Toxicological Aspects of Hemoprotein Regulation

描述（由申请人提供）：拟议研究的目的是 为了阐明失活，降解和周转的机制， 神经元型一氧化氮合酶（nNOS），一种细胞色素P450样血红素蛋白， 由于暴露于异生物质，包括药物和环境毒物。 核心假设是，这种作用降低了形成NO的能力， 参与许多生理功能重要生物调节分子， 会引起某种化学毒性。最近表明 guanabenz（WytensinTM）是一种抗高血压药物， 在体外以代谢依赖性方式存在，并导致nNOS蛋白的损失， 体内活性。同样，香烟烟雾中含有的化合物会导致 体内阴茎nNOS的代谢失活和丢失。这些发现 可能是重要的，因为NO在阴茎勃起和抗高血压中起关键作用 药物以及吸烟已知会导致阳痿。几 代谢为基础的活化剂，包括胍那苄，被发现会导致 nNOS的蛋白水解降解增强，部分导致nNOS的损失 体内蛋白质发现这种降解涉及基于hsp90的分子伴侣， 泛素化和蛋白酶体降解。因此，当前的目标 建议是了解nNOS是如何被灭活和共价改变， 外源性物质以及nNOS的稳态水平随后如何受到影响。 这将不仅涉及降解过程的研究， nNOS的修复或组装的相反力量。我们将利用现代化学 和生物技术在体外，细胞和体内模型，以解决 这些目标。具体的目的是：（1）确定 代谢型激活剂引起的nNOS失活，（2）表征 使nNOS易于泛素化的结构决定因素， 蛋白酶体降解，（3）表征负责 （4）观察不同浓度的NNOS对大鼠海马神经元的影响。 烟草烟雾成分对nNOS的利用知识的目的 1-3.这些研究应该会让我们更好地理解药物是如何 抑制和调节nNOS，从而设计出更安全的药物 而没有不希望的毒理学副作用，例如阳痿。相反地， 同样的知识可以用来设计更安全、更有效的抑制剂， 用于多种神经系统疾病的药理学用途。

DESCRIPTION (provided by applicant): The objective of the proposed research is to elucidate the mechanisms of inactivation, degradation, and turnover of neuronal nitric oxide synthase (nNOS), a cytochrome P450-like hemoprotein, caused by exposure to xenobiotics, including drugs and environmental toxicants. The central hypothesis is that such effects diminish the ability to form NO, an important bioregulatory molecule involved in many physiological functions, and cause some forms of chemically induced toxicities. It was recently shown that guanabenz (WytensinTM), an antihypertensive agent, inactivates penile nNOS in vitro in a metabolism dependent manner and causes a loss of nNOS protein and activity in vivo. Similarly, cigarette smoke contains compounds that lead to metabolism-based inactivation and loss of penile nNOS in vivo. These findings may be important as NO plays a key role in penile erection and antihypertensive agents as well as cigarette smoking are known to cause impotence. Several metabolism-based in activators, including guanabenz, were found to cause the enhanced proteolytic degradation of nNOS that, in part, causes the loss of nNOS protein in vivo. This degradation was found to involve hsp9o-based chaperones, ubiquitination, and proteasomal degradation. Thus, the aims of the current proposal are to understand how nNOS is inactivated and covalently altered by xenobiotics and how the steady state levels of nNOS are subsequently affected. This will involve the study of not only the degradation process but also the opposing forces of repair or assembly of nNOS. We will utilize modern chemical and biological techniques in vitro, cellular, and in vivo models to address these aims. The specific aims are: (1) To determine the molecular mechanism of nNOS inactivation caused by metabolism-based in activators, (2) To characterize the structural determinants that render nNOS susceptible to ubiquitination and proteasomal degradation, (3) To characterize the processes responsible for activation or maintenance of nNOS function, (4) To characterize the effect of tobacco smoke constituents on nNOS with the use of knowledge gained in Aims 1-3. These studies should lead to a better understanding of how drugs inactivate and regulate nNOS and thereby lead to the design of safer drugs without undesired toxicological side effects, such as impotence. Conversely, the same knowledge could be used to design safer and more effective inhibitors of NOS for pharmacological use in a variety of neurological diseases.