Structural and Functional Specificity of Rab GTPases

Rab GTP 酶的结构和功能特异性

• 批准号: 7195450
• 负责人: GUANGPU LI
• 金额: $ 24.53万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-25 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7195450
• 关键词: enzyme mechanism; enzyme substrate complex; guanosinetriphosphatase activating protein; guanosinetriphosphatases; membrane fusion; membrane transport proteins; molecular assembly /self assembly; polymerization; protein biosynthesis; protein engineering; protein protein interaction; protein transport; receptor binding; site directed mutagenesis; vesicle /vacuole

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.

描述（由申请人提供）：长期目标是采用多学科方法来全面了解GTPase周期如何在各种生物系统中被利用来控制特定的不同细胞功能，包括信号转导，细胞骨架组织和细胞膜内运输。目前的研究项目以Rab5与其效应物Rabaptin5的相互作用为结构基础，重点研究Rab5 GTPases的功能特异性。Rab GTPase家族在人类基因组中包含大约40个不同的成员（如果包括同工异构体，则有60个），每个成员都与一组特定的效应物相互作用，并在膜运输系统中促进特定的膜融合或运输功能。因此，一个重要的问题是每个Rab如何在识别其特定效应时与其他Rab区别开来。这一功能特异性问题仍有待在分子和结构水平上得到解决，这不仅会对GTPase领域产生广泛影响，而且会对蛋白质-蛋白质识别机制产生广泛影响。本项目将基于我们最近的Rab5-Rabaptin5晶体结构，研究Rab5-Rabaptin5相互作用的机制及其在核内体融合和膜运输中的作用。除了Rabaptin5，其他Rab5的效应物（EEA1和Rabenosyn5）也将在下面的实验中进行研究和比较。目的1定义Rab5-Rabaptin5复合物的相互作用界面，并通过诱变、生化结合实验和结构研究阐明这种rab5 -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Rab5来测试Rab的特异性。目的3探讨Rabaptin5在早期核内体融合中的作用机制。基于晶体结构提出了Rabaptin5四聚化的新机制，并将在功能分析中进行测试。Aim 4通过在细胞中表达Rab5-或Rab4-结合域或Rab5/Rab4结合缺陷片段，特异性地破坏内源性Rabaptin5与Rab5或Rab4的相互作用，解决了Rabaptin5在胞吞过程中Rab5-和Rab4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与膜转运和Rab GTPases有关的各种疾病，包括心脏病、癌症、皮肤和视网膜疾病。