Structural and Functional Specificity of Rab GTPases

长期目标是采用多学科的方法来彻底了解 GTP酶循环在各种生物系统中被用来控制特定不同的细胞功能， 包括信号转导、细胞骨架组织和细胞内膜运输。海流 研究项目集中在RAB GTP酶与RABS与其相互作用的功能特异性 效应器RabaptinS作为结构基础。Rab GTP酶家族包含约40个不同的成员(60个 异构体)存在于人类基因组中，每个异构体都与一组特定的效应器相互作用，并促进 膜转运系统中特定的膜融合或转运功能。因此，一个重要的 问题是，每个RAB如何在识别其特定效应器方面与其他RAB区分开来。此功能 特异性问题仍有待在分子和结构层面上解决，这应该会产生广泛的影响 不仅在GTPase领域，而且在蛋白质-蛋白质识别机制上也是如此。在这 Rab5-Rabaptin5相互作用机制及其在内体融合和膜中的作用 贩运将根据我们最近的Rab5-Rabaptin5晶体结构进行调查。除了……之外 RabaptinS、融合中的其他RABS效应器(EEA1和RabenSyns)也将在#年进行研究和比较 下面的实验。目的1确定Rabs-RabaptinS复合体的相互作用界面并阐明 通过诱变、生化结合分析和结构分析确定Rab-效应器相互作用的特异性 学习。目标2是通过将另一个RAb转换为功能RAB来测试RAb的特异性 基于结构的工程学。目的3探讨Rabaptin S在早期内吞体内的作用机制 核聚变。从晶体结构出发，提出了RabaptinS四聚反应的新机理 并将在功能分析中进行测试。目的4探讨RabaptinS在生物发生和发育中的作用 通过表达RABS-或RAB-4-来维持RABS-和RAB-4阳性的内吞作用 结合结构域或Rab5/Rab4结合细胞中的缺陷片段以特异性干扰内源性 RabaptinS与RABS或Rab4的相互作用。这个项目应该有助于我们理解各种 涉及膜转运和Rab GTP酶的疾病，包括心脏病、癌症、皮肤和 视网膜疾病。

The long-term goal is to employ a multidisciplinary approachto gain a thorough understanding of how the GTPase cycle is utilized in various biological systems to control specifically diverse cellular functions, including signal transduction, cytoskeleton organization, and intracellular membrane trafficking. The current research project focuses on the functional specificity of Rab GTPases with the RabS interaction with its effector RabaptinS as the structural basis. The Rab GTPase family contains about 40 distinct members (60 if isoforms are included) in the human genome and each interacts with a set of specific effectors and promotes a specific membrane fusion or transport function in the membrane trafficking system. Thus an important question is how each Rab distinguishes from other Rabs in recognizing its specific effectors. This functional specificity issue remains to be resolved at molecular and structural levels, which should have a broad impact not only on the GTPase field but also on the protein-protein recognition mechanisms in general. In this project, the mechanism of Rab5-Rabaptin5 interaction and its role in endosome fusion and membrane trafficking will be investigated, based on our recent Rab5-Rabaptin5 crystal structure. In addition to RabaptinS, other RabS effectors in fusion (EEA1 and RabenosynS) will also be investigated and compared in the experiments below. Aim 1 is to define the interaction interface of RabS-RabaptinS complex and elucidate the specificity of this Rab-effector interaction via mutagenesis, biochemical binding assays and structural studies. Aim 2 is to test the Rab specificity by converting another Rab into functional RabS through structure-based engineering. Aim 3 addresses the mechanism of RabaptinSfunction in early endosome fusion. A new mechanism involving RabaptinS tetramerization is proposed based on the crystal structure and will be tested in functional assays. Aim 4 addresses the role of RabaptinS in the biogenesis and maintenance of RabS- and Rab4-positive endosomes during endocytosis via expressing its RabS- or Rab4- binding domain or Rab5/Rab4-binding defective fragments in the cell to disrupt specifically the endogenous RabaptinS interaction with RabS or Rab4. This project should contribute to our understanding of a variety of diseases involving membrane trafficking and Rab GTPases, including heart diseases, cancers, skin and retinal diseases.