Neuronal NOS, Nitroarenes, and Neurotoxicity

描述（由申请人提供）：为了理解nNOS如何被特定化学物质差异调节和/或调制，解开神经元型一氧化氮合酶（nNOS）的结构秘密已成为一个重要目标。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，我们假设在硝基芳烃存在下，nNOS从纯一氧化氮（NO*）和L-瓜氨酸合酶转化为过氧亚硝酸盐（ONOO-）和L-瓜氨酸合酶。ONOO-是nNOS同时产生NO* 和超氧阴离子自由基（O2-）时形成的非常有效和反应性的氧化剂。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保持足够的电子流到血红素，以产生其正常产物NO* 和L-瓜氨酸。同时生产的NO* 和O2-在紧密接近导致ONOO-形成通过这两个自由基的组合在一个接近扩散控制的反应速率。沿着产生的ONOO-与硝基芳烃的部分还原中间体一起，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导致活性中间体（如ONOO-，O2-，H2 O2，NOx）和活性还原代谢物的产生，介导中枢神经系统内的毒性。此外，我们将确定如何nNOS的酶活性可以调节硝基芳烃，O2-，和活性还原代谢产物的硝基芳烃，如亚硝基和N-羟基物种。为实现这一目标，我们的近期具体目标是： 目的#1：通过使用重组表达和纯化的nNOS和nNOS构建体来剖析从nNOS到硝基芳烃如1，3-DNB的电子转移。 目标二：为了检验NOS与神经毒性硝基芳烃（包括1，3- DNB）的相互作用导致NOS活性调节、O2产生刺激以及通过成为ONOO生成酶而获得功能的假设。

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.