| The release of neurotransmitters is the basis of performance of the normal function and transmission of information of the nervous system, and the release of neurotransmitters is finished mainly through the process of vesicle exocytosis, and the process involves a variety of proteins, lipid molecules and other substances, which is a very complicated process. At present, the regulation and the molecular mechanism of vesicle exocytosis and endocytosis process is becoming a hot research of scientists, which has a very important theoretical significance and broad application prospects to reveal these important life processes, such as the nerve signal transmission, learning and memory, cell secretion, cell growth, membrane structure and functional proteins' embedding.SNARE protein is the major molecule mediating membrane vesicles and cell fusion or other organelles. SNARE core protein complex is the molecular basis for membrane fusion to provide energy for membrane fusion. Snapin protein was first found in nerve cells as the SNAP-25binding protein in SNARE complex, and the molecular weight of it is about15kDa. TrkA receptor is a tyrosine kinase receptor, just like other tyrosine kinase receptors, after the tyrosine in TrkA receptor intracellular domain phosphorylated, it could be recognized by other signal molecular to provide the protein recruitment sites for the signal transduction. Hepatocyte growth factor receptor tyrosine kinase Met is also a tyrosine kinase receptor, and after its tyrosine kinase domain compared with the same region of TrkA, there is a37%homology, and some studies have shown the interaction between Snapin with the Met, Therefore, we speculate Snapin protein and TrkA should be interacted with each other. The early experiments of our work have used the method of yeast two-hybrid system to verify the interaction between TrkA intracellular domain(TrkAICD) and Snapin, The further study of the interaction between Snapin and TrkA was carried out by other means of molecular biology in this paper.1. The TrkAICD-Snapin interaction was tested by co-immunoprecipitaiton studies.2. The pEYFP-Snapin and pECFP-TrkAICD were constructed for FRET experiment, and the FRET results confirmed the interaction between TrkAICD and Snapin. 3. The pGEX-6p-1-SN(Snapin N-terminal domain) and pGEX-6p-1-SC(Snapin C-terminal domain) were constructed for GST pull down experiment, and the C-terminal domain was identified the domain associated with TrkA by GST pull down.These results provide useful clues and laid a certain experiment foundation for the study on whether Snapin is phosphorylated after associating with TrkA or not,the molecular mechanism of signal transduction as well as whether endocytosis is affected by the interaction between Snapin and TrkA, thereby regulating the release of neuro transmitters.TRAF6(Tumor necrosis factor receptor-associated factor6) is a member of TNF receptor-associated factor family, and also an ubiquitin ligase involved in the proteasome degradation pathway of proteins, as well as a signal regulatory molecular adaptor in many physiological pathway, related to gene transcription, cell proliferation and apoptosis. Congenital and acquired immunity, bone metabolism, lymph node development, breast development or even the skin and central nervous system development are inextricably linked with TRAF6.RanBPM (Ran binding protein in microtubule organizing center) was found as the Ran-binding protein at first, named after participating in microtubule nucleation phenomenon, which is a highly conserved and widely expressed protein, maybe related to membrane receptors Trk and p75NTR-related downstream signaling molecules. In our previous work and the literature reported, TrkA and RanBPM,p75NTR and TRAF6were shown related in the signaling pathway. In the signaling pathway, the network are often formed by the pathways cross-talk with each other impacting the cell metabolism, growth and differentiation. And informatics analysis shows that there is one TRAF6binding site existing in RanBPM. Based on the above considerations, we consider it is necessary to verify the interaction between RanBPM and TRAF6and its specific function, which may impact a specific signal transduction pathway.During the previous work of our laboratory, the interaction between RanBPM and TRAF6was identified through the yeast two-hybrid system, over-expression immunoprecipitation and endogenous immunoprecipitation. At the same time the SPRY domain of RanBPM was identified as the region associated with TRAF6. The luciferase reporter gene experiment proved that RanBPM can inhibit the TRAF6-induced NF-κB signal transduction pathway. In this paper, the focus of this part of the experiment is that based on the above experimental results, the advanced study on the interaction between TRAF6and RanBPM will be further carried out.1. Five pCMV-HA eukaryotic expression vectors of TRAF6'different domains were constructed according to the analysis results of TRAF6'domains to confirm that the C terminal of TRAF6is the necessary region associated with RanBPM.2. Three sets of experiments were designed to study the effect of overexpressed RanBPM on the endogenous TRAF6'autoubiquition and the overexpressed TRAF6autoubiquition as well as the different expression levels of RanBPM how to affect the degree of TRAF6'autoubiquition. The conclusion was accordant with that RanBPM inhibited TRAF6'autoubiquition.3. RanBPM affects on IκBα, one inhibitor of NF-κB signal pathway. The result is the expression level of IκBα rised with that the expression level of RanBPM is higher. The result showed the inhibition of RanBPM to NF-κB signal pathway, which is identified by our early experiments..4. The immunofluorescence experiment method was taken to study the RanBPM-TRAF6interaction's affection on p65into nucleus, and the resulis was that RanBPM inhibited p65muclei translocation which is another evidence to certify the RanBPM-TRAF6interaction's inhibiting NF-κB signal pathway.5. The flow cytometric result revealed that RanBPM could inhibit the apotosis caused by TRAF6, furthermore the inhibition would be strengthened by the expression level of RanBPM improved.The results of this research has enriched the study content of NF-κB signal transduction mechanism, at the same time it provided theoretical and experimental basis for the advanced study of the mechanism of growth,apoptosis and differentiation caused by NF-κB signal pathway.
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