Neuronal NOS, Nitroarenes, and Neurotoxicity

描述(申请人提供)：揭开神经元型一氧化氮合酶(NNOS)的结构秘密已成为了解nNOS如何被特定化学物质差异调节和/或调节的重要目标。NNOS与硝基芳烃等环境污染物的相互作用，导致活性中间体的产生和毒性，是本提案的主题。为了探讨1，3-二硝基苯(1，3-DNB)的神经毒性机制，我们假设在硝基芳烃存在的情况下，nNOS从纯一氧化氮(NO*)和L瓜氨酸合成酶转化为过氧亚硝酸根(ONOO-)和L瓜氨酸合成酶。ONOO-是当nNOS同时产生NO*和超氧阴离子自由基(O2-)时形成的一种非常有效的活性氧化剂。O2-是由nNOS介导的硝基芳烃还原和随后的再氧化形成的。同时，nNOS维持足够的电子流向血红素，以产生其正常产物NO*和L瓜氨酸。NO*和O2-在近距离同时产生，通过这两个自由基的结合，以接近扩散控制的反应速度立即导致ONOO-的形成。与硝基芳烃的部分还原中间体一起产生的ONOO-被认为在与1，3-DNB接触有关的神经毒性中发挥了作用。该项目的长期目标是确定硝基芳烃的代谢如何在中枢神经系统内调节毒性，从而产生反应性中间体(如ONOO-、O2-、H2O2、NOx)和活性还原代谢物。此外，我们将确定硝基芳烃、O2-和硝基芳烃的活性还原代谢产物(如亚硝基和N-羟基)如何调节nNOS的酶活性。为了实现这一目标，我们当前的具体目标是： 目的#1：利用重组表达和纯化的nNOS和nNOS结构分析nNOS向1，3-DNB等硝基芳烃的电子传递。 目的#2：验证一氧化氮合酶(NOS)与神经毒性硝基芳烃(包括1，3-DNB)相互作用，调节一氧化氮合酶(NOS)活性，刺激O2-产生，并通过成为ONOO产生酶而获得功能的假说。

DESCRIPTION (provided by applicant): Unraveling the structural secrets of neuronal nitric oxide synthase (nNOS) has become an important goal for the purpose of understanding how nNOS can be differentially regulated and/or modulated by specific chemicals. The interaction of nNOS with environmental pollutants such as nitroarenes, resulting in the production of reactive intermediates and toxicity, is the subject of this proposal. In order to investigate the mechanisms of 1,3-dinitrobenzene (1,3-DNB)-mediated neurotoxicity, we hypothesize that in the presence of nitroarenes, nNOS is converted from a purely nitric oxide (NO*) and L-citrulline synthase to a peroxynitrite (ONOO-) and L-citrulline synthase. ONOO- is a very potent and reactive oxidant formed when nNOS simultaneously produces NO* and superoxide anion radical (O2-). O2- is formed by the nNOS-mediated reduction and subsequent reoxidation of nitroarenes. Concomitantly, nNOS maintains adequate electron flow to the heme to produce its normal products, NO* and L-citrulline. The simultaneous production of both NO* and O2- in close proximity leads immediately to ONOO- formation via the combination of these two radicals at a near diffusion-controlled reaction rate. The ONOO- that is produced, along with partially-reduced intermediates of the nitroarene, are proposed to play a role in the neurotoxicity associated with exposure to 1,3-DNB. The long-term objective of this project is to determine how metabolism of nitroarenes, resulting in the production of reactive intermediates (such as ONOO-, O2-, H2O2, NOx), and active reduced metabolites, mediate toxicity within the central nervous system. Further, we will determine how enzymatic activity of nNOS can be regulated by nitroarenes, O2-, and active reduced metabolites of nitroarenes such as the nitroso- and N-hydroxy-species. Toward this goal, our immediate specific aims are: Aim #1: To dissect electron transfer from nNOS to nitroarenes such as 1,3-DNB by using recombinantly-expressed and purified nNOS and nNOS constructs. Aim #2: To test the hypothesis that interaction of NOS with neurotoxic nitroarenes, including 1,3- DNB, results in modulation of NOS activity, stimulation of O2- production, and a gain of function by becoming a ONOO-generating enzyme.