MECHANISMS OF NO-MEDIATED PROTEIN S-NITROSYLATION IN EAE

EAE 中无介导蛋白质 S-亚硝基化机制

• 批准号: 6990479
• 负责人: OSCAR A BIZZOZERO
• 金额: $ 27.1万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6990479
• 关键词: experimental allergic encephalomyelitis; immunocytochemistry; laboratory rat; mass spectrometry; molecular pathology; multiple sclerosis; myelinopathy; nerve /myelin protein; nitric oxide; peroxynitrites; spinal cord; thiols

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.

描述（由申请人提供）：我们的长期目标是确定和描述导致炎症性脱髓鞘疾病中髓磷脂破坏的机制。一氧化氮（NO）与多发性硬化症（MS）和实验性过敏性脑脊髓炎（EAE）的病理生理有关，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/小胶质细胞释放出高水平的一氧化氮。然而，一氧化氮导致髓磷脂分解的机制尚不清楚。基于最近对其他系统中亚硝化损伤的分子和细胞后果的研究，以及我们实验室的一些重要发现，我们假设NO可以从炎性病变扩散到相当远的距离，导致广泛的髓鞘分解。我们认为，这一过程的基础是N203对重要髓鞘蛋白的s -亚硝基化，N203是一种强亚硝基化剂，在髓鞘等富含脂质的环境中，一氧化氮可以很容易地自氧化形成。本文所述的实验将详细描述髓磷脂蛋白在非渗透性NO供体培养的大鼠脊髓切片中s-亚硝基化的机制，该机制产生的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诱导的硫基相关修饰（s-硫基化和蛋白质二硫化物的形成）的存在，(2)通过质谱法鉴定主要的s-亚硝基化髓磷脂蛋白。(3)测试几种化合物防止/恢复这些有害蛋白质修饰的能力。此外，将研究脊髓中s -亚硝基化蛋白在EAE过程中的发生情况，并使用蛋白质组学方法确定这些物种的身份。最后，通过免疫细胞化学评估s -亚硝基化蛋白在病变组织中的分布，直接验证游离NO诱导的亚硝基化蛋白损伤可以发生在距离炎性病变一定距离的地方，并且独立于过氧亚硝酸盐的产生。拟议的研究将产生关键信息，可以支持炎症脱髓鞘过程中no介导的髓磷脂破坏的新途径。阐明蛋白修饰的机制，髓磷脂中受NO影响的分子的鉴定，以及这些修饰在广泛使用的多发性硬化症动物模型的中枢神经系统中的发生，对于理解这种毁灭性疾病的病理生理至关重要。