Structural and Functional Specificity of Rab GTPases

Rab GTP 酶的结构和功能特异性

• 批准号: 7681169
• 负责人: GUANGPU LI
• 金额: $ 23.81万
• 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/7681169
• 关键词: Acids; Address; Amino Acids; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Biological Process; C-terminal; Cell Surface Receptors; Cell physiology; Cells; Chemicals; Collaborations; Complement; Complex; Coupled; Crystallography; Cytoskeleton; Data; Disease; Down-Regulation; Early Endosome; Endocytosis; Endosomes; Engineering; Family; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heart Diseases; Human Genome; Hydrolysis; Intracellular Membranes; Knowledge; Lead; Maintenance; Membrane Fusion; Membrane Protein Traffic; Molecular; Molecular Conformation; Mutagenesis; Mutate; N-terminal; Nutrient; Population; Process; Protein Binding Domain; Protein Isoforms; Proteins; Receptor Signaling; Recycling; Research Personnel; Research Project Grants; Retinal Diseases; Role; Signal Transduction; Skin Cancer; Solutions; Specificity; Structure; Surface; System; Testing; Translating; Translations; analog; base; biological systems; dimer; extracellular; interdisciplinary approach; member; mutant; novel; programs; rab GTP-Binding Proteins; receptor recycling; research study; uptake

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彻底了解各种生物系统中GTPase周期的使用方式，以控制特定多样化的细胞功能，包括信号转导，细胞骨架组织和细胞内细胞内的细胞功能膜贩运。当前的研究项目着重于RAB GTPases与Rab5相互作用与效应子Rabaptin5作为结构基础的功能特异性。 RAB GTPase家族在人类基因组中包含大约40个不同的成员（如果包括同工型，则包含60个成员），并且每个成员都与一组特定的效应子相互作用，并促进了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如何在识别其特定效应子时与其他RAB的区分。这种功能特异性问题仍有待解决的分子和结构水平尚待解决，这不仅应该对GTPase场产生广泛的影响，而且还应对蛋白质 - 蛋白质识别机制产生广泛的影响。在这个项目中，将根据我们最近的Rab5-Rabaptin5晶体结构研究Rab5-Rabaptin5相互作用的机制及其在内体融合和膜运输中的作用。除rabaptin5外，还将在以下实验中研究并进行比较。目的1是定义Rab5-Rabaptin5复合物的相互作用界面，并通过诱变，生化结合测定和结构研究来阐明这种Rab-peffector相互作用的特异性。 AIM 2是通过通过基于结构的工程将另一个RAB转换为功能性RAB5来测试RAB特异性。 AIM 3解决了早期内体融合中Rabaptin5功能的机制。基于晶体结构提出了一种涉及Rabaptin5四聚体的新机制，并将在功能测定中进行测试。 AIM 4通过表达其Rab5-或rab4-结合结构域或Rab5/rab5/rab4结合结构域或细胞中细胞中的Rab5-或Rab4结合结构域或RAB5/RAB4结合段的作用来解决Rabaptin5在内吞作用期间Rab5-和Rab4阳性内体的生物发生和维持中的作用与rab5或rab4。该项目应有助于我们理解各种涉及膜运输和RAB GTPASE的疾病，包括心脏病，癌症，皮肤和视网膜疾病。