Protein array analysis of oligomerization-induced changes in alpha-synuclein protein-protein interactions points to an interference with Cdc42 effector proteins
Protein array analysis of oligomerization-induced changes in alpha-synuclein protein-protein interactions points to an interference with Cdc42 effector proteins
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DOI：
10.1016/j.neuroscience.2008.02.049
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发表时间：
2008-07-17
期刊：
影响因子：
3.3
通讯作者：
Gillardon, F.
中科院分区：
文献类型：
--
作者：
Schnack, C.;Danzer, K. M.;Gillardon, F.
α-突触核蛋白的聚集可能在帕金森氏病患者和转基因动物模型的神经病理中起作用。天然未折叠的α-突触核蛋白与多种蛋白质结合，由于α-突触核蛋白错误折叠而引起的构象变化可能改变生理相互作用。在目前的研究中，我们使用了带有5000个重组人类蛋白的蛋白质阵列来大规模分析单体和寡聚α-突触核蛋白的相互作用。单体α-突触核蛋白与阵列化cAMP调节的磷蛋白19结合，结合似乎被α-突触核蛋白寡聚破坏。与重组α-突触核蛋白寡聚体孵育后，几种GTP酶激活蛋白和CDC42效应蛋白被鉴定为结合伙伴。蛋白质数据库搜索发现，在一些相互作用元件中存在一个CDC42/RAC相互作用结合域。为了证明体内相关性，我们分析了从α-突触核蛋白(A30P)转基因小鼠中提取的脑干蛋白。使用与CDC42/RAC相互结合结构域结合的珠子的下拉分析导致内源性α-突触核蛋白寡聚体的丰富。Cdc42效应蛋白也与脑干裂解液中的α-突触核蛋白共沉淀，并通过双重免疫染色与α-突触核蛋白聚集体在脑切片上共定位。通过对转基因小鼠脑中突触体组分的双向凝胶电泳分析，我们检测到了额外的Septin 6亚型，它是CDC42效应蛋白的下游靶标。最近在遗传修饰物筛选中发现了一些小的GTP酶，以抑制酵母中的α-突触核蛋白毒性。我们的数据表明，小的GTPase信号转导通路的组件可能直接被α-突触核蛋白寡聚体靶向，这可能导致信号缺陷和神经退化。(C)2008年IBRO。爱思唯尔有限公司出版。保留所有权利。
Aggregation of alpha-synuclein may contribute to neuropathology in Parkinson's disease patients and in transgenic animal models. Natively unfolded alpha-synuclein binds to various proteins and conformational changes due to alpha-synuclein misfolding may alter physiological interactions. In the present study, we used protein arrays spotted with 5000 recombinant human proteins for a large scale interaction analysis of monomeric versus oligomeric alpha-synuclein. Monomeric alpha-synuclein bound to arrayed cAMP regulated phosphoprotein 19 and binding appears to be disrupted by alpha-synuclein oligomerization. Incubation with recombinant alpha-synuclein oligomers lead to the identification of several GTPase activating proteins and Cdc42 effector proteins as binding partners. Protein database searches revealed a Cdc42/Rac interactive binding domain in some interactors. To demonstrate in vivo relevance, we analyzed brainstem protein extracts from alpha-synuclein(A30P) transgenic mice. Pull-down assays using beads conjugated with a Cdc42/Rac interactive binding domain lead to an enrichment of endogenous alpha-synuclein oligomers. Cdc42 effector proteins were also co-immunoprecipitated with alpha-synuclein from brainstem lysates and were colocalized with alpha-synuclein aggregates in brain sections by double immunostaining. By two-dimensional gel electrophoretic analysis of synaptosomal fractions from transgenic mouse brains we detected additional isoforms of septin 6, a downstream target of Cdc42 effector proteins.Small GTPases have recently been identified in a genetic modifier screen to suppress alpha-synuclein toxicity in yeast. Our data indicate that components of small GTPase signal transduction pathways may be directly targeted by alpha-synuclein oligomers which potentially leads to signaling deficits and neurodegeneration. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.