Vites Olga, Rhee Jeong-Seop, Schwarz Martin, Rosenmund Christian, Jahn Reinhard
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany.
J Biol Chem. 2004 Jun 18;279(25):26251-6. doi: 10.1074/jbc.M404079200. Epub 2004 Apr 14.
Snapin, a 15-kDa protein, has been identified recently as a binding partner of SNAP-25. Moreover, snapin is regulated by phosphorylation and enhances synaptotagmin binding to SNAREs. Furthermore, snapin and C-terminal snapin fragments have been effective in changing the release properties of neurons and chromaffin cells. Here we have reinvestigated the role of snapin using both biochemical and electrophysiological approaches. Snapin is ubiquitously expressed at low levels with no detectable enrichment in the brain or in synaptic vesicles. Using non-equilibrium and equilibrium assays including pulldown experiments, co-immunoprecipitations, and CD and fluorescence anisotropy spectroscopy, we were unable to detect any specific interaction between snapin and SNAP-25. Similarly, overexpression of a C-terminal snapin fragment in hippocampal neurons failed to influence any of the analyzed parameters of neurotransmitter release. Initial biochemical characterization of recombinant snapin revealed that the protein is a stable dimer with a predominantly alpha-helical secondary structure. We conclude that the postulated role of snapin as a SNARE regulator in neurotransmitter release needs reconsideration, leaving the true function of this evolutionarily conserved protein to be discovered.
Snapin是一种15千道尔顿的蛋白质，最近被鉴定为SNAP-25的结合伴侣。此外，Snapin受磷酸化调节，并增强突触结合蛋白与SNAREs的结合。此外，Snapin和C端Snapin片段可有效改变神经元和嗜铬细胞的释放特性。在这里，我们使用生化和电生理方法重新研究了Snapin的作用。Snapin在各处低水平表达，在大脑或突触小泡中未检测到富集。使用包括下拉实验、免疫共沉淀以及圆二色光谱和荧光各向异性光谱在内的非平衡和平衡分析，我们未能检测到Snapin与SNAP-25之间的任何特异性相互作用。同样，在海马神经元中过表达C端Snapin片段未能影响神经递质释放的任何分析参数。重组Snapin的初步生化特征表明，该蛋白质是一种稳定的二聚体，主要具有α螺旋二级结构。我们得出结论，Snapin作为神经递质释放中SNARE调节剂的假定作用需要重新考虑，这一进化保守蛋白的真正功能有待发现。
