Influence of Membrane Surface Charge and Post-Translational Modifications to Myelin Basic Protein on Its Ability To Tether the Fyn-SH3 Domain to a Membrane in Vitro
Influence of Membrane Surface Charge and Post-Translational Modifications to Myelin Basic Protein on Its Ability To Tether the Fyn-SH3 Domain to a Membrane in Vitro
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DOI：
10.1021/bi8022587
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发表时间：
2009-03-24
期刊：
影响因子：
2.9
通讯作者：
Boggs, Joan M.
中科院分区：
文献类型：
--
作者：
Homchaudhuri, Lopamudra;Polverini, Eugenia;Boggs, Joan M.
髓磷脂碱性蛋白（MBP）是中枢神经系统髓磷脂的高度后修饰的多功能结构组分，粘附于磷脂膜并组装细胞骨架蛋白，并且先前已显示在体外结合SH 3结构域并将它们拴系于膜表面[Polverini，E.，等。（2008）Biochemistry，47，267-282]。由于分子模拟表明，Fyn-SH 3结构域具有负表面电荷密度，即使在结合MBP配体后，我们已经研究了负膜表面电荷的影响和MBP的翻译后修饰对Fyn-SH 3结构域与膜相关MBP的相互作用的影响。使用的沉淀分析与多层囊泡组成的中性磷脂酰胆碱（PC）和带负电荷的磷脂酰肌醇（PI），我们表明，通过增加PI的比例增加膜的负表面电荷减少Fyn-SH 3域的量，结合到膜相关的MBP，由于静电排斥。当磷脂酰肌醇之一PI（4）P或PI（4，5）P-2以相等比例取代PI时，在所用条件下没有Fyn-SH 3结构域与MBP结合。MBP的翻译后修饰减少了其净正电荷，即，磷酸化或精氨酸脱亚氨基化，增加了Fyn-SH 3从膜表面排斥的程度，这种作用进一步受到脂质电荷的调节。该研究表明，由于蛋白质或脂质修饰引起的膜负表面电荷的变化，这可能发生在细胞信号传导期间，可以调节Fyn-SH 3结构域与膜结合MBP的结合，从而可以调节少突胶质细胞膜表面Fyn的活性。
Myelin basic protein (MBP) is a highly post-translationally modified, multifunctional structural component of central nervous system myelin, adhering to phospholipid membranes and assembling cytoskeletal proteins, and has previously been shown to bind SH3 domains in vitro and tether them to a membrane surface [Polverini, E., et at. (2008) Biochemistry, 47, 267-282]. Since molecular modeling shows that the Fyn-SH3 domain has a negative surface charge density even after binding the MBP ligand, we have investigated the influence of negative membrane surface charge and the effects of post-translational modifications to MBP on the interaction of the Fyn-SH3 domain with membrane-associated MBP. Using a sedimentation assay with multilamellar vesicles consisting of neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol (PI), we demonstrate that increasing the negative surface charge of the membrane by increasing the proportion of PI reduces the amount of Fyn-SH3 domain that binds to membrane-associated MBP, due to electrostatic repulsion. When one of the phosphoinositides, PI(4)P or PI(4,5)P-2 was substituted for PI in equal proportion, none of the Fyn-SH3 domain bound to MBP under the conditions that were used. Post-translational modifications of MBP which reduced its net positive charge, i.e., phosphorylation or arginine deimination, increased the degree of repulsion of Fyn-SH3 from the membrane surface, an effect further modulated by the lipid charge, This study suggests that changes in membrane negative Surface charge due to protein or lipid modifications, which could occur during cell signaling, can regulate the binding of the Fyn-SH3 domain to membrane-associated MBP and thus Could regulate the activity of Fyn at the oligodendrocyte membrane surface.