MECHANISMS OF NO-MEDIATED PROTEIN S-NITROSYLATION IN EAE

描述（由申请人提供）：我们的长期目标是确定和表征导致炎性脱髓鞘疾病中髓鞘破坏的机制。一氧化氮（NO）与多发性硬化（MS）和实验性过敏性脑脊髓炎（EAE）的病理生理学有关，因为在各种促炎细胞因子诱导一氧化氮合成酶后，巨噬细胞/小胶质细胞释放高水平的这种气体。然而，NO导致髓磷脂分解的机制还远不清楚。基于最近的调查有关的分子和细胞的亚硝化损伤在其他系统中的后果，和一些重要的发现，从我们的实验室，我们假设，NO可以传播在相当远的距离从炎症病变引起广泛的髓鞘解压缩。我们提出，这一过程的基础是重要的髓鞘蛋白的S-亚硝基化N203，一个强大的亚硝基化剂，可以很容易地形成一个富含脂质的环境中，如髓鞘的NO自氧化。本提案中描述的实验将详细描述与非渗透性NO供体孵育的大鼠脊髓切片中髓鞘蛋白的S-亚硝基化机制，其产生的NO水平与EAE和MS中发现的水平相当。这些实验还将（1）检查其他NO诱导的巯基相关修饰的存在（S-硫醇化和蛋白质二硫化物的形成），（2）通过质谱法鉴定主要的S-亚硝基化髓鞘蛋白质，和（3）测试几种化合物防止/逆转这些有害蛋白质修饰的能力。此外，将研究EAE过程中脊髓中S-亚硝基化蛋白的发生，并使用蛋白质组学方法确定这些物种的身份。最后，S-亚硝基化蛋白在受影响的组织中的分布将通过免疫细胞化学进行评估，以直接测试由游离NO诱导的亚硝化蛋白损伤可以在离炎性病变一定距离处发生并且独立于过氧亚硝酸根生成的假设。这些研究将产生重要的信息，可以支持炎症脱髓鞘过程中NO介导的髓鞘破坏的新途径。阐明蛋白质修饰的机制，识别受NO影响的髓鞘中的分子，以及在广泛使用的MS动物模型的CNS中发生这些修饰对于理解这种破坏性疾病的病理生理学是必不可少的。

DESCRIPTION (provided by applicant): Our long-term goal is to identify and characterize the mechanisms that lead to the destruction of myelin in inflammatory demyelinating disorders. Nitric oxide (NO) has been implicated in the pathophysiology of both multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), as high levels of this gas are released by macrophages/microglia following induction of nitric oxide synthetase by various proinflammatory cytokines. However, the mechanism by which NO leads to myelin breakdown is far from clear. Based on recent investigations regarding the molecular and cellular consequences of nitrosative damage in other systems, and a number of important findings from our laboratory, we hypothesize that NO can spread at considerable distances from inflammatory lesions to cause extensive myelin decompaction. We propose that underlying this process is the S-nitrosylation of important myelin proteins by N203, a strong nitrosylating agent that can be readily formed by NO autooxidation in a lipid-rich environment like the myelin sheath. The experiments described in this proposal will characterize in detail the mechanism of S-nitrosylation of myelin proteins in rat spinal cord slices incubated with a non-permeable NO-donor, which generates levels of NO comparable to those found in EAE and MS. These experiments will also (1) examine the existence of other NO-induced thiol-related modifications (S-thiolation and formation of protein disulfides), (2) identify the major S-nitrosylated myelin proteins by mass-spectrometry, and (3) test several compounds for their ability to prevent/revert these deleterious protein modifications. In addition, the occurrence of S-nitrosylated proteins in spinal cord during the course of EAE will be investigated and the identity of these species determined using a proteomic approach. Finally, the distribution of S-nitrosylated proteins in the affected tissue will be assessed by immunocytochemistry to directly test the hypothesis that nitrosative protein damage induced by free NO can take place at some distance from the inflammatory lesions and independently of peroxynitrite generation. The proposed studies will generate crucial information that could support a new pathway for the NO-mediated destruction of myelin during inflammatory demyelination. The elucidation of the mechanism of protein modification, the identification of the molecules in myelin that are affected by NO, and the occurrence of these modifications in the CNS of a widely-used animal model of MS are essential for understanding the pathophysiology of this devastating disorder.