Hepatic degradation of cytochrome P450 enzymes

项目总结/摘要： 肝内质网（ER）锚定的单调蛋白，细胞色素P450（P450）， 代谢内源性和外源性物质的酶，即药物、致癌物、毒素、天然和化学产品。 这些试剂通过增加形成或通过失活和/或蛋白水解损失来调节肝P450含量 降解，导致具有临床意义的药物相互作用（DDI）。DDI通常源于P450的改变 药物介导的P450稳定（即乙醇（EtOH））引起的ER相关降解（ERAD），或 增强药物介导的P450降解（即葡萄柚汁）。肝脏P450 ERAD涉及两个主要的 途径：泛素（Ub）依赖性蛋白酶体降解（UPD）和自噬-溶酶体降解 （ALD）。一些P450（CYP 3A 4，主要的人类肝脏/肠道P450）引起UPD，其他（CYP 2B 1）引起ALD 而另一些（乙醇代谢CYP 2 E1）则会引起两者。这种差异P450蛋白水解的决定因素 分类是未知的，它们的识别是我们未来研究的主要目标。似然决定因素 包括（i）ER膜中的P450均聚化;（ii）脂质紊乱（Id）与脂质 有序（lo;脂筏）ER-微结构域，对去污剂提取（DRM）有抗性;（iii）ER或 细胞质P450聚集和随后由自噬受体p62/Sequestosome募集 和NBR-1（Braca 1基因的邻居）;（iv）除了泛素化以外的特异性翻译后修饰 (i.e.磷酸化、乙酰化）;和（v）赋予ALD差异分选的特异性结构域 与UPD相比，两种密切相关的正构或异构P450。我们建议采用各种实验 方法例如：共聚焦荧光显微术，双分子荧光互补， 荧光共振能量转移（FRET），细胞内化学交联，严格亲和 用羊驼纳米抗体进行免疫纯化（AIP），蛋白质组学（LC-MS/MS）分析， 蛋白质相互作用和相互作用识别通过邻近标记，以及翻译后 修饰、p62-/NBR-1-缺失突变体和基因消融细胞、P450-嵌合体和融合蛋白，以及 相关遗传（ATG 5-/-、p62-/-、NBR-1-/-）小鼠模型原代培养大鼠和人肝细胞和细胞 线我们认为，阐明P450 ERAD过程的这些基本方面是重要的，因为 它们不仅将促进我们对基本P450生物学/生理学的理解，而且还将对 P450依赖性治疗和病理生理学，因此具有临床相关性。了解 P450水平的分子决定因素对于P450药物底物的精确给药和 阐明内源性P450底物在生理学和病理生理学中的作用。我们相信， 从这些研究中获得的结果将普遍适用于其他细胞蛋白。

PROJECT SUMMARY/ABSTRACT: The hepatic endoplasmic reticulum (ER)-anchored monotopic proteins, cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content via increased formation or loss via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions (DDIs). DDIs often stem from altered P450 ER-associated degradation (ERAD) elicited by drug-mediated P450 stabilization i.e. ethanol (EtOH), or enhanced drug-mediated P450 degradation (i.e. grapefruit juice). Hepatic P450 ERAD involves two major pathways: Ubiquitin (Ub)-dependent proteasomal degradation (UPD) and autophagic-lysosomal degradation (ALD). Some P450s (CYP3A4, the major human liver/intestinal P450) incur UPD, others (CYP2B1) incur ALD and yet others (EtOH-metabolizing CYP2E1) incur both. The determinants of this differential P450 proteolytic sorting are unknown and their identification are major goals of our future research. Plausible determinants include (i) P450-homomerization in the ER-membrane; (ii) localization in lipid-disordered (ld) versus lipid- ordered (lo; lipid rafts) ER-microdomains, resistant to detergent extraction (DRMs); (iii) propensity for ER or cytoplasmic P450 aggregation and subsequent recruitment by the autophagic receptors p62/Sequestosome and NBR-1 (neighbor of Braca 1 gene); (iv) specific post-translational modifications other than ubiquitination (i.e. phosphorylation, acetylation); and (v) specific structural domains that confer differential sorting into ALD versus UPD to two closely related orthologous or isoformic P450s. We propose to employ various experimental approaches such as: Confocal fluorescence microscopy, bimolecular fluorescence complementation, fluorescence resonance energy transfer (FRET), in-cell chemical crosslinking, rigorous affinity immunopurification (AIP) with alpaca nanobodies, proteomic (LC-MS/MS) analyses, LC-MS/MS analyses of protein interactions and interactant identification through proximity labeling, as well as post-translational modifications, p62-/NBR-1-deletion mutants and gene ablated cells, P450-chimeras and fusion proteins, and relevant genetic (ATG5-/-, p62-/-, NBR-1-/-) mouse models primary cultured rat and human hepatocytes and cell lines. Elucidation of these fundamental aspects of P450 ERAD processes, we believe, are important because they would not only advance our understanding of basic P450 biology/physiology, but also critically impact on P450-dependent therapeutics and pathophysiology, and thus are clinically relevant. Understanding the molecular determinants of P450 levels is critical for precision dosing of P450 drug substrates and for unraveling the role of endogenous P450 substrates in physiology and pathophysiology. We believe the insights gained from these studies will be universally applicable to other cellular proteins.