MECHANISMS OF LIGHT-MEDIATED PROTEIN DEGRADATION

光介导的蛋白质降解机制

• 批准号: 7515190
• 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/7515190
• 关键词: Animals; Bioluminescence; Brain; Cells; Chimeric Proteins; Circadian Rhythms; Cultured Cells; Depth; Drosophila genus; Enzymes; Family; Flavoproteins; Hour; In Vitro; Length; Light; Luciferases; Mammalian Cell; Mediating; Methods; None or Not Applicable; Photoreceptors; Plants; Proteins; Range; 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）：隐花色素是黄素蛋白家族，在植物和动物中充当蓝光感光器。果蝇隐花色素作为一种深脑非眼光感受器，负责昼夜节律的光夹带。在体内，隐花色素介导生物钟基因产物 Timeless 的光依赖性降解；通过一种单独的机制，它还会在光照后触发自身降解。我们通过将生物发光酶荧光素酶与全长隐花色素融合，建立了一种体外测定法，用于研究细胞培养物中光依赖性隐花色素的降解。超过 80% 的荧光素酶活性在光照一小时内消失。 将光触发的降解赋予与隐花色素融合的功能蛋白是研究细胞中特定蛋白功能的潜在非常有效的方法。我们将确定介导光依赖性降解所需的隐花色素的最小片段，并确定可通过与隐花色素融合而变得对光不稳定的蛋白质的范围。我们将应用这种方法来研究整个果蝇的蛋白质。我们将鉴定光介导的蛋白质降解所需的蛋白质，并尝试使用这些组件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，并且还将为研究特定蛋白质的体外和体内功能提供有价值的新方法。 项目叙述：隐花色素是在植物和动物中发现的蓝光光感受器。果蝇隐花色素在体内和体外经历光依赖性降解。含有隐花色素的融合蛋白也会经历光依赖性降解。我们建议进行实验，利用这种现象作为研究蛋白质功能的通用工具。