MECHANISMS OF LIGHT-MEDIATED PROTEIN DEGRADATION

描述（由申请人提供）：隐花色素是植物和动物中作为蓝光光感受器发挥功能的黄素蛋白家族。果蝇隐花色素作为一种脑深部的非眼光感受器，参与昼夜节律的光诱导。在体内，隐花色素介导生物钟基因产物Timeless的光依赖性降解;通过单独的机制，它也在光照后引发自身降解。我们已经建立了一个体外试验研究光依赖性隐花色素降解细胞培养，融合生物发光酶荧光素酶全长隐花色素。超过80%的荧光素酶活性在光暴露的一小时内丧失。 赋予与隐花色素融合的功能蛋白质光触发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隐花色素的最小片段，并确定通过与隐花色素融合可以使蛋白质对光不稳定的范围。我们将把这种方法应用于研究整个苍蝇的蛋白质。我们将鉴定光介导的蛋白质降解所需的蛋白质，并尝试使用这些组分将该系统移植到哺乳动物细胞培养中。这些研究结果将增强我们对光介导的蛋白质降解机制的理解，并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都经历光依赖性降解。含有隐花色素的融合蛋白也经历光依赖性降解。我们建议实验采用这种现象作为研究蛋白质功能的一般工具。

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.