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: 8323488
• 负责人: MAXIM SOKOLOV
• 金额: $ 31.74万
• 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/8323488
• 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; Health; Heterotrimeric GTP-Binding Proteins; Homologous Gene; Knockout Mice; Knowledge; Light; Light Adaptations; 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; 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(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。在这项研究方案中，我们讨论了一个大型伴侣复合体CCT及其辅助伴侣蛋白光导蛋白样蛋白1(PhLP1)在视觉异源三聚体G蛋白转导蛋白的折叠和组装中的作用。我们的工作假设是，CCT和PhLP1是将转导蛋白的β亚基折叠成其特有的七叶片β-螺旋桨结构所必需的。我们还提出了PhLP1的一个密切的同源物，phosducin，在转导蛋白-β的折叠过程中与PhLP1协同作用，并调节转导蛋白在光感受器细胞中的运输。为了验证我们的假设，我们建立了几个转基因小鼠模型，包括那些光感受器中PhLP1/CCT功能受到抑制的小鼠，以及表达突变的硫代蛋白的小鼠。这些动物模型为研究CCT、PhLP1和Phosducin在体内的功能提供了独特的机会。在具体目标1中，我们将利用PhLP1/CCT功能受抑制的小鼠来探索这些伴侣蛋白在光感受器中的生理意义，并确定其功能受抑制导致视网膜变性的分子机制。在特定目标2中的研究将探讨光导蛋白和PhLP1伴侣蛋白协同作用的机制，并证明操纵光感受器中伴侣蛋白活性的可行性，目的是对抗由于蛋白质折叠异常而导致的视网膜退化。在特定的目标3中，我们将确定磷酸还原蛋白上丝氨酸54和丝氨酸71磷酸化位点的生理作用，它们以光依赖的方式调节其与转导蛋白的相互作用。利用没有这些磷酸化位点的表达硫代蛋白的小鼠，我们将在体内确定每个位点在调节转导蛋白运输到杆状外节方面的意义。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，并阐明必要的视觉信号蛋白的折叠和组装机制。对这一机制的了解对于制定治疗由分子伴侣功能障碍和蛋白质折叠异常引起的神经退行性失明疾病的策略具有重要意义。我们预计，我们的结果还将显著提高我们对G蛋白信号及其细胞调控的理解。与公共健康相关：拟议的研究旨在阐明分子伴侣--帮助新合成的多肽具有适当的二级结构的蛋白质--在视网膜感光神经元中视觉异源三聚体G蛋白转导蛋白的折叠中所起的作用。神经元中蛋白质的异常折叠是神经退行性疾病的一个公认的原因，因此，本研究将促进我们对神经退行性疾病的机制的理解，包括由转导蛋白异常折叠引起的疾病。我们预计，我们的结果还将显著提高我们对G蛋白介导的信号及其细胞调控的理解。