Assembly and Trafficking of Heterotrimeric G Proteins in Vertebrate Photoreceptor

脊椎动物感光器中异三聚体 G 蛋白的组装和运输

• 批准号: 7697239
• 负责人: MAXIM SOKOLOV
• 金额: $ 32.96万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7697239
• 关键词: Acceleration; Address; Animal Model; Biological Assay; Cell Culture Techniques; Cessation of life; Characteristics; Complex; Dark Adaptation; Data; Development; Disease; Dominant-Negative Mutation; GTP-Binding Proteins; Goals; Guanylate Cyclase; Heterotrimeric GTP-Binding Proteins; Homologous Gene; Knockout Mice; Knowledge; Light; Lighting; Mediating; Membrane; Modeling; Molecular; Molecular Chaperones; Monitor; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Phosphorylation Site; Photoreceptors; Physiological; Physiology; Positioning Attribute; Process; Protein Family; Proteins; Research; Research Design; Research Proposals; Retina; Retinal; Retinal Degeneration; Rod Outer Segments; Role; Serine; Signal Transduction; Signaling Protein; Site; Structure; Testing; Time; Transducin; Transgenic Mice; Vertebrate Photoreceptors; Visual; Work; beta Subunit Transducin; cell growth regulation; chaperonin; chaperonin CCT; endoplasmic reticulum stress; genetic manipulation; improved; in vivo; mouse model; mutant; overexpression; phosducin; phosducin-like protein; polypeptide; postnatal; protein expression; protein folding; protein misfolding; public health relevance; response; retinal rods; trafficking

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.

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感光体中的作用。在这项研究建议中，我们解决了大型伴侣蛋白复合蛋白CCT及其共伴侣，磷蛋白样蛋白1（PHLP1）的作用，在视觉异三聚二聚体G蛋白tronsducin的折叠和组装中的作用。我们的工作假设是，CCT和PHLP1是将传ducin的β亚基折叠成其特征性的七叶β-蛋白酶结构所必需的。我们还提出，phlp1，硫代蛋白的紧密同源物在跨核蛋白β折叠过程中与PHLP1共同起作用，并调节转传中的转丁基蛋白在光感受器细胞中的运输。为了检验我们的假设，我们生成了几种转基因小鼠模型，包括在感光体中具有抑制PHLP1/CCT功能的那些模型，以及表达突变体phosducin的小鼠。这些动物模型提供了研究CCT，PHLP1和phosducin在体内的功能的独特机会。在特定目标1中，我们将使用具有抑制PHLP1/CCT功能的小鼠来探索这些伴侣在感光体中这些伴侣的生理意义，并确定由于其功能抑制引起的视网膜变性的分子机制。特定目标2中的研究将探讨磷素和PHLP1伴侣协同作用的机制，并证明操纵感光体中伴侣活性的可行性，目的是应对异常的蛋白质折叠引起的视网膜变性。在特定的目标3中，我们将确定丝氨酸54和丝氨酸71磷酸化位点的生理作用，这些磷酸化位点以光依赖性方式调节其与跨蛋白的相互作用。利用表达磷酸素的小鼠，没有这些磷酸化位点，我们将在体内确定每个位点在调节跨牙菌素对杆外段的运输中的重要性。拟议的研究将揭示真核伴侣蛋白，CCT在脊椎动物感光体中的功能，并阐明基本视觉信号蛋白的折叠和组装机理。这种机制的知识对于针对由分子伴侣故障和异常蛋白质折叠引起的神经退行性盲目疾病的策略很重要。我们预计我们的结果还将显着提高我们对G蛋白信号传导及其细胞调节的理解。公共卫生相关性：拟议的研究旨在阐明分子伴侣蛋白的作用 - 蛋白质，这些蛋白质有助于新合成的多肽假定适当的二级结构 - 在视网膜的光感受器神经元中的视觉杂核G蛋白传染蛋白（传统）中。神经元中蛋白质的异常折叠是神经退行性疾病的确定原因。因此，拟议的研究将提高我们对神经退行性疾病的机制的理解，包括由异常折叠引起的跨多霉素引起的。我们预计我们的结果还将显着提高我们对G蛋白介导的信号传导及其细胞调节的理解。