Department of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 440-746, South Korea; Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, South Korea.
Department of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 440-746, South Korea.
Biochem Biophys Res Commun. 2014 Jul 18;450(1):831-6. doi: 10.1016/j.bbrc.2014.06.064. Epub 2014 Jun 21.
Fusion of synaptic vesicles with the presynaptic plasma membrane in the neuron is mediated by soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) proteins. SNARE complex formation is a zippering-like process which initiates at the N-terminus and proceeds to the C-terminal membrane-proximal region. Previously, we showed that this zippering-like process is regulated by several polyphenols, leading to the arrest of membrane fusion and the inhibition of neuroexocytosis. In vitro studies using purified SNARE proteins reconstituted in liposomes revealed that each polyphenol uniquely regulates SNARE zippering. However, the unique regulatory effect of each polyphenol in cells has not yet been examined. In the present study, we observed SNARE zippering in neuronal PC12 cells by measuring the fluorescence resonance energy transfer (FRET) changes of a cyan fluorescence protein (CFP) and a yellow fluorescence protein (YFP) fused to the N-termini or C-termini of SNARE proteins. We show that delphinidin and cyanidin inhibit the initial N-terminal nucleation of SNARE complex formation in a Ca(2+)-independent manner, while myricetin inhibits Ca(2+)-dependent transmembrane domain association of the SNARE complex in the cell. This result explains how polyphenols exhibit botulinum neurotoxin-like activity in vivo.
突触小泡与神经元中突触前质膜的融合是由可溶性 N-乙基马来酰亚胺敏感的融合蛋白附着蛋白受体（SNARE）蛋白介导的。SNARE 复合物的形成是一个拉链样的过程，从 N 端开始，向 C 端膜近侧区域进行。以前，我们表明，这个拉链样的过程受到几种多酚的调节，导致膜融合的停滞和神经递质释放的抑制。在使用纯化的 SNARE 蛋白在脂质体中重建的体外研究中，发现每种多酚都独特地调节 SNARE 的拉链。然而，每种多酚在细胞中的独特调节作用尚未得到检验。在本研究中，我们通过测量融合到 SNARE 蛋白的 N 端或 C 端的青色荧光蛋白（CFP）和黄色荧光蛋白（YFP）的荧光共振能量转移（FRET）变化，观察到神经元 PC12 细胞中的 SNARE 拉链。我们表明，飞燕草素和矢车菊素以钙离子非依赖性的方式抑制 SNARE 复合物形成的初始 N 端核化，而杨梅素抑制 SNARE 复合物在细胞中的钙离子依赖性跨膜结构域的结合。这一结果解释了多酚如何在体内表现出类似于肉毒杆菌神经毒素的活性。
