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

表征模拟 18.5-kDa 髓磷脂碱性蛋白 (MBP) 脱亚胺化和磷酸化的取代对其在髓磷脂样膜中的结构和动态的影响

• 批准号: 320907857
• 负责人: Professor Dr. Dariush Hinderberger
• 金额: --
• 依托单位: Arbeitsbereich Physikalische und Theoretische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/320907857?language=en
• 关键词: Characterizing; Effect; Substitutions; Mimicking; Deimination

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.

多发性硬化症(MS)是一种病因不明、临床变异性高的严重衰弱的神经系统疾病。这种疾病的特征是中枢神经系统脱髓鞘，即包裹神经轴突的保护性、富含脂质的髓鞘丧失，使它们能够有效地传递脉冲，导致斑块形成、神经衰弱和进行性退化。这种疾病的最初诱因尚不清楚，也没有任何治愈方法。髓鞘碱性蛋白(MBP)家族维持髓鞘结构和动态平衡，其中18.5 kDa的亚型在成人脑中占主导地位。该蛋白具有内在的无序性和多功能，与少突胶质细胞的细胞质小叶结合，与大量其他蛋白质结合，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，其靶向性和关联性受到翻译后修饰(PTM)的调节。在MS中，PTMS减少了很大一部分MBP的净正电荷(从+19降至+13)，并有助于物理脱髓鞘和诱导自身免疫。18.5 kDa的MBP异构体(家族原型)更喜欢在髓鞘中进行第三级接触和自组装，从而使其成为一种多功能的网络蛋白。这项建议旨在描述和理解PTM引起的这些相互作用的变化，这些相互作用直接改变MBP的结构和髓鞘中的自组装。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，必要时结合其他方法。我们的目标是形成MBP的构象集合及其在髓鞘中分布的详细模型，以了解位点特异性修饰(在这种情况下是模仿蛋白质中的脱亚胺和磷酸化的电荷减少)如何调节其与膜的结合、其三级结构集合以及髓鞘样膜中的动力学。为此，将使用EPR/IRRA组合光谱方法表达和研究18.5 kDa MBP的8个重组变体(在小鼠中有168个残基)，代表不同的功能形式(对于用氮氧化物双自旋标记后的EPR)。在MBP与膜重组后，主要是MBP的三级结构将通过多种EPR方法的组合进行研究，并辅之以IRRAS方法，该方法允许研究脂质/空气界面的取向和二级结构变化。总体目标是将类似PTM的变化、脂膜组成和二价金属离子的有效性与不同MBP亚型的不同髓鞘紧致能力直接相关。我们希望阐明多发性硬化症脱髓鞘早期的分子基础，并帮助寻找新的治疗方法。

项目成果

Interaction of Myelin Basic Protein with Myelin-like Lipid Monolayers at Air-Water Interface.

• DOI: 10.1021/acs.langmuir.8b00321
• 发表时间: 2018-05
• 期刊: Langmuir : the ACS journal of surfaces and colloids
• 作者: Katharina Widder;Jennica Träger;Andreas Kerth;G. Harauz;D. Hinderberger

Effect of Cholesterol and Myelin Basic Protein (MBP) Content on Lipid Monolayers Mimicking the Cytoplasmic Membrane of Myelin

胆固醇和髓磷脂碱性蛋白 (MBP) 含量对模拟髓磷脂细胞质膜的脂质单层的影响

• DOI: 10.3390/cells9030529
• 发表时间: 2020
• 期刊: Cells
• 影响因子: 6
• 作者: Jennica Träger;Katharina Widder;Andreas Kerth;George Harauz;Dariush Hinderberger
• 通讯作者: Dariush Hinderberger

Myelin basic protein (MBP) charge variants show different sphingomyelin-mediated interactions with myelin-like lipid monolayers.

髓磷脂碱性蛋白 (MBP) 电荷变体表现出不同的鞘磷脂介导的与髓磷脂样脂质单层的相互作用

• DOI: 10.1016/j.bbamem.2019.183077
• 发表时间: 2020
• 期刊: Biochimica et biophysica acta. Biomembranes
• 作者: Katharina Widder;George Harauz;Dariush Hinderberger
• 通讯作者: Dariush Hinderberger