Amyloid Beta A4 Precursor Protein-binding Family B Member 1 (FE65) Interactomics Revealed Synaptic Vesicle Glycoprotein 2A (SV2A) and Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 (SERCA2) as New Binding Proteins in the Human Brain
Amyloid Beta A4 Precursor Protein-binding Family B Member 1 (FE65) Interactomics Revealed Synaptic Vesicle Glycoprotein 2A (SV2A) and Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 (SERCA2) as New Binding Proteins in the Human Brain
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DOI：
10.1074/mcp.m113.029280
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发表时间：
2014-02-01
影响因子：
7
通讯作者：
Mueller, Thorsten
中科院分区：
文献类型：
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作者：
Nensa, Fabian M.;Neumann, Martin H. D.;Mueller, Thorsten
FE65 是一种胞质接头蛋白，也是淀粉样前体蛋白的重要结合伴侣。依赖于淀粉样前体蛋白中的 Thr668 磷酸化（影响淀粉样蛋白前体蛋白加工），FE65 经历核易位，从而将信号从细胞膜传递到细胞核。由于这种易位可能与阿尔茨海默病有关，并且 FE65 由三个蛋白质-蛋白质相互作用结构域组成，能够结合并影响各种其他蛋白质和下游信号通路，因此 FE65 相互作用组的鉴定是阿尔茨海默病研究的核心兴趣。在这项研究中，我们使用人类死后大脑样本作为重组表达 FE65 的蛋白质库，通过下拉/质谱方法鉴定了 121 种蛋白质作为新的潜在 FE65 相互作用蛋白质。免疫共沉淀分析进一步验证了 FE65 与候选 SV2A 和 SERCA2 的相互作用。与此同时，我们研究了 FE65/FE65L1 双敲除小鼠原代海马神经元的全细胞蛋白质组。值得注意的是，相对于野生型对照神经元，经验证的 FE65 结合蛋白在 FE65 敲除小鼠衍生的神经元中也存在差异丰富。 SERCA2 是细胞钙稳态的重要参与者，被发现在双敲除神经元中上调。事实上，HEK293T 细胞中 FE65 的敲低也会引起对毒胡萝卜素的敏感性升高，毒胡萝卜素是一种专门针对 SERCA2 活性的应激源。因此，我们的结果表明 FE65 参与细胞内钙稳态的调节。单独转染 FE65 在细胞核中引起典型的点状表型，而共转染 SV2A 显着降低了 FE65 点阳性细胞的百分比，表明 SV2A 在调节 FE65 细胞内靶向中可能发挥作用。鉴于 SV2A 在突触前具有信号传导功能，其对 FE65 细胞内定位的影响表明 SV2A/FE65 相互作用可能在突触信号转导中发挥作用。
FE65 is a cytosolic adapter protein and an important binding partner of amyloid precursor protein. Dependent on Thr668 phosphorylation in amyloid precursor protein, which influences amyloidogenic amyloid precursor protein processing, FE65 undergoes nuclear translocation, thereby transmitting a signal from the cell membrane to the nucleus. As this translocation may be relevant in Alzheimer disease, and as FE65 consists of three protein-protein interaction domains able to bind and affect a variety of other proteins and downstream signaling pathways, the identification of the FE65 interactome is of central interest in Alzheimer disease research. In this study, we identified 121 proteins as new potential FE65 interacting proteins in a pulldown/mass spectrometry approach using human post-mortem brain samples as protein pools for recombinantly expressed FE65. Co-immunoprecipitation assays further validated the interaction of FE65 with the candidates SV2A and SERCA2. In parallel, we investigated the whole cell proteome of primary hippocampal neurons from FE65/FE65L1 double knockout mice. Notably, the validated FE65 binding proteins were also found to be differentially abundant in neurons derived from the FE65 knockout mice relative to wild-type control neurons. SERCA2 is an important player in cellular calcium homeostasis, which was found to be up-regulated in double knockout neurons. Indeed, knock-down of FE65 in HEK293T cells also evoked an elevated sensitivity to thapsigargin, a stressor specifically targeting the activity of SERCA2. Thus, our results suggest that FE65 is involved in the regulation of intracellular calcium homeostasis. Whereas transfection of FE65 alone caused a typical dot-like phenotype in the nucleus, co-transfection of SV2A significantly reduced the percentage of FE65 dot-positive cells, pointing to a possible role for SV2A in the modulation of FE65 intracellular targeting. Given that SV2A has a signaling function at the presynapse, its effect on FE65 intracellular localization suggests that the SV2A/FE65 interaction might play a role in synaptic signal transduction.