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Mode of actions and functions of the Rab3A and SNARE systems in neurotransmitter release

Rab3A 和 SNARE 系统在神经递质释放中的作用模式和功能

基本信息

批准号：
11670118
负责人：
SHIRATAKI Hiromichi
金额：
$ 2.3万
依托单位：
DOKKYO UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670118/
关键词：
Neurotransmitter Rab3A system SNARE system Small GTPase Rab系 Rab3A SNARE Tomosyn

项目摘要

It has been revealed that the Rab3A, a small GTPase, and SNARE systems are involved in neurotransmitter release. The Rab3A system consists of its target protein, which interacts with the GTP-bound form of Rab3A but not with the GDP-bound form of Rab3A, and its several regulatory proteins. We have identified Rabphilin-3 as a target protein for Rab3A and revealed the possibility that Rabphilin-3 functions as a Ca^<2+>-sensor in the presynapse. On the other hand, available evidence suggests that the SNARE system functions at the downstream of the Rab3A system and is implicated in neurotransmitter release. We have identified Tomosyn as a regulatory protein of the SNARE system. It is possible that Tomosyn is a central player which links between the Rab3A and SNARE systems. However, the mechanism by which the Rab3A system regulates the SNARE system in neurotransmitter release remains to be clarified. Therefore, in this study we attempted to clarify the mechanism. We revealed that there were at least three isoforms of Tomosyn, b-, m-, and s-Tomosyns and that all of the tomosyn isoforms interacted through their C-terminal VAMP-like regions with syntaxin-1 but not with other syntaxin isoforms such as syntaxin-2, -3, and -4. Moreover, we identified a novel syntaxin-1-binding protein. On the basis of its biochemical properties, it was possible that the syntaxin-1-binding protein was a cytoskeleton-related protein. One mole of the syntaxin-1-binding protein bound to about 0.8 mole of syntaxin-1 and the dose giving the half maximal binding of syntaxin-1 to the syntaxin-1-binding protein was about 90 nM.The interaction of the syntaxin-1-binding protein with syntaxin-1 was inhibited by Munc18 in a dose-dependent manner. In future we attempt to purify the syntaxin-1-binding protein, determine its primary structure, and reveal its function in neurotransmitter release.

研究表明，Rab3A（一种小型 GTP 酶）和 SNARE 系统参与神经递质释放。 Rab3A 系统由其靶蛋白及其几种调节蛋白组成，该靶蛋白与 Rab3A 的 GTP 结合形式相互作用，但不与 Rab3A 的 GDP 结合形式相互作用。我们已将Rabphilin-3鉴定为Rab3A的靶蛋白，并揭示了Rabphilin-3在突触前充当Ca^2+-传感器的可能性。另一方面，现有证据表明 SNARE 系统在 Rab3A 系统下游发挥作用，并与神经递质释放有关。我们已经确定 Tomosyn 是 SNARE 系统的调节蛋白。 Tomosyn 可能是连接 Rab3A 和 SNARE 系统的核心角色。然而，Rab3A系统调节SNARE系统神经递质释放的机制仍有待阐明。因此，在本研究中，我们试图阐明其机制。我们发现 Tomosyn 至少存在三种异构体：b-、m-和 s-Tomosyn，并且所有 Tomosyn 异构体都通过其 C 端 VAMP 样区域与 Syntaxin-1 相互作用，但不与其他 Syntaxin 异构体（如 Syntaxin-2、-3 和 -4）相互作用。此外，我们还发现了一种新的 Syntaxin-1 结合蛋白。根据其生化特性，突触蛋白-1结合蛋白可能是细胞骨架相关蛋白。一摩尔突触蛋白-1结合蛋白与约0.8摩尔突触蛋白-1结合，突触蛋白-1与突触蛋白-1结合蛋白的半最大结合的剂量为约90nM。突触蛋白-1结合蛋白与突触蛋白-1的相互作用被Munc18以剂量依赖性方式抑制。未来我们尝试纯化 Syntaxin-1 结合蛋白，确定其一级结构，并揭示其在神经递质释放中的功能。