Structural and Functional Specificity of Rab GTPases

描述(由申请人提供)：长期目标是了解Rab GTP酶在细胞和生物体中的生理和病理作用。本项目的重点是两个密切相关的Rab GTP酶(Rab22和Rab5)，它们定位于早期的内体，调节内体的分选和内吞作用。具体地说，Rab22是从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。神经生长因子信号转导功能障碍在阿尔茨海默病、唐氏综合征等神经退行性疾病中起重要作用。我们发现，通过RNAi或显性负性突变体抑制Rab22功能显著减少NGF诱导的VGF表达和PC12细胞突起生长，而显性负性Rab5突变体的刺激作用相反。此外，激活的NGF受体(PTrkA)被内吞并定位于含有Rab22的内小体。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。本项目包含三个特定的目的，研究Rab22在PC12细胞和神经元中促进NGF信号转导和细胞分化的机制，以及确定Rab22在促进NGF信号转导和细胞分化方面与Rab5的结构差异。Aim 1将测试通过RNAi敲除Rab22是否可以消除NGF-pTrkA信号内体的形成，从而降低PC12细胞中pTrkA的水平和持续时间以及下游效应因子(Rap1和ERKs)的活性。此外，这些实验将确定在NGF诱导的ERKs激活、VGF表达和轴突生长中起作用的Rab22特异性效应器。目的2检测Rab22基因敲除是否可以阻断背根神经节(DRG)神经元NGF-pTrkA信号内体的逆行转运和信号转导，从而导致细胞凋亡。Aim 3将测试Rab5是否以及哪些突变可能导致Rab22在与效应器的相互作用中的特异性以及在NGF信号和细胞分化中的功能。综上所述，该项目将有助于了解Rab GTPase家族在Rab22和Rab5的结构和功能特异性方面的进化。此外，它还提出了一个新的概念，即早期内小体相关的Rab22对于促进NGF信号转导和神经元分化的信号内小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。

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.