用于定量测定脑细胞膜蛋白棕榈酰化的酰基-聚乙二醇交换凝胶迁移分析
Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins.
作者信息
Speca David J, Diaz Elva
机构信息
Department of Pharmacology, UC Davis School of Medicine.
Department of Pharmacology, UC Davis School of Medicine;
出版信息
J Vis Exp. 2020 Mar 29(157). doi: 10.3791/61018.
Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.
突触后致密区（PSD）内突触α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPARs）水平的活性依赖性改变被认为是学习和记忆的一种细胞机制。棕榈酰化以活性依赖的方式调节许多突触蛋白的定位和功能，包括AMPA受体、辅助因子和突触支架。我们鉴定出了一个由四个基因组成的突触分化诱导基因（SynDIG）家族（SynDIG1 - 4），它们编码与AMPARs相关并调节突触强度的脑特异性跨膜蛋白。SynDIG1在靠近跨膜区域的第191和192位的两个半胱氨酸残基处被棕榈酰化，这对于活性依赖的兴奋性突触发育很重要。在这里，我们描述了一种创新的生化方法，即酰基-聚乙二醇交换凝胶迁移（APEGS）测定法，用于研究任何感兴趣蛋白质的棕榈酰化状态，并在小鼠脑裂解物中用SynDIG蛋白家族证明了其效用。
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