Guscott Benjamin, Balklava Zita, Safrany Stephen T, Wassmer Thomas
School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
School of Pharmacy, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, U.K.
Biosci Rep. 2016 Apr 15;36(2). doi: 10.1042/BSR20160040. Print 2016.
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.
目前，调节PIKfyve复合物活性的机制正在逐渐明晰。PIKfyve复合物由磷酸肌醇激酶PIKfyve（也称为FAB1）、VAC14和FIG4组成，是生成磷脂酰肌醇3,5-二磷酸[PI(3,5)P2]所必需的。PIKfyve的功能对于内体/溶酶体系统的稳态是必需的，并且在神经元功能和完整性方面至关重要，因为PIKfyve复合物中的功能丧失突变会导致小鼠模型和人类患者发生神经退行性变。我们最近的研究表明，淀粉样前体蛋白（APP）的细胞内结构域（APP是阿尔茨海默病病因学中的核心分子）与VAC14结合并增强PIKfyve的功能。在本研究中，我们利用这一最新进展创建了一种易于使用的工具，用于增加细胞中PIKfyve的活性。我们将APP细胞内结构域（AICD）与HIV TAT结构域融合，HIV TAT结构域是一种可穿透细胞的肽，能使蛋白质穿透细胞。所得的TAT-AICD融合蛋白可穿透细胞并引发PI(3,5)P2的增加。使用PI(3,5)P2特异性的GFP-ML1Nx2探针，我们表明可穿透细胞的AICD改变了PI(3,5)P2的动态变化。TAT-AICD还能提供部分保护，防止PIKfyve受到药物抑制。这三条证据均表明AICD在细胞中激活了PIKfyve复合物，这一发现对于我们理解阿尔茨海默病神经退行性变的机制具有重要意义。
