Drug-Mediated Alteration of Cytochrome P450

描述（由申请人提供）：拟议研究的长期目标是阐明药物和外源介导的细胞色素P450酶失活、降解和转化的机制。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guanabenz和烟草，是基于代谢的NOS失活剂，并导致NOS蛋白的共价改变、增强的转换和损失。鉴于NOS功能和蛋白质的丧失可以解释与这些药物相关的一些毒性，我们想知道药物是如何引起NOS的强化转换的。我们已经确定药物会引起假体血红素改变或四氢生物蝶呤氧化，而这种改变是NOS降解的触发因素。我们发现这些改变通过一种涉及hsp90和hsp70的伴侣依赖机制使NOS的泛素化和蛋白酶体降解变得不稳定。我们还发现了一种潜在的修复途径，其中细胞蛋白，包括伴侣蛋白，促进血红素插入血红素缺陷的载脂蛋白nos。我们现在可以利用迄今为止的发现来确定伴侣如何选择修复或泛素化药物改变的NOS。因此，我们提出以下具体目标：(1)表征NOS的稳定形式与hsp70-和hsp90-伴侣和合作伙伴的相互作用，(2)确定hsp70和hsp90在NOS泛素化中的作用，(3)分离和表征促进血红素进入载脂蛋白NOS的血红素插入机制。我们将在各种体外和细胞系统中利用siRNA、免疫纯化、生化和LC-MS/MS技术来实现这些目标。我们将展示NOS和伴侣之间的分子相互作用如何导致确定和可预测的生物反应，最终决定药物的药理学和毒理学特征。这将有助于设计安全有效的药物来控制NOS，以及减少与NOS相关的药物不良反应的策略。我们还讨论了血红素假体如何插入NOS以及细胞如何维持NOS蛋白质量控制的基本生物学过程。利用这种质量控制机制可以提供一种特异性去除蛋白质的新方法。总的来说，这项工作进一步加深了我们对药物代谢的理解，特别是那些长期使用的药物，如何改变正常的生物过程，从而产生有害和有益的药物作用。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。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to elucidate the mechanisms of drug- and 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 NOS and cause the covalent alteration, enhanced turnover, and loss of NOS protein. In that the loss of NOS function and protein may explain some of the toxicities associated with these drugs, we wondered how drugs cause the enhanced turnover of NOS. We have established that the drugs cause prosthetic heme alteration or tetrahydrobiopterin oxidation and that such alterations are triggers for degradation of NOS. Furthermore, we discovered that these alterations labilize NOS for ubiquitination and proteasomal degradation by a chaperone-dependent mechanism involving hsp90 and hsp70. We have also uncovered a potential repair pathway where cellular proteins, including chaperones, facilitate insertion of heme into heme-deficient apo-NOS. We can now utilize the discoveries to date to determine how chaperones select for repair or ubiquitination of drug-altered NOS. Thus, we propose the following specific aims: (1) To characterize the interaction of labilized forms of NOS with hsp70- and hsp90- chaperones and cochaperones, (2) To determine the role of hsp70 and hsp90 in the ubiquitination of NOS, (3) To isolate and characterize the heme insertion machinery that facilitates heme entry into apo-NOS. We will utilize siRNA, immunopurification, biochemical, and LC-MS/MS techniques in a variety of in vitro and cellular systems to address these aims. We will show how the molecular interactions between NOS and chaperones lead to defined and predictable biological responses that ultimately determine the pharmacological and toxicological profiles of drugs. This will aid in the design of safe and effective drugs to control NOS as well as strategies to decrease adverse drug effects related to NOS. We also address the fundamental biological processes of how the heme prosthetic group is inserted into NOS and how cells maintain NOS protein quality control. Taking advantage of this quality control mechanism may provide a new method to specifically remove proteins for therapeutic benefit. Overall, this work furthers our understanding of how the metabolism of drugs, especially those used chronically, can alter the normal biological processes to give rise to adverse as well as beneficial drug effects. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. PUBLIC HEALTH RELEVANCE: This research furthers our understanding of how metabolism of drugs alters biological processes that lead to adverse as well as beneficial drug effects. Ultimately, these studies may provide a way to predict, evaluate, and refine, the efficacy and safety of drugs and other xenobiotics. The discoveries made through this line of study indicate that a new way to target proteins for degradation may have important therapeutic uses.