Neuronal NOS, Nitroarenes, and Neurotoxicity

描述（由申请人提供）：解开神经元一氧化氮合酶（nNOS）的结构秘密已经成为了解nNOS如何被特定化学物质差异调节和/或调节的重要目标。nNOS与环境污染物（如硝基芳烃）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。为了研究1,3-二硝基苯（1,3- dnb）介导的神经毒性机制，我们假设在硝基arenes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盐（ONOO-）和l -瓜氨酸合成酶。ONOO-是一种非常有效的活性氧化剂，当nNOS同时产生NO*和超氧阴离子自由基（O2-）时形成。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。同时产生相近的NO*和O2-，通过这两个自由基以接近扩散控制的反应速率结合，立即形成ONOO-。ONOO-与部分还原的硝基芳烃中间体一起产生，被认为在与暴露于1,3- dnb相关的神经毒性中发挥作用。该项目的长期目标是确定硝基芳烃的代谢如何介导中枢神经系统的毒性，从而产生活性中间体（如ONOO-、O2-、H2O2、NOx）和活性还原性代谢物。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，我们近期的具体目标是：

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.