Chaperone recognition of xenobiotic-altered NO Synthase P450

描述（由申请人提供）：拟议研究的长期目标是阐明外源介导的细胞色素P450酶的失活，降解和转换机制。一氧化氮合酶（NOS）是调控程度最高的细胞色素P450酶，在多种生物过程中发挥关键作用，包括调节胃肠道运动和肝脏药物代谢。我们已经发现药物，如鸟那苯和烟草，是神经元NOS （nNOS）的代谢基础失活剂，并通过泛素蛋白酶体途径导致nNOS P450蛋白的共价改变、增强的转换和损失。NOS的丧失是与这些药物相关的毒性机制。我们已经确定，活性位点构象的改变使nNOS“不稳定”，然后被Hsp70和Hsp90伴侣识别，并被CHIP（伴侣相关的泛素连接酶）泛素化，导致被稳定的nNOS的特异性蛋白酶体降解。我们计划利用这些发现和我们最近对nNOS和nNOS¿Hsp70¿CHIP复合物的电子显微镜（EM）研究的突破性成功，更好地了解伴侣如何通过以下具体目标识别不稳定的nNOS P450：(1)利用单颗粒阴性染色EM和低温EM技术表征nNOS稳定和稳定状态的结构；(2)利用EM和LC-MS/MS技术表征nNOS与Hsp70和Hsp90的伴侣复合物的结构；(3)利用细胞渗透性巯基可切割交联剂和LC-MS/MS方法分离和表征与稳定的nNOS相关的伴侣、共伴侣和其他蛋白质。这项工作将首次阐明全长nNOS、nNOS¿伴侣复合物的结构，并确定被伴侣识别的nNOS的特定构象状态。这些研究将有助于更好地理解伴侣蛋白如何识别不稳定的nNOS形式并维持蛋白质质量。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性药物的有效性和安全性。此外，了解不稳定的nNOS的识别机制和质量控制可能为特异性去除蛋白质提供新的方法。这种效用的一个例子是我们最近在神经退行性疾病模型中通过激活伴侣蛋白来去除蛋白质聚集体的研究（Nature Chemical Biology 9: 112- 118,2013）。

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).