Characterization of the physiological turnover of native and inactivated cytochromes P450 3A in cultured rat hepatocytes: A role for the cytosolic AAA ATPase p97?
Characterization of the physiological turnover of native and inactivated cytochromes P450 3A in cultured rat hepatocytes: A role for the cytosolic AAA ATPase p97?
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DOI：
10.1021/bi700340n
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发表时间：
2007-07-03
期刊：
影响因子：
2.9
通讯作者：
Correia, Maria Almira
中科院分区：
文献类型：
--
作者：
Faouzi, Saadia;Medzihradszky, Katalin F.;Correia, Maria Almira
哺乳动物肝细胞色素P450（P450）是内质网（ER）锚定的血红素蛋白，参与许多异种和内源性代谢。P450表现出广泛的半衰期，利用自噬溶酶体（ALD）和泛素依赖性26 S蛋白酶体（UPD）降解途径。尽管酿酒酵母中自杀失活的肝CYP 3A和“天然”CYP 3A 4通过UPD降解，但天然肝CYP 3A在其生理环境中的周转尚未阐明。在本文中，我们使用蛋白酶体（MG-132和MG-262）和ALD [NH 4Cl和3-甲基腺嘌呤（3-MA）]抑制剂来表征天然地塞米松诱导的CYP 3A在培养的原代大鼠肝细胞中的降解，以检查其特定的降解途径。脉冲追踪免疫沉淀分析显示，6小时内基础S-35-CYP 3A损失52%，这是稳定的两种蛋白酶体抑制剂。相比之下，用ALD抑制剂NH 4Cl或3-MA均未检测到相应的CYP 3A稳定。此外，MG-262诱导的CYP 3A稳定化与其多聚泛素化相关，从而证实天然CYP 3A也通过UPD降解。为了确定这一过程中的具体参与者，在培养的肝细胞中用多聚甲醛（PFA）原位交联细胞蛋白。免疫印迹分析后PFA交联的CYP 3A免疫沉淀揭示了存在的p97，胞质AAA ATP酶的泛素化ER蛋白的提取和交付的蛋白酶体降解。这种天然CYP 3A-p97相互作用在CYP 3A自杀性失活（加速UPD）和/或蛋白酶体抑制后大大放大，并通过蛋白质组学和共聚焦免疫荧光显微镜分析证实。这些发现清楚地揭示了天然CYP 3A经历UPD，并暗示了p97在此过程中的作用。
Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno- and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in Saccharomyces cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal (MG-132 and MG-262) and ALD [NH4Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase with immunoprecipitation analyses revealed a basal 52% S-35-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH4Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were crosslinked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-cross-linking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.