MECHANISMS OF LIGHT-MEDIATED PROTEIN DEGRADATION

光介导的蛋白质降解机制

• 批准号: 7665302
• 负责人: Russell N. Van Gelder
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7665302
• 关键词: Animals; Bioluminescence; Brain; Cell Culture Techniques; Cells; Chimeric Proteins; Circadian Rhythms; Drosophila genus; Enzymes; Family; Flavoproteins; Hour; In Vitro; Length; Light; Luciferases; Mammalian Cell; Mediating; Methods; Photoreceptors; Plants; Proteins; Research Design; System; circadian pacemaker; cryptochrome; fly; in vitro Assay; in vivo; protein degradation; protein function; research study; tool

DESCRIPTION (provided by applicant): Cryptochromes are a family of flavoproteins that function as blue light photoreceptors in plants and animals. Drosophila Cryptochrome functions as a deep-brain, non-ocular photoreceptor for photic entrainment of circadian rhythms. In vivo, Cryptochrome mediates light-dependent degradation of the circadian clock gene product Timeless; by a separate mechanism, it also triggers its own degradation following light exposure. We have established an in vitro assay for studying light-dependent Cryptochrome degradation in cell culture, by fusing the bioluminescence enzyme luciferase to full-length Cryptochrome. Greater than 80% of luciferase activity is lost within one hour of light exposure. The conferral of light-triggered degradation to a functional protein fused to Cryptochrome is a potentially very powerful way to study the function of specific proteins in cells. We will determine the minimal fragment of Cryptochrome necessary to mediate light-dependent degradation, and determine the range of proteins which can be made light-labile by fusion with Cryptochrome. We will apply this method to the study of proteins in whole flies. We will identify proteins required for light- mediated protein degradation, and attempt to use these components to port this system to mammalian cell culture. Results from these studies will enhance our understanding of the mechanisms of light-mediated protein degradation, and will additionally provide a valuable new method for studying specific proteins' function in vitro and in vivo. Project Narrative: Cryptochromes are blue light photoreceptors found in plants and animals. Drosophila Cryptochrome undergoes light-dependent degradation in vivo and in vitro. Fusion proteins containing Cryptochrome also undergo light-dependent degradation. We propose experiments to employ this phenomenon as a general tool for studying protein function.

描述（由申请人提供）：加密染色体是一系列黄酮蛋白，它们在植物和动物中充当蓝光光感受器。果蝇加密色素充当昼夜节律的光夹带的深脑，非眼光感受器。在体内，加密色素介导了昼夜节律基因产物永恒的光依赖性降解。通过单独的机制，它还触发了光照后的降解。我们已经建立了一种通过将生物发光酶荧光素酶融合到全长加密染料中，用于研究细胞培养中光依赖性的加密弥漫降解的体外测定。超过80％的荧光素酶活性在光线暴露后一小时内流失。 光触发的降解与融合与密码色素的功能蛋白的降解是研究细胞中特定蛋白质功能的潜在非常强大的方法。我们将确定介导光依赖性降解所需的密码片段的最小片段，并确定可以通过与隐形色素融合而制成的蛋白质范围。我们将将这种方法应用于整个果定中蛋白质的研究。我们将确定光介导的蛋白质降解所需的蛋白质，并尝试使用这些成分将该系统移植到哺乳动物细胞培养物中。这些研究的结果将增强我们对光介导的蛋白质降解机制的理解，并另外为研究特定蛋白质在体外和体内的功能提供了宝贵的新方法。 项目叙述：隐形色素是植物和动物中发现的蓝光光感受器。果蝇加密色原会在体内和体外经历光依赖性降解。含有密封块的融合蛋白也会经历光依赖性降解。我们建议实验将这种现象用作研究蛋白质功能的一般工具。