Assembly and Trafficking of Heterotrimeric G Proteins in Vertebrate Photoreceptor

描述（由申请人提供）：我们的长期研究目标是了解分子伴侣在脊椎动物光感受器中的作用。在这项研究中，我们解决了一个大的伴侣蛋白复合物，CCT，和它的共伴侣蛋白，糖蛋白样蛋白1（PhLP 1），在折叠和组装的视觉异源三聚体G蛋白，转导蛋白的作用。我们的工作假设是，CCT和PhLP 1所需的折叠的β亚基的转导成其特征性的七叶片β螺旋桨结构。我们还提出，一个密切的同源物的PhLP 1，acidducin，与PhLP 1在折叠过程中的transformin-beta，并调节感光细胞中的transformin的贩运。为了验证我们的假设，我们产生了几个转基因小鼠模型，包括那些抑制PhLP 1/CCT功能的光感受器，和小鼠表达突变体pepducin。这些动物模型提供了一个独特的机会，在体内研究CCT，PhLP 1和Bducin的功能。在具体目标1中，我们将使用PhLP 1/CCT功能抑制的小鼠来探索这些分子伴侣在光感受器中的生理意义，并确定由其功能抑制引起的视网膜变性的分子机制。特定目标2中的研究将解决Ducin和PhLP 1分子伴侣协同作用的机制，并证明操纵光感受器中分子伴侣活性的可行性，目的是抵消由于异常蛋白质折叠引起的视网膜变性。在具体目标3中，我们将确定丝氨酸54和丝氨酸71磷酸化位点在调节其与转导蛋白的相互作用中的生理作用。使用表达无这些磷酸化位点的转导蛋白的小鼠，我们将在体内确定每个位点在调节转导蛋白向视杆细胞外节运输中的意义。这些研究将揭示真核生物伴侣蛋白CCT在脊椎动物光感受器中的功能，并阐明重要视觉信号蛋白的折叠和组装机制。这一机制的知识是非常重要的发展战略，对神经退行性致盲性疾病所造成的分子伴侣功能障碍和异常蛋白质折叠。我们希望我们的研究结果也将显着提高我们对G蛋白信号转导及其细胞调控的理解。公共卫生相关性：拟议的研究旨在阐明分子伴侣的作用-蛋白质，帮助新合成的多肽承担适当的二级结构-在折叠的视觉异源三聚体G蛋白，transducin，在视网膜的感光神经元。神经元中蛋白质的异常折叠是神经退行性疾病的既定原因;因此，所提出的研究将促进我们对神经退行性疾病的机制的理解，包括由transducin的异常折叠引起的神经退行性疾病。我们希望我们的研究结果也将显着提高我们对G蛋白介导的信号转导及其细胞调控的理解。

DESCRIPTION (provided by applicant): Our long-term research objective is to understand the roles of molecular chaperones in vertebrate photoreceptors. In this research proposal, we address the role of a large chaperonin complex, CCT, and its co-chaperone, phosducin-like protein 1 (PhLP1), in the folding and assembly of the visual heterotrimeric G protein, transducin. Our working hypothesis is that CCT and PhLP1 are required for the folding of the beta subunit of transducin into its characteristic seven-blade beta-propeller structure. We also propose that a close homolog of PhLP1, phosducin, acts in concert with PhLP1 during folding of transducin-beta, and regulates trafficking of transducin in the photoreceptor cell. To test our hypotheses, we generated several transgenic mouse models, including those with the suppressed PhLP1/CCT function in photoreceptors, and mice expressing mutant phosducin. These animal models provide a unique opportunity to study the functions of CCT, PhLP1 and phosducin in vivo. In Specific Aim 1, we will use mice with the suppressed PhLP1/CCT function to explore the physiological significance of these chaperones in photoreceptors and to determine the molecular mechanism of the retinal degeneration caused by suppression of their function. Studies within Specific Aim 2 will address the mechanism of phosducin and PhLP1 chaperone synergy, and demonstrate the feasibility of manipulating chaperone activity in photoreceptors, with a purpose to counteract retinal degeneration due to the aberrant protein folding. In Specific Aim 3, we will determine the physiological roles of the serine 54 and serine 71 phosphorylation sites on phosducin that regulate its interaction with transducin in a light-dependent manner. Using mice expressing phosducin without these phosphorylation sites, we will determine in vivo the significance of each site in regulating trafficking of transducin to the rod outer segments. The proposed studies will reveal the function of the eukaryotic chaperonin, CCT, in vertebrate photoreceptors, and shed light on the mechanism of folding and assembly of essential visual signaling proteins. The knowledge of this mechanism is important for developing strategies against neurodegenerative blinding diseases caused by molecular chaperone malfunction and aberrant protein folding. We expect that our results will also significantly improve our understanding of G protein signaling and its cellular regulation. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to elucidate the roles of molecular chaperones - proteins that help the newly synthesized polypeptides to assume proper secondary structure - in the folding of the visual heterotrimeric G protein, transducin, in the photoreceptor neurons of the retina. Aberrant folding of proteins in neurons is an established cause of neurodegenerative diseases; therefore, the proposed studies will advance our understanding of the mechanisms of neurodegenerative diseases, including those caused by aberrant folding of transducin. We expect that our results will also significantly improve our understanding of G protein-mediated signaling and its cellular regulation.