Peroxynitrite, nitrotyrosine and HSP90 in neuronal death

描述（由申请人提供）：过氧亚硝酸盐（ONOO-）与肌萎缩性侧索硬化症（ALS）、帕金森病、阿尔茨海默病、中风和脊髓损伤有关。虽然预防过氧亚硝酸盐的形成在所有这些情况下都可以起到保护作用，但在诊断时，过氧亚硝酸盐产生的损害已经存在。潜在靶点的多样性阻碍了揭示过氧亚硝酸盐介导神经变性的机制的努力。该项目的长期目标是阐明氧化剂过氧亚硝酸盐激活的神经元死亡途径。在过去的15年里，我们的团队和其他人对PC12细胞和运动神经元进行了实验，结果表明过氧亚硝酸盐可能会通过对一小群蛋白质的选择性硝化作用引发细胞死亡。我们发现，细胞内递送过氧亚硝酸盐修饰的热休克蛋白90 （HSP90）足以重现过氧亚硝酸盐诱导的运动神经元细胞死亡。相反，经过氧亚硝酸盐处理的HSP70、肌动蛋白、微管蛋白或白蛋白的释放对细胞没有毒性。我们假设硝化的HSP90在过氧亚硝酸盐诱导的细胞死亡中起关键作用，并且硝化的HSP90足以激活细胞死亡途径。用过氧亚硝酸盐处理像HSP90这样的纯蛋白质会产生多种修饰，可以通过质谱法识别。利用一种新开发的方法，将非天然氨基酸（如硝基酪氨酸）掺入重组HSP90中，我们可以首次测试在特定位点的硝化是否会对蛋白质产生毒性的功能获得。我们在体内几种病理条件下开发的单克隆抗体鉴定了硝化的HSP90，突出了所提出的研究的意义和影响；这些情况包括肾排斥、心脏病、中风、脊髓损伤以及ALS患者和小鼠模型的脊髓运动神经元。我们的研究将影响氧化应激诱导的细胞死亡机制的理解，通过提供一个新的工作模型来研究特异性蛋白质修饰，而不是一般的氧化损伤，如何激活高度调节的死亡途径。此外，这些研究的完成将为一氧化氮和过氧亚硝酸盐刺激细胞死亡的具体机制提供详细的见解，并对一系列广泛的神经退行性和炎症状况产生影响。

DESCRIPTION (provided by applicant): Peroxynitrite (ONOO-) has been implicated in amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, stroke, and spinal cord injury. Although preventing peroxynitrite formation can be protective in all of these conditions, the damage produced by peroxynitrite is already present at the time of diagnosis. The multiplicity of potential targets has hampered efforts to uncover the mechanisms by which peroxynitrite mediates neurodegeneration. The long-term goal of this project is to elucidate the neuronal death pathways activated by the oxidant peroxynitrite. Experimental results from our group and others over the last 15 years using PC12 cells and motor neurons in culture suggest that peroxynitrite may trigger cell death via the selective nitration of a surprisingly small group of proteins. We found that the intracellular delivery of peroxynitrite-modified heat shock protein 90 (HSP90) was sufficient to recapitulate peroxynitrite-induced cell death in motor neurons. Conversely, the release of HSP70, actin, tubulin or albumin treated with peroxynitrite was not toxic to the cells. We hypothesize that nitrated HSP90 is a critical player in peroxynitrite-induced cell death and that nitration of HSP90 is sufficient to activate cell death pathways. Treatment of a pure protein like HSP90 with peroxynitrite produces multiple modifications that can be identified by mass spectrometry. Using a newly developed method for incorporation of non-natural amino acids such as nitrotyrosine into recombinant HSP90, we can, for the first time, test whether nitration at a specific site confers a toxic gain-of-function on a protein. The significance and impact of the proposed investigations is highlighted by the identification of the nitrated HSP90 with monoclonal antibodies we developed in several pathological conditions in vivo; these conditions include kidney rejection, heart disease, stroke, spinal cord injury, and spinal motor neurons from patients and mouse models of ALS. Our investigations will impact the understanding of cell death mechanisms induced by oxidative stress by providing a new working model for examining how specific protein modifications, rather than general oxidative damage, can activate highly regulated death pathways. In addition, completion of these investigations will provide detailed insights into the specific mechanisms of cell death stimulated by nitric oxide and peroxynitrite with implications for a wide array of neurodegenerative and inflammatory conditions. PUBLIC HEALTH RELEVANCE: A variety of neuropathological conditions involve inflammation associated with the formation of reactive nitrogen species and nitrotyrosine. The role of tyrosine nitration in the pathogenesis of these conditions is unknown and controversial. The identification of a target molecule, such as HSP90, may result in the development of new therapeutic strategies aimed to prevent the effects of tyrosine nitration.