Chaperone recognition of xenobiotic-altered NO Synthase P450

异种生物改变的 NO 合酶 P450 的伴侣识别

• 批准号: 9060951
• 负责人: YOICHI OSAWA
• 金额: $ 39.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9060951
• 关键词: Active Sites; Address; Androgen Receptor; Animals; Arginine; Binding; Biological; Biological Process; Biology; Carbon Tetrachloride; Cells; Cerebral Palsy; ChIP-on-chip; Chemical Exposure; Chemicals; Cleaved cell; Clinical; Complex; Cross-Linking Reagents; Crosslinker; Cytochrome P450; Disease model; Drug Targeting; Electron Microscopy; Enzymes; Epidermal Growth Factor Receptor; Event; Excision; Functional disorder; Funding; Gastrointestinal Motility; Goals; Grant; Guanabenz; Health; Heme; Impotence; In Vitro; Lead; Length; Liver; Mass Spectrum Analysis; Mediating; Metabolism; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Models; Myoglobin; Nature; Negative Staining; Neurodegenerative Disorders; Neuronal Injury; Neurons; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxygenases; Penile Erection; Pharmaceutical Preparations; Physiological; Play; Procedures; Process; Proteins; Quality Control; Regulation; Research; Safety; Scheme; Site; Staging; Stroke; Structure; Sulfhydryl Compounds; System; Techniques; Therapeutic Uses; Time; Tobacco; Toxic effect; Ubiquitin; Ubiquitin-Proteasomal Pathway; Ubiquitination; Work; Xenobiotics; base; conformational alteration; crosslink; cryogenics; drug metabolism; environmental chemical; inhibitor/antagonist; microscopic imaging; molecular modeling; neurotransmission; novel; particle; polyglutamine; protein aggregate; protein degradation; protein protein interaction; receptor; stroke treatment; success; tetrahydrobiopterin; therapeutic protein; ubiquitin ligase

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to elucidate the mechanisms of xenobiotic- mediated inactivation, degradation, and turnover of cytochrome P450 enzymes. Nitric oxide synthase (NOS), the most highly regulated cytochrome P450 enzyme, plays a key role in a variety of biological processes, including regulation of gastrointestinal motility and liver drug metabolism. We have discovered that drugs, such as guanabenz and tobacco, are metabolism- based inactivators of neuronal NOS (nNOS) and lead to the covalent alteration, enhanced turnover, and loss of nNOS P450 protein via the ubiquitin proteasomal pathway. The loss of NOS is a mechanism of toxicity associated with these drugs. We have established that alteration of the active site conformation 'labilizes' the nNOS, which is then recognized by Hsp70 and Hsp90 chaperones, and is ubiquitinated by CHIP, a chaperone-associated ubiquitin ligase, resulting in the specific proteasomal degradation of the labilized nNOS. We plan on utilizing these discoveries and our recent ground-breaking success with electron microscopy (EM) studies on nNOS and nNOS¿Hsp70¿CHIP complexes to better understand how chaperones recognize labilized nNOS P450 through the following specific aims: (1) To characterize the structures of the stabilized and labilized states of nNOS with the use of single particle negative stain EM and cryogenic-EM techniques, (2) To characterize the structure of nNOS chaperone complexes with Hsp70 and Hsp90 by EM as well as LC-MS/MS techniques, (3) To isolate and characterize the chaperones, co-chaperones and other proteins that associate with labilized nNOS by use of a cell permeable thiol-cleavable crosslinker and LC- MS/MS methods. This work would be the first to elucidate the structure of full-length nNOS, nNOS¿chaperone complexes, as well as determine the specific conformational states of nNOS that are recognized by chaperones. These studies should lead to a better understanding of how chaperones recognize labilized forms of nNOS and maintain protein quality. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. Moreover, understanding the mechanism of recognition of labilized nNOS and quality control may provide a new method to specifically remove proteins for therapeutic benefit. An example of such utility is our recent study on removal of protein aggregates through activation of chaperones in a neurodegenerative disease model (Nature Chemical Biology 9: 112-118, 2013).

描述(申请人提供)：拟议研究的长期目标是阐明异种生物介导的细胞色素P450酶的失活、降解和周转的机制。一氧化氮合酶(NOS)是细胞色素P450中最受调控的酶，在多种生物学过程中发挥着关键作用，包括调节胃肠动力和肝脏药物代谢。我们发现，药物，如愈创木酚和烟草，是基于代谢的神经元型一氧化氮合酶(NNOS)失活剂，并通过泛素蛋白酶体途径导致nNOS P450蛋白的共价改变、增加的周转和丢失。一氧化氮合酶的丢失是这些药物的毒性机制之一。我们已经证实，活性部位构象的改变使nNOS被Hsp70和Hsp90伴侣识别，并被伴侣相关的泛素连接酶ChIP泛素化，导致标记的nNOS特异性的蛋白酶体降解。我们计划利用这些发现和我们最近对nNOS和nNOS？Hsp70芯片复合体的电子显微镜(EM)研究的开创性成功，通过以下具体目标更好地了解伴侣如何识别标记的nNOS P450：(1)利用单粒子负染色EM和低温EM技术表征nNOS稳定和稳定状态的结构，(2)用EM和LC-MS/MS技术表征nNOS与Hsp70和Hsp90的伴侣复合体的结构，(3)分离和表征伴侣。通过使用细胞可渗透的硫醇可裂解交联剂和LC-MS/MS方法，与标记的nNOS相关联的辅伴侣和其他蛋白质。这项工作将首次阐明全长nNOS、nNOS伴侣复合体的结构，并确定伴侣识别的nNOS的特定构象状态。这些研究应该有助于更好地理解伴侣如何识别nNOS的稳定形式并保持蛋白质质量。最终，这些研究可能提供一种方法来预测、评估和改进药物和其他外源药物的有效性和安全性。此外，了解标记的nNOS的识别机制和质量控制可能提供一种新的方法来特异性地去除蛋白质以达到治疗的目的。这种效用的一个例子是我们最近在一种神经退行性疾病模型中通过激活伴侣蛋白来移除蛋白质聚集体的研究(自然化学生物学9：112-118,2013)。