Structural and Functional Specificity of Rab GTPases

描述（由申请人提供）：长期目标是采用多学科方法，全面了解GT3循环如何在各种生物系统中用于控制特定的多种细胞功能，包括信号转导、细胞骨架组织和细胞内膜运输。本研究以Rab 5与其效应子Rabaptin 5的相互作用为结构基础，重点研究Rab GTP酶的功能特异性。在人类基因组中，Rab GT3家族包含约40个不同的成员（如果包括同种型，则为60个），每个成员与一组特异性效应物相互作用，并促进膜运输系统中的特异性膜融合或转运功能。因此，一个重要的问题是，每个Rab如何区别于其他Rab识别其特定的效应器。这种功能特异性问题仍有待解决，在分子和结构水平，这应该有广泛的影响，不仅对GTdR领域，而且在一般的蛋白质-蛋白质识别机制。在本项目中，Rab 5-Rabaptin 5相互作用的机制及其在内体融合和膜运输中的作用将基于我们最近的Rab 5-Rabaptin 5晶体结构进行研究。除了Rabaptin 5之外，还将在以下实验中研究和比较融合中的其他Rab 5效应物（EEA 1和Rabenosyn 5）。目的1通过诱变、生化结合试验和结构研究确定Rab 5-Rabaptin 5复合物的相互作用界面，并阐明Rab 5-效应物相互作用的特异性。目的2是通过基于结构的工程将另一种Rab转化为功能性Rab 5来测试Rab特异性。目的3阐明Rabaptin 5在早期核内体融合中的作用机制。基于晶体结构提出了一种涉及Rabaptin 5四聚体的新机制，并将在功能测定中进行测试。目的4通过在细胞中表达Rabaptin 5-或Rab 4-结合结构域或Rab 5/Rab 4-结合缺陷片段以特异性破坏内源性Rabaptin 5与Rab 5或Rab 4的相互作用，解决Rabaptin 5在内吞作用期间在Rab 5-和Rab 4-阳性内体的生物发生和维持中的作用。该项目将有助于我们了解涉及膜运输和Rab GTP酶的各种疾病，包括心脏病，癌症，皮肤和视网膜疾病。

DESCRIPTION (provided by applicant): The long-term goal is to employ a multidisciplinary approach to 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 Rab5 interaction with its effector Rabaptin5 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 Rabaptin5, other Rab5 effectors in fusion (EEA1 and Rabenosyn5) will also be investigated and compared in the experiments below. Aim 1 is to define the interaction interface of Rab5-Rabaptin5 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 Rab5 through structure-based engineering. Aim 3 addresses the mechanism of Rabaptin5 function in early endosome fusion. A new mechanism involving Rabaptin5 tetramerization is proposed based on the crystal structure and will be tested in functional assays. Aim 4 addresses the role of Rabaptin5 in the biogenesis and maintenance of Rab5- and Rab4-positive endosomes during endocytosis via expressing its Rab5- or Rab4- binding domain or Rab5/Rab4-binding defective fragments in the cell to disrupt specifically the endogenous Rabaptin5 interaction with Rab5 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.