by this reconstituted system was evaluated. Degradation of CYP2E1 required ATP; ATP-gammaS, a nonhydrolyzable analogue of ATP, did not catalyze CYP2E1 degradation by the cytosol fraction, indicating that ATP hydrolysis is required. Geldanamycin, a specific inhibitor of hsp90, inhibited the degradation of microsomal CYP2E1 by the cytosol fraction. Control experiments indicated that geldanamycin was not a substrate/ligand of CYP2E1 nor did it prevent microsomal lipid peroxidation, a process which increases CYP2E1 turnover. Inhibition by geldanamycin was prevented by molybdate. Both of these compounds have been shown to promote alterations in hsp90 structure and to modulate hsp90-protein interactions. The proteasome activity in the cytosol, as assayed by the cleavage of a fluorogenic peptide, was enhanced when ATP was added and inhibited by 30-40% by geldanamycin, effects that are similar, although less pronounced, to the degradation of CYP2E1 by the cytosol. Purified 20S proteasome could catalyze degradation of CYP2E1; however, in an assay using equal peptidase activity, the cytosol fraction was much more effective than the 20S proteasome in promoting CYP2E1 degradation. Immunodepletion of hsp90 from the cytosol resulted in prevention of the degradation of CYP2E1, a reaction that was reversed by the addition of pure hsp90 to this cytosol. These results suggest that in addition to the proteasome, the cytosol fraction contains other factors that modulate the efficiency of CYP2E1 degradation. The sensitivity to geldanamycin and molybdate and the immunodepletion experiments suggest that hsp90 is one of these factors that interact with CYP2E1 and/or with the proteasome to promote the degradation of this microsomal P450.
细胞色素P450 2E1（CYP2E1）的一种主要调节模式是在转录后水平，因为许多低分子量化合物可稳定该酶，使其免受蛋白酶体复合物的蛋白水解作用。在包含人肝微粒体的体外系统中，微粒体中CYP2E1的降解需要加入人肝细胞溶质部分，该反应对蛋白酶体复合物抑制剂敏感。尚不清楚微粒体膜中的CYP2E1如何被胞质蛋白酶体识别。由于分子伴侣在蛋白质折叠和降解中起作用，因此评估了热休克蛋白在该重组系统中对CYP2E1降解的可能作用。CYP2E1的降解需要ATP；ATP-γS是一种不可水解的ATP类似物，它不能催化CYP2E1被胞质部分降解，这表明需要ATP水解。格尔德霉素是hsp90的特异性抑制剂，它抑制了微粒体CYP2E1被胞质部分降解。对照实验表明，格尔德霉素不是CYP2E1的底物/配体，也不能防止微粒体脂质过氧化，而脂质过氧化会增加CYP2E1的周转。钼酸盐可阻止格尔德霉素的抑制作用。这两种化合物均已显示可促进hsp90结构的改变并调节hsp90-蛋白质相互作用。当加入ATP时，通过荧光肽的裂解测定的胞质中蛋白酶体活性增强，而被格尔德霉素抑制30-40%，这些效应与CYP2E1被胞质部分降解相似，尽管不太明显。纯化的20S蛋白酶体可以催化CYP2E1的降解；然而，在使用相等肽酶活性的测定中，胞质部分在促进CYP2E1降解方面比20S蛋白酶体有效得多。从胞质中免疫去除hsp90可防止CYP2E1的降解，向该胞质中加入纯hsp90可使该反应逆转。这些结果表明，除蛋白酶体外，胞质部分还含有其他调节CYP2E1降解效率的因子。对格尔德霉素和钼酸盐的敏感性以及免疫去除实验表明，hsp90是这些与CYP2E1和/或蛋白酶体相互作用以促进这种微粒体P450降解的因子之一。
