Murray B P, Correia M A
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.
Arch Biochem Biophys. 2001 Sep 1;393(1):106-16. doi: 10.1006/abbi.2001.2482.
Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs. We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. Biophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structurally damaged. To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Delta, hrd2-1, and hrd3Delta) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al. (1996) Mol. Biol. Cell 7, 2029; Hampton and Bhakta (1997) Proc. Natl. Acad. Sci. USA 94, 12,944). Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub-ligase complex. Furthermore, studies in a S. cerevisiae pep4Delta strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function. The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains. Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins.
细胞色素P450，CYP3A4，是人类肝脏内质网（ER）中占主导地位的血红素蛋白酶，负责60%以上临床相关药物的代谢。我们之前已经表明，通过对CYP3A4及其大鼠肝脏内质网同源物CYPs 3A的蛋白质部分进行血红素修饰，基于机制的自杀失活会导致它们依赖泛素（Ub）的26S蛋白酶体降解（Korsmeyer等人，（1999年）《生物化学与生物物理学报》365卷，31页；Wang等人，（1999年）《生物化学与生物物理学报》365卷，45页）。鉴于血红素修饰的CYP3A蛋白在结构上受到破坏，这并不奇怪。为了确定天然酶的周转是否同样利用了这条途径，我们在野生型酿酒酵母和突变菌株（hrd1Delta、hrd2 - 1和hrd3Delta）中异源表达了这种蛋白质，这些菌株之前已被证明在多跨膜内质网蛋白3 - 羟基 - 3 - 甲基戊二酰辅酶A还原酶（异构体Hmg2p）的依赖Ub的26S蛋白酶体降解方面存在缺陷，Hmg2p是甾醇生物合成中的限速酶，我们还在缺乏内质网相关Ub结合酶Ubc6p和/或Ubc7p的菌株中进行了研究（Hampton等人，（1996年）《分子生物学细胞》7卷，2029页；Hampton和Bhakta，（1997年）《美国国家科学院院刊》94卷，12944页）。我们的研究结果表明，与Hmg2p的降解一样，天然CYP3A4的降解也需要Hrd2p（26S蛋白酶体19S帽复合物的一个亚基）和Ubc7p，在较小程度上还需要Hrd3p，它是内质网相关Ub连接酶复合物中的一个成分。与Hmg2p的降解不同，天然CYP3A4的降解似乎并不绝对需要Hrd1p，Hrd1p是内质网相关Ub连接酶复合物的另一个成分。此外，在已被证明在羧肽酶Y的液泡降解方面存在缺陷的酿酒酵母pep4Delta菌株中的研究表明，CYP3A4的降解在很大程度上也不依赖液泡（溶酶体）的蛋白水解功能。我们还平行研究了另外两种天然内质网蛋白Sec61p和Sec63p（内质网转运体的正常成分）的降解情况，发现在缺乏HRD2和UBC7的菌株中它们在一定程度上得到了稳定。这些研究结果共同证明了内质网蛋白正常降解过程中显著的机制多样性。
