Characterizing the Effect of Substitutions Mimicking Deimination and Phosphorylation of 18.5-kDa Myelin Basic Protein (MBP) on its Structure and Dynamics in Myelin-Like Membranes

多发性硬化症（MS）是一种起源不明、临床变异性高的严重衰弱性神经系统疾病。该疾病的标志是中枢神经系统脱髓鞘，即包裹神经轴突并使它们能够有效地传递冲动的保护性富脂髓鞘的丧失，导致斑块形成、神经衰弱和进行性变性。这种疾病的最初诱因尚不清楚，也没有任何治疗方法。髓鞘结构和稳态由髓鞘碱性蛋白（MBP）家族维持，其中18.5-kDa同种型在成人脑中占主导地位。这种蛋白质具有内在的无序性和多功能性，它粘附在少突胶质细胞的胞质小叶上，与大量其他蛋白质缔合，在所有相互作用（包括分子识别片段和开关）中经历部分诱导折叠，并且其靶向和缔合受到翻译后修饰（PTM）的调节。在MS中，PTM减少了大部分MBP的净正电荷（从+19到+13），并有助于物理脱髓鞘和诱导自身免疫。18.5 kDa MBP同种型（家族原型）在髓磷脂中具有优选的三级接触和自组装，使其成为一种多功能网络蛋白。该提案旨在表征和理解PTM诱导的这些相互作用的变化，直接改变髓鞘的MBP结构和自组装。我们使用基于电子顺磁共振（EPR）和红外反射吸收（IRRA）光谱的方法，必要时与其他方法相结合。我们的目标是形成一个详细的模型的构象整体的MBP和它的分布在髓鞘，了解位点特异性的修改，在这种情况下，电荷减少模仿脱亚胺和磷酸化的蛋白质，调节其协会与膜，其三级结构整体和动力学髓鞘样膜。为此，8个重组变体的18.5 kDa的MBP（168个残基在小鼠），代表不同的功能形式，将表达和研究使用组合EPR/IRRA光谱方法（EPR后，双自旋标记与氮氧化物）。用膜重建MBP后，主要是MBP三级将通过多种EPR方法的组合进行研究，并通过IRRAS方法进行补充，该方法允许研究脂质/空气界面处的取向和二级结构变化。总体目标是将PTM模拟变化、脂质膜组成和二价金属离子的可用性与不同MBP同种型的不同髓鞘压缩能力直接相关。我们希望阐明MS脱髓鞘早期步骤的分子基础，并帮助寻找新的治疗方法。

Multiple sclerosis (MS) is a severely debilitating neurological disease of unknown origin and high clinical variability. The disease hallmark is central nervous system demyelination, i.e. loss of the protective, lipid-rich myelin sheath that enwraps nerve axons and enables them to transmit impulses efficiently leading to formation of plaques, neurological debilitation, and progressive degeneration. Neither the initial trigger for the disease is known, nor is there any cure. Myelin structure and homeostasis are maintained by the myelin basic protein (MBP) family, of which the 18.5-kDa isoform predominates in adult brain. This protein has been shown to be intrinsically disordered and multifunctional, it adheres the cytoplasmic leaflets of oligodendrocytes, associates with a large number of other proteins, undergoes partial induced folding in all interactions (including molecular recognition fragments and switches), and its targeting and associations are modulated by post-translational modifications (PTMs). In MS, PTMs reduce the net positive charge of a large fraction of MBP (from +19 to +13) and contribute to physical demyelination and induction of autoimmunity. The 18.5-kDa MBP isoform (the family-prototype) has preferred tertiary contacts and self-assembles in myelin, allowing it to be a versatile networking protein. This proposal aims at characterizing and understanding PTM-induced changes in these interactions that directly alter MBP structure and self-assembly in myelin. We use an approach based on Electron Paramagnetic Resonance (EPR) and Infrared Reflection-Absorption (IRRA) spectroscopies, combined where necessary with other approaches. The goal is to form a detailed model of the conformational ensemble of MBP and its distribution within myelin, to understand how site-specific modifications, in this case charge reductions mimicking deimination and phosphorylation in the protein, regulate its associations with membranes, its tertiary structure ensemble and dynamics in myelin-like membranes. To this end, 8 recombinant variants of 18.5-kDa MBP (168 residues in mouse), representing different functional forms, will be expressed and studied using the combined EPR/IRRA spectroscopic approaches (for EPR after double spin-labeling with nitroxides). After reconstitution of MBP with membranes, mainly the MBP tertiary will be studied by a combination of diverse EPR approaches and is complemented by the IRRAS method that allows studying orientation and secondary structure changes at the lipid/air interface. The overall goal is to directly correlate the PTM-mimicking changes, the lipid membrane composition and the availability of divalent metal ions with the different myelin-compacting capabilities of the different MBP isoforms. We hope to shed light on the molecular bases on the early steps of demyelination in MS and to aid the search for new approaches to therapy.