Liao Mingxiang, Faouzi Saadia, Karyakin Andrey, Correia Maria Almira
Dept. of Cellular and Molecular Pharmacology, Box 2280, University of California-San Francisco, CA 94143-2280, USA.
Mol Pharmacol. 2006 Jun;69(6):1897-904. doi: 10.1124/mol.105.021816. Epub 2006 Mar 23.
The monotopic, endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) undergo variable proteolytic turnover. CYP3A4, the dominant human liver drug-metabolizing enzyme, is degraded via a ubiquitin (Ub)-dependent 26S proteasomal pathway after heterologous expression in Saccharomyces cerevisiae. This turnover involves the Ub-conjugating enzyme Ubc7p and the 19S proteasomal subunit Hrd2p but is independent of Hrd1p/Hrd3p, a major Ub-ligase (E3) involved in ER protein degradation. We now show that CYP3A4 ERAD also involves the Ubc7p-ER anchor Cue1p, because CYP3A4 is significantly stabilized at the stationary growth phase in Cue1p-deficient yeast. To determine whether the other major Ub-ligase Doa10p or Rsp5p involved in ER protein degradation functions in CYP3A4 ERAD, wild type and Doa10p- or Rsp5p-deficient yeast strains were also similarly examined. No appreciable CYP3A4 stabilization was detected in either Doa10p- or Rsp5p-deficient yeast, thereby excluding these E3s and revealing that CYP3A4 ERAD involves a novel or yet to be identified E3. Similar studies also revealed that the Cdc48p-Ufd1p-Hrd4p complex, responsible for the translocation of polyubiquitinated ER proteins was critical for CYP3A4 ERAD. We previously reported that grafting of the C-terminal (CT) CYP3A4 heptapeptide onto the CYP2B1 C terminus switched its proteolytic susceptibility from predominantly vacuolar to proteasomal degradation. To determine the relevance of this CT heptapeptide to CYP3A4 ERAD, CYP3A4 degradation after CT heptapeptide-deletion (CYP3A4DeltaCT) was similarly examined in yeast. These findings revealed that CYP3A4DeltaCT was also degraded by Ubc7p-26S proteasomal pathway, thereby indicating that this CT heptapeptide is not critical for CYP3A4 proteasomal degradation. Thus, unlike CYP2B1, CYP3A4 harbors additional/multiple structural degrons for its recruitment into the Ubproteasomal pathway.
单一位点、内质网（ER）锚定的细胞色素P450（P450s）经历可变的蛋白水解周转。CYP3A4是主要的人肝脏药物代谢酶，在酿酒酵母中异源表达后通过泛素（Ub）依赖性26S蛋白酶体途径降解。这种周转涉及Ub缀合酶Ubc7p和19S蛋白酶体亚基Hrd2p，但独立于参与ER蛋白降解的主要Ub连接酶（E3）Hrd1p/Hrd3p。我们现在表明，CYP3A4内质网相关降解（ERAD）也涉及Ubc7p-ER锚定蛋白Cue1p，因为在Cue1p缺陷型酵母的稳定生长期CYP3A4显著稳定。为了确定参与ER蛋白降解的其他主要Ub连接酶Doa10p或Rsp5p是否在CYP3A4 ERAD中起作用，也类似地检测了野生型以及Doa10p或Rsp5p缺陷型酵母菌株。在Doa10p或Rsp5p缺陷型酵母中均未检测到明显的CYP3A4稳定化，从而排除了这些E3，并揭示CYP3A4 ERAD涉及一种新的或尚未鉴定的E3。类似的研究还表明，负责多泛素化ER蛋白转运的Cdc48p-Ufd1p-Hrd4p复合物对CYP3A4 ERAD至关重要。我们之前报道过，将CYP3A4 C末端（CT）七肽嫁接到CYP2B1 C末端会将其蛋白水解敏感性从主要的液泡降解转变为蛋白酶体降解。为了确定该CT七肽与CYP3A4 ERAD的相关性，在酵母中类似地检测了CT七肽缺失后（CYP3A4DeltaCT）的CYP3A4降解。这些发现表明CYP3A4DeltaCT也通过Ubc7p-26S蛋白酶体途径降解，从而表明该CT七肽对CYP3A4蛋白酶体降解并不关键。因此，与CYP2B1不同，CYP3A4具有额外的/多个结构降解基序，用于其被招募到泛素-蛋白酶体途径中。
