Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein.
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein.
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鉴定与单体和寡聚 α-突触核蛋白结合的突触体蛋白。
DOI：
10.1371/journal.pone.0116473
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Jensen PH
中科院分区：
文献类型：
--
作者：
Betzer C;Movius AJ;Shi M;Gai WP;Zhang J;Jensen PH
单体α-突触核蛋白（α-synuclein，αSN）在神经末梢中含量丰富，推测其在突触囊泡翻转中发挥生理作用。在帕金森病（PD）和路易体痴呆（DLB）中，αSN在终末、轴突和体树突隔室中以聚集的可溶性寡聚体和路易包涵体和路易神经突中以不溶性细丝的形式蓄积。与αSN基因突变相关的常染色体显性遗传力表明与聚集αSN相关的功能获得。我们已经进行了蛋白质组学筛选，以确定αSN相互作用组在脑突触体。将猪脑突触体分级分离，溶解于非变性去污剂中，并使用纯化的重组人αSN单体或寡聚体作为诱饵进行免疫共沉淀。使用LC-LTQ-orbitrap串联质谱法鉴定分离的αSN结合蛋白，并使用Windows客户端应用程序Skyline Targeted Proteomic Environment通过峰面积进行定量。数据可通过ProteomeXchange获得，标识符为PXD 001462。为了定量优先结合，通过比较与单体和寡聚体的结合来计算平均倍数增加。我们鉴定了10个优先结合单体的蛋白质，76个优先结合寡聚体的蛋白质和一组92个不显示αSN任何优先构象的蛋白质。使用针对单体αSN相互作用物（Abl相互作用物1和髓鞘蛋白脂质蛋白）和寡聚体相互作用物（谷氨酸脱羧酶2、突触蛋白1、神经胶质细胞酸性蛋白和VAMP-2）的抗体，通过免疫沉淀在人和猪脑提取物中验证蛋白质组学数据。我们证明了αSN构象选择性配体的存在，并列出了蛋白质，其身份和功能将有助于模拟正常和病理性αSN依赖性过程。
Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson’s disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.
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