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，彻底了解GTPase循环如何在各种生物系统中被用来控制特别不同的细胞功能，包括信号转导、细胞骨架组织和细胞膜运输。目前的研究重点是以Rab5与其效应子Rabaptin5的相互作用为结构基础的Rab GTP酶的功能特异性。Rab GTPase家族在人类基因组中包含约40个不同的成员(如果包括异构体，则为60个)，每个成员都与一组特定的效应器相互作用，促进膜运输系统中特定的膜融合或运输功能。因此，一个重要的问题是，每个RAB如何在识别其特定的效应器方面与其他RAB区分开来。这一功能特异性问题仍有待在分子和结构水平上解决，这不仅应该对GTPase领域产生广泛的影响，而且还应该对蛋白质-蛋白质识别机制产生广泛的影响。在这个项目中，我们将基于我们最近的Rab5-Rabaptin5的晶体结构来研究Rab5-Rabaptin5相互作用的机制及其在内体融合和膜转运中的作用。除了Rabaptin5，还将在下面的实验中研究和比较融合中的其他Rab5效应器(EEA1和RabenSyn5)。目的1确定Rab5-Rabaptin5复合体的相互作用界面，并通过诱变、生化结合分析和结构研究阐明这种相互作用的特异性。目标2是通过基于结构的工程将另一个Rab转化为功能Rab5来测试Rab的特异性。目的3探讨Rabaptin5在内体早期融合中的作用机制。基于晶体结构，提出了一种涉及Rabaptin5四聚反应的新机制，并将在功能分析中进行验证。目的4通过在细胞中表达Rab5或Rab4结合结构域或Rab5/Rab4结合缺陷片段，特异性地阻断内源性Rabaptin5与Rab5或Rab4的相互作用，探讨Rabaptin5在Rab5和Rab4阳性内吞作用中的生物发生和维持中的作用。这个项目应该有助于我们了解涉及膜转运和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.