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.
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