Hepatic degradation of cytochrome P450 enzymes

细胞色素 P450 酶的肝脏降解

基本信息

批准号：
10227957
负责人：
Maria Almira Correia
金额：
$ 45.22万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-04-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10227957
关键词：
Acetylation Address Affinity Alcohols Alpaca Biological Biology CYP1A2 gene CYP2B1 gene CYP2B6 gene CYP2D6 gene CYP2E1 gene CYP3A4 gene Carcinogens Cell Line Cell physiology Cells Cellular Stress Chemical Agents Chemicals Chimera organism Chimeric Proteins Clinical Complement Coupled Cytochrome P450 Detergents Disease Dissection Dose Drug Interactions Drug toxicity Endoplasmic Reticulum Endoplasmic Reticulum Degradation Pathway Enzymes Ethanol Exposure to Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Functional disorder Funding Furanocoumarins Genes Genetic Goals Grapefruit juice Health Hepatic Hepatocyte Human Intestines Label Lipids Literature Liver Mediating Membrane Membrane Microdomains Metabolism Modeling Molecular Mus Orthologous Gene Oryctolagus cuniculus Participant Pathway interactions Pharmaceutical Preparations Phosphorylation Physiology Post-Translational Protein Processing Process Protein Analysis Protein Isoforms Proteins Proteomics Rattus Reporting Resistance Role Route Sorting - Cell Movement Structure System Therapeutic Toxin UBA Domain Ubiquitin Ubiquitination Water Xenobiotics clinically relevant clinically significant crosslink inhibitor/antagonist insight lipid disorder molecular recognition mouse model mutant nanobodies overexpression preference protein degradation receptor recruit response stem trafficking

项目摘要

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.

项目摘要/摘要：肝内质网 (ER) 锚定的单位蛋白、细胞色素 P450 (P450s) 是代谢内源性和外源性物质（即药物、致癌物、毒素、天然和化学产品）的酶。这些药物通过增加形成或失活和/或蛋白水解的损失来调节肝脏 P450 含量降解，导致临床上显着的药物相互作用（DDI）。 DDI 通常源于 P450 的改变由药物介导的 P450 稳定作用（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 P450 ERAD 涉及两个主要途径：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（CYP3A4，主要的人类肝脏/肠道 P450）引发 UPD，其他 P450（CYP2B1）引发 ALD 还有一些（EtOH 代谢 CYP2E1）会同时发生这两种情况。 P450 蛋白水解差异的决定因素排序是未知的，它们的识别是我们未来研究的主要目标。合理的决定因素包括 (i) ER 膜中的 P450 均聚化； (ii) 脂质紊乱 (ld) 与脂质紊乱 (ld) 的定位有序（lo；脂筏）ER 微结构域，耐洗涤剂萃取 (DRM)； (iii) ER 倾向或细胞质 P450 聚集以及随后由自噬受体 p62/Sequestosome 募集和 NBR-1（Braca 1 基因的邻居）； (iv) 除泛素化之外的特定翻译后修饰 (即磷酸化、乙酰化)； (v) 赋予 ALD 差异排序的特定结构域与两个密切相关的直系同源或同工 P450 的 UPD 相比。我们建议采用各种实验方法例如：共焦荧光显微镜、双分子荧光互补、荧光共振能量转移（FRET）、细胞内化学交联、严格亲和力使用羊驼纳米抗体进行免疫纯化 (AIP)、蛋白质组 (LC-MS/MS) 分析、LC-MS/MS 分析通过邻近标记以及翻译后进行蛋白质相互作用和相互作用物识别修饰、p62-/NBR-1-缺失突变体和基因消除细胞、P450-嵌合体和融合蛋白，以及相关遗传（ATG5-/-、p62-/-、NBR-1-/-）小鼠模型原代培养的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非常重要，因为它们不仅会增进我们对基本 P450 生物学/生理学的理解，而且还会对 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 药物底物的精确剂量和揭示内源性 P450 底物在生理学和病理生理学中的作用。我们相信洞察力从这些研究中获得的成果将普遍适用于其他细胞蛋白质。