Chaperone recognition of xenobiotic-altered NO Synthase P450

描述（由申请方提供）：拟定研究的长期目标是阐明外源性物质介导的细胞色素P450酶失活、降解和转换的机制。一氧化氮合酶（NOS）是细胞色素P450酶中调节最高的一种，在胃肠道运动和肝脏药物代谢等多种生物学过程中起着关键作用。我们已经发现，药物，如胍那苄和烟草，是基于代谢的神经元NOS（nNOS）失活剂，并且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。我们已经建立了活性位点构象的改变使nNOS“不稳定”，然后由Hsp 70和Hsp 90分子伴侣识别，并由CHIP（分子伴侣相关的泛素连接酶）泛素化，导致不稳定的nNOS的特异性蛋白酶体降解。我们计划利用这些发现以及我们最近在nNOS和nNOS <$Hsp70 <$CHIP复合物的电子显微镜（EM）研究方面取得的突破性成功，以更好地了解分子伴侣如何通过以下特定目标识别不稳定的nNOS P450：（1）利用单粒子负染电镜和冷冻电镜技术对nNOS的稳定态和不稳定态的结构进行表征，（2）利用电镜和LC-MS/MS技术对nNOS分子伴侣与Hsp 70和Hsp 90复合物的结构进行了表征。（3）利用细胞可渗透的巯基交联剂和LC-MS/MS技术对nNOS分子伴侣、辅助分子伴侣和其他蛋白质进行了分离和表征。这项工作将是第一个阐明全长nNOS，nNOS？分子伴侣复合物的结构，以及确定分子伴侣识别的nNOS的特定构象状态。这些研究应该导致更好地了解分子伴侣如何识别nNOS的不稳定形式并保持蛋白质质量。最终，这些研究可能会提供一种预测，评估和改进药物和其他外源性物质的有效性和安全性的方法。此外，了解不稳定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获得治疗益处。这种效用的一个实例是我们最近关于在神经变性疾病模型中通过激活分子伴侣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).