Department of Cellular & Molecular Pharmacology, Bioengineering & Therapeutic Sciences and the Liver Center, University of California, San Francisco, California 94158-2517, USA.
J Biol Chem. 2010 Nov 12;285(46):35866-77. doi: 10.1074/jbc.M110.167189. Epub 2010 Sep 6.
CYP3A4 is a dominant human liver cytochrome P450 enzyme engaged in the metabolism and disposition of >50% of clinically relevant drugs and held responsible for many adverse drug-drug interactions. CYP3A4 and its mammalian liver CYP3A orthologs are endoplasmic reticulum (ER)-anchored monotopic proteins that undergo ubiquitin (Ub)-dependent proteasomal degradation (UPD) in an ER-associated degradation (ERAD) process. These integral ER proteins are ubiquitinated in vivo, and in vitro studies have identified the ER-integral gp78 and the cytosolic co-chaperone, CHIP (C terminus of Hsp70-interacting protein), as the relevant E3 Ub-ligases, along with their cognate E2 Ub-conjugating enzymes UBC7 and UbcH5a, respectively. Using lentiviral shRNA templates targeted against each of these Ub-ligases, we now document that both E3s are indeed physiologically involved in CYP3A ERAD/UPD in cultured rat hepatocytes. Accordingly, specific RNAi resulted in ≈80% knockdown of each hepatic Ub-ligase, with a corresponding ≈2.5-fold CYP3A stabilization. Surprisingly, however, such stabilization resulted in increased levels of functionally active CYP3A, thereby challenging the previous notion that E3 recognition and subsequent ERAD of CYP3A proteins required ab initio their structural and/or functional inactivation. Furthermore, coexpression in HepG2 cells of both CYP3A4 and gp78, but not its functionally inactive RING-finger mutant, resulted in enhanced CYP3A4 loss greater than that in corresponding cells expressing only CYP3A4. Stabilization of a functionally active CYP3A after RNAi knockdown of either of the E3s, coupled with the increased CYP3A4 loss on gp78 or CHIP coexpression, suggests that ERAD-associated E3 Ub-ligases can influence clinically relevant drug metabolism by effectively regulating the physiological CYP3A content and consequently its function.
CYP3A4 是一种主要的人类肝细胞色素 P450 酶，参与代谢和处置 >50%的临床相关药物，并负责许多不良药物相互作用。CYP3A4 及其哺乳动物肝 CYP3A 同源物是内质网 (ER) 锚定的单拓扑蛋白，在 ER 相关降解 (ERAD) 过程中经历泛素 (Ub)-依赖性蛋白酶体降解 (UPD)。这些完整的 ER 蛋白在体内被泛素化，体外研究已经确定 ER 整合的 gp78 和细胞质共伴侣 CHIP（Hsp70 相互作用蛋白的 C 端）是相关的 E3 Ub 连接酶，以及它们各自的 E2 Ub 连接酶 UBC7 和 UbcH5a。使用针对每种 Ub 连接酶的慢病毒 shRNA 模板，我们现在证明这两种 E3 实际上都参与了培养的大鼠肝细胞中的 CYP3A ERAD/UPD。因此，特异性 RNAi 导致每种肝 Ub 连接酶的表达≈80%下调，相应的 CYP3A 稳定性增加约 2.5 倍。然而，令人惊讶的是，这种稳定导致功能性活性 CYP3A 的水平增加，从而挑战了先前的观点，即 CYP3A 蛋白的 E3 识别和随后的 ERAD 需要它们的结构和/或功能失活。此外，在 HepG2 细胞中共表达 CYP3A4 和 gp78，但不是其功能无效的 RING 指突变体，导致 CYP3A4 丢失增加大于仅表达 CYP3A4 的相应细胞。在用 E3 中的任一种进行 RNAi 敲低后，功能性活性 CYP3A 的稳定，加上 gp78 或 CHIP 共表达时 CYP3A4 的增加丢失，表明 ERAD 相关的 E3 Ub 连接酶可以通过有效调节生理 CYP3A 含量及其功能来影响临床相关药物代谢。
