A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity.
A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity.
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DOI：
10.1042/bsr20160040
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发表时间：
2016
影响因子：
4
通讯作者：
Wassmer T
中科院分区：
文献类型：
--
作者：
Guscott B;Balklava Z;Safrany ST;Wassmer T
在这项工作中，我们开发并验证了一种细胞渗透性工具，用于研究阿尔茨海默病中心分子--淀粉样前体蛋白--的细胞内功能。我们发现它调节PIKfyve激酶复合体的活性。调节PIKfyve复合体活性的机制目前正在形成。PIKfyve复合体由磷脂酰肌醇激酶PIKfyve(也称为FAB1)、VAC14和FIG4组成，是生产磷脂酰肌醇3，5-二磷酸[PI(3，5)P2]所必需的。PIKfyve功能是内/溶酶体系统内稳态所必需的，与神经元的功能和完整性密切相关，因为PIKfyve复合体功能突变的丢失会导致小鼠模型和人类患者的神经退化。我们最近的工作表明，淀粉样前体蛋白(APP)的细胞内结构域与VAC14结合并增强PIKfyve功能。淀粉样前体蛋白是阿尔茨海默病病因的核心分子。在目前的研究中，我们利用这一最新进展来创建一个易于使用的工具来增加细胞中的PIKfyve活性。我们将APP胞内域(AICD)融合到HIV TAT结构域，这是一种细胞渗透肽，允许蛋白质穿透细胞。由此产生的TAT-AICD融合蛋白是细胞通透性的，并触发PI(3，5)P2的增加。使用PI(3，5)P2特异性GFP-ML1Nx2探针，我们发现细胞通透性AICD改变了PI(3，5)P2的动力学。TAT-AICD还对PIKfyve的药理抑制提供部分保护。所有这三条证据都表明，AICD激活了细胞中的PIKfyve复合体，这一发现对于我们理解阿尔茨海默病神经退化的机制很重要。
In this work we developed and validated a cell permeable tool to study the intracellular function of a central molecule in Alzheimer's disease, the amyloid precursor protein. We showed that it regulates the activity of the PIKfyve kinase complex. The mechanisms for regulating PIKfyve complex activity are currently emerging. The PIKfyve complex, consisting of the phosphoinositide kinase PIKfyve (also known as FAB1), VAC14 and FIG4, is required for the production of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]. PIKfyve function is required for homoeostasis of the endo/lysosomal system and is crucially implicated in neuronal function and integrity, as loss of function mutations in the PIKfyve complex lead to neurodegeneration in mouse models and human patients. Our recent work has shown that the intracellular domain of the amyloid precursor protein (APP), a molecule central to the aetiology of Alzheimer's disease binds to VAC14 and enhances PIKfyve function. In the present study, we utilize this recent advance to create an easy-to-use tool for increasing PIKfyve activity in cells. We fused APP intracellular domain (AICD) to the HIV TAT domain, a cell-permeable peptide allowing proteins to penetrate cells. The resultant TAT–AICD fusion protein is cell permeable and triggers an increase in PI(3,5)P2. Using the PI(3,5)P2 specific GFP-ML1Nx2 probe, we show that cell-permeable AICD alters PI(3,5)P2 dynamics. TAT–AICD also provides partial protection from pharmacological inhibition of PIKfyve. All three lines of evidence show that the AICD activates the PIKfyve complex in cells, a finding that is important for our understanding of the mechanism of neurodegeneration in Alzheimer's disease.
影响因子：
11.4
作者：
Michell, Robert H.;Dove, Stephen K.
通讯作者：
Dove, Stephen K.