Toxicological Aspects of Hemoprotein Regulation

描述(由申请人提供)：建议研究的目标是 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样血球蛋白， 由于接触到包括药物和环境毒物在内的外来物质而引起的。 中心假说是，这种影响降低了形成NO的能力，并 重要的生物调节分子，参与许多生理功能，以及 导致某些形式的化学中毒。最近有证据表明， 抗高血压药Ganabenz(WytensinTM)灭活阴茎nNOS 在体外代谢依赖的方式，并导致nNOS蛋白和 活体内活动。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 可能很重要，因为NO在阴茎勃起和抗高血压中起着关键作用 众所周知，毒剂和吸烟都会导致阳萎。几个 以新陈代谢为基础的激活剂，包括吧那苯肼，被发现导致 促进nNOS的蛋白分解降解，部分导致nNOS的丢失 体内的蛋白质。这种降解被发现涉及基于hsp90的伴侣， 泛素化和蛋白酶体降解。因此，当前的目标是 建议是了解nNOS是如何失活和共价改变的 外源生物以及nNOS的稳态水平随后如何受到影响。 这将不仅涉及对退化过程的研究，而且还涉及对 修复或组装nNOS的相反力量。我们将使用现代化学品 以及生物技术在体外、细胞和体内模型中的应用 这些目标。其具体目的是：(1)确定其分子机制。 代谢激活剂引起的nNOS失活，(2)表征 使nNOS容易泛素化的结构决定因素和 蛋白酶体降解，(3)表征负责的过程 激活或维持nNOS功能，(4)表征 利用AIMS获得的知识研究烟草烟雾成分对nNOS的影响 1比3。这些研究应该有助于更好地理解药物是如何 使nNOS失活和调节，从而导致设计更安全的药物 没有不良的毒理副作用，如阳萎。相反， 同样的知识可以用来设计更安全、更有效的抑制剂。 一氧化氮合酶在多种神经疾病中的药理作用。

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.