NITRIC OXIDE, PLP ACYLATION & THE PATHOPHYSIOLOGY OF MS

我们的长期目标是了解控制中枢神经系统髓鞘形成和维持的分子机制，以及在多发性硬化症(MS)中导致其崩溃的因素。尽管激活的巨噬细胞和小胶质细胞产生的一氧化氮(NO)和过氧亚硝酸盐被认为是MS中髓鞘和少突胶质细胞破坏的原因，但这些药物在髓鞘中的分子靶点尚未确定。我们推测，丰富的髓鞘蛋白脂蛋白(PLP)的结构和脂肪酸酰化都受到NO和过氧亚硝酸盐的影响，这可能导致髓鞘不稳定。我们的具体目标是：1-利用内源产生的18/O标记脂肪酸来表征PLP的脂肪酰化机制。这项最新开发的同位素技术不仅可以测量酰化速率，还可以测量被修饰的最小蛋白质数量。快速髓鞘形成大鼠的脑白质切片将与[~3H]棕榈酸酯和H2/18/O孵育，在各种代谢毒物和酶抑制剂的存在下，以确定PLP酰化是否(A)需要ATP，(B)需要形成酰基-CoA，(C)主要使用从头合成的脂肪酸，以及(D)由单独的蛋白质脂肪酰基转移酶催化。2-评估一氧化氮和过氧亚硝酸盐对PLP脂肪酰化的影响。我们将使用组织切片和上述双标记技术来确定病理浓度的内源性NO、外源性NO和过氧亚硝酸根对PLP和脂质酰化的影响。此外，还将进行各种代谢和结构研究，以确定氮和氧自由基改变蛋白质酰化的机制(S)。3-确定从MS脑中分离出的各种PLP物种的结构和脂肪酰化是否正常。从对照组和MS脑制备的髓鞘和非髓鞘膜中存在的主要PLP、DM-20和16KDA蛋白脂将被分离并进行全面的化学和质谱分析。本申请中提出的研究将为多发性硬化症中髓鞘破坏的机制提供直接的信息，同时，它们将有助于我们理解PLP的生物学及其唯一的翻译后修饰。

Our long-term goal is to understand the molecular mechanisms that control the formation and maintenance of CNS myelin and the factors that lead to its breakdown in multiple sclerosis (MS). Although the production of nitric oxide (NO) and peroxynitrite by activated macrophages and microglial cells is considered responsible for the destruction of myelin and oligodendrocytes in MS, the molecular targets of these agents in myelin have not yet been identified. We hypothesize that both the structure and fatty acid acylation of the abundant myelin proteolipid protein (PLP) are affected by NO and peroxynitrites, and that this may lead to myelin instability. Our specific aims are: 1- To characterize the mechanism of fatty acylation of PLP using endogenously generated 18/O-labeled fatty acids. This recently developed isotopomeric technique measures not only the acylation rate but also the minimal amount of proteins that are modified. Brain white matter slices from rapidly myelinating rats will be incubated with [3H]palmitate and H2/18/O, in the presence of a variety of metabolic poisons and enzyme inhibitors to ascertain whether PLP acylation (a) needs ATP, (b) requires the formation acyl-CoA, (c) using primarily fatty acids synthesized de novo, and (d) is catalyzed by a separate protein fatty acyltransferase. 2- To assess the effects of nitric oxide and peroxynitrite on the fatty acylation of PLP. We will determine the effects of pathological concentrations of endogenously-generated NO, exogenously-produced NO and peroxynitrite on acylation on PLP and lipids using tissue slices and the double-label technique described above. In addition, a variety of metabolic and structural studies will be carried out to identify the mechanism(s) by which nitrogen and oxygen free radicals could alter protein acylation. 3- To determine whether or not the structure and fatty acylation of the various PLP species isolated from MS brains are normal. The major PLP, DM-20 and 16 KdA proteolipid present in myelin and non-myelin membranes prepared from control and MS brains will be isolated and subjected to a comprehensive chemical and mass-spectrometric analysis. The studies proposed in this application will provide direct information into the mechanisms of myelin destruction that takes place in MS, and at the same time, they will aid our understanding of the biology of PLP and its only post-translational modification.