Neuronal NOS, Nitroarenes, and Neurotoxicity

神经元 NOS、硝基芳烃和神经毒性

• 批准号: 7128139
• 负责人: Richard Timothy Miller
• 金额: $ 20.59万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7128139
• 关键词: citrulline; electron transport; enzyme mechanism; free radical oxygen; laboratory rat; neurotoxicology; nitration; nitric oxide synthase; nitrobenzene; peroxynitrites; recombinant proteins

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.

描述（由申请人提供）：阐明神经元一氧化氮合酶（NNOS）的结构秘密已成为了解如何通过特定化学物质对NNO进行差异调节和/或调节的目的的重要目标。 NNO与硝化环境污染物（导致反应性中间体和毒性的产生）的相互作用是该提议的主题。为了研究1,3-二硝基苯（1,3-DNB）介导的神经毒性的机制，我们假设在硝化烯烃的存在下，NNOS从纯氧化氧化物（NO*）和L- citrulline合成酶转化为过氧亚硝酸盐（ONOO-）和L-citrulline合酶。当NNOS同时产生NO*和超氧化阴离子自由基（O2-）时，Onoo-是一种非常有效和反应性氧化剂。 O2-由NNOS介导的还原和随后再氧化的氮气形成。同时，NNOS保持足够的电子流向血红素的流量，以生产其正常产品，NO*和L-Citrulline。在接近近端的NO*和O2同时产生的同时产生通过这两个自由基以接近扩散控制的反应速率的结合立即导致形成。提议生产的Onoo-以及部分降低的硝化中间体，提议在与1,3-DNB暴露有关的神经毒性中发挥作用。该项目的长期目标是确定硝化剂的代谢如何导致反应性中间体的产生（例如ONOO-，O2-，H2O2，NOX）和主动降低代谢产物的代谢，并在中枢神经系统内介导毒性。此外，我们将确定如何通过硝酸，O2-和主动降低硝酸代谢产物（如硝基和N-羟基物种）的代谢产物来调节NNO的酶活性。为了实现这一目标，我们的直接特定目标是： 目标＃1：通过使用重组表达和纯化的NNOS和NNOS构建体来剖析从NNOS到硝化烯（例如1,3-DNB）的电子转移。 目标＃2：测试假说，即NOS与包括1,3- DNB在内的神经毒性氮的相互作用，导致NOS活性的调节，O2-产生的刺激以及通过成为一种产生的酶来获得功能。