Structural and Functional Specificity of Rab GTPases

Rab GTP 酶的结构和功能特异性

• 批准号: 8237266
• 负责人: GUANGPU LI
• 金额: $ 27.97万
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8237266
• 关键词: Alzheimer' s Disease; Apoptosis; Axon; Biochemical; Biogenesis; Biological Assay; Cell Differentiation process; Cell Survival; Cells; Characteristics; Chimera organism; Data; Development; Diagnosis; Dominant-Negative Mutation; Down Syndrome; Early Endosome; Endocytosis; Endosomes; Eukaryota; Evolution; Family; Fluorescence Microscopy; Goals; Hereditary Sensory Neuropathy; Human; Immunoblot Analysis; Intracellular Membranes; Label; Lead; Mediating; Membrane Protein Traffic; Monitor; Mutation; NGFR Protein; Nerve Growth Factors; Neurites; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Organism; PC12 Cells; Phosphotransferases; Phylogenetic Analysis; Physiological; Play; Property; Proteins; Quantum Dots; RNA Interference; Recruitment Activity; Regulation; Role; Screening procedure; Signal Transduction; Signal Transduction Pathway; Site-Directed Mutagenesis; Sorting - Cell Movement; Specific qualifier value; Specificity; Spinal Ganglia; Testing; Therapeutic; Vertebrates; Yeasts; autonomic neuropathy; base; cell growth regulation; computerized data processing; man; member; mutant; neuronal cell body; neuronal survival; novel; rab GTP-Binding Proteins; research study; trafficking

DESCRIPTION (provided by applicant): The long-term goal is to understand the physiological and pathological roles of Rab GTPases in cells and organisms. This project is focused on two closely related Rab GTPases (Rab22 and Rab5) that are localized in early endosomes and regulate endosomal sorting and endocytosis. Specifically the hypothesis that Rab22 is evolved from Rab5 to accommodate the need for regulation of specialized cell differentiation processes in higher eukaryotes, such as nerve growth factor (NGF)-mediated neuronal differentiation and survival, will be investigated. Malfunction in NGF signal transduction plays an important role in neurodegenerative diseases such as Alzheimer's disease and Down syndrome. We found that inhibition of Rab22 function via RNAi or dominant negative mutants dramatically reduces NGF-induced VGF expression and neurite outgrowth in PC12 cells, in contrast to the stimulatory effect of dominant negative Rab5 mutants. Furthermore, the activated NGF receptor (pTrkA) is endocytosed and localized to Rab22-containing endosomes. The preliminary data suggest an important role for Rab22 in the biogenesis and function of NGF-pTrkA signaling endosomes that promote cell differentiation and survival. This project contains three specific aims to investigate the mechanism of Rab22 in NGF signal transduction and cell differentiation in PC12 cells as well as neurons and to determine the structural differences that distinguish Rab22 from Rab5 in terms of promoting NGF signal transduction and cell differentiation. Aim 1 will test if Rab22 knockdown via RNAi may abrogate the formation of NGF-pTrkA signaling endosomes and consequently diminish the level and duration of pTrkA in PC12 cells and the activities of downstream effectors (Rap1 and ERKs). Furthermore, the experiments will determine Rab22- specific effectors responsible for the function in NGF-induced activation of ERKs, VGF expression, and neurite outgrowth. Aim 2 will test if Rab22 knockdown may abrogate the retrograde trafficking and signaling of NGF- pTrkA signaling endosomes in dorsal root ganglia (DRG) neurons and lead to apoptosis. Aim 3 will test if and what mutations in Rab5 may give rise to Rab22 characteristics in terms of specificity in interactions with effectors and function in NGF signaling and cell differentiation. In summary, this project will contribute to understanding the evolution of the Rab GTPase family in terms of structural and functional specificity of Rab22 and Rab5. Furthermore, it develops a novel concept that the early endosome-associated Rab22 is critical for the biogenesis and function of signaling endosomes that promote NGF signaling and neuron differentiation, which will contribute to understanding the mechanism of neurodegenerative diseases and developing potential therapeutics.

描述（由申请人提供）：长期目标是了解 Rab GTPases 在细胞和生物体中的生理和病理作用。该项目重点关注两种密切相关的 Rab GTPases（Rab22 和 Rab5），它们位于早期内体中并调节内体分选和内吞作用。具体而言，将研究 Rab22 从 Rab5 进化而来的假设，以适应高等真核生物中特殊细胞分化过程的调节需要，例如神经生长因子 (NGF) 介导的神经元分化和存活。 NGF 信号转导故障在阿尔茨海默病和唐氏综合症等神经退行性疾病中起着重要作用。我们发现，通过 RNAi 或显性失活突变体抑制 Rab22 功能，可显着降低 PC12 细胞中 NGF 诱导的 VGF 表达和神经突生长，这与显性失活 Rab5 突变体的刺激作用相反。此外，激活的 NGF 受体 (pTrkA) 被内吞并定位到含有 Rab22 的内体。初步数据表明 Rab22 在促进细胞分化和存活的 NGF-pTrkA 信号内体的生物发生和功能中发挥着重要作用。该项目包含三个具体目标，即研究Rab22在PC12细胞和神经元中NGF信号转导和细胞分化的机制，并确定Rab22与Rab5在促进NGF信号转导和细胞分化方面的结构差异。目标 1 将测试通过 RNAi 敲低 Rab22 是否可以消除 NGF-pTrkA 信号传导内体的形成，从而降低 PC12 细胞中 pTrkA 的水平和持续时间以及下游效应器（Rap1 和 ERK）的活性。此外，实验将确定 Rab22 特异性效应器在 NGF 诱导的 ERK 激活、VGF 表达和神经突生长中发挥作用。目标 2 将测试 Rab22 敲低是否可以消除背根神经节 (DRG) 神经元中 NGF-pTrkA 信号内体的逆行运输和信号传导并导致细胞凋亡。目标 3 将测试 Rab5 中的突变是否以及哪些突变可能会在与效应器相互作用的特异性以及 NGF 信号传导和细胞分化中的功能方面产生 Rab22 特征。总之，该项目将有助于理解 Rab GTPase 家族在 Rab22 和 Rab5 的结构和功能特异性方面的进化。此外，它提出了一个新的概念，即早期内体相关的 Rab22 对于促进 NGF 信号传导和神经元分化的信号内体的生物发生和功能至关重要，这将有助于理解神经退行性疾病的机制和开发潜在的治疗方法。