Structural and Functional Specificity of Rab GTPases

Rab GTP 酶的结构和功能特异性

• 批准号: 7481033
• 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/7481033
• 关键词: Acids; Address; Amino Acids; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Biological Process; C-terminal; Cell physiology; Cell surface; Cells; Chemicals; Collaborations; Complement; Complex; Coupled; Crystallography; Cytoskeleton; Data; Disease; Down-Regulation; Early Endosome; Endocytosis; Endosomes; Engineering; Family; Goals; Guanosine Diphosphate; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heart Diseases; Helix (Snails); Human Genome; Hydrolysis; Intracellular Membranes; Knowledge; Lead; Maintenance; Membrane Fusion; Membrane Protein Traffic; Molecular; Molecular Conformation; Mutagenesis; Mutate; N-terminal; Numbers; 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; dimer; ear helix; extracellular; interdisciplinary approach; member; mouse Gdi2 protein; 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全面了解如何在各种生物系统中利用 GTP 酶循环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内膜运输。目前的研究项目重点研究 Rab GTPase 的功能特异性，以 Rab5 与其效应子 Rabaptin5 的相互作用作为结构基础。 Rab GTPase 家族在人类基因组中包含约 40 个不同的成员（如果包括同工型，则为 60 个），每个成员与一组特定的效应子相互作用，并促进膜运输系统中的特定膜融合或转运功能。因此，一个重要的问题是每个 Rab 在识别其特定效应器时如何与其他 Rab 区分开来。这种功能特异性问题仍有待在分子和结构水平上解决，这不仅会对 GTP 酶领域产生广泛影响，而且还会对一般的蛋白质-蛋白质识别机制产生广泛影响。在本项目中，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内体融合和膜运输中的作用。除了Rabaptin5之外，融合中的其他Rab5效应子（EEA1和Rabenosyn5）也将在下面的实验中进行研究和比较。目标 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阐明这种 Rab-效应子相互作用的特异性。目标 2 是通过基于结构的工程将另一个 Rab 转化为功能性 Rab5 来测试 Rab 特异性。目标 3 探讨 Rabaptin5 在早期内体融合中的功能机制。基于晶体结构提出了一种涉及 Rabaptin5 四聚化的新机制，并将在功能测定中进行测试。目标 4 通过在细胞中表达 Rab5 或 Rab4 结合结构域或 Rab5/Rab4 结合缺陷片段来特异性破坏内源性 Rabaptin5 与 Rab5 或 Rab4 的相互作用，从而解决在内吞过程中 Rabaptin5 在 Rab5 和 Rab4 阳性核内体的生物发生和维持中的作用。该项目应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。