MECHANISMS OF LIGHT-MEDIATED PROTEIN DEGRADATION

描述(申请人提供)：隐花色素是黄素蛋白家族，在动植物中起蓝光感受器的作用。果蝇隐花色素是一种大脑深层的、非眼部的感光器，用于昼夜节律的光携带。在体内，隐花色素介导生物钟基因产物Timless的光依赖降解；通过另一种单独的机制，它也在光暴露后触发自身的降解。我们通过将生物发光酶荧光素酶与全长隐色素融合，建立了一种研究细胞培养中光依赖的隐色素降解的体外方法。超过80%的荧光素酶活性在光照后一小时内消失。将光引发的降解转化为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一种潜在的非常有效的方法。我们将确定介导光依赖降解所需的隐色素的最小片段，并确定通过与隐色素融合而使其光不稳定的蛋白质的范围。我们将把这种方法应用到整个果蝇的蛋白质研究中。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这个系统移植到哺乳动物细胞培养中。这些研究结果将加深我们对光介导蛋白质降解机制的理解，并将为研究特定蛋白质在体外和体内的功能提供一种有价值的新方法。 项目简介：隐花色素是一种蓝光感受器，存在于植物和动物中。果蝇隐色素在体内和体外都经历了光依赖的降解。含有隐花色素的融合蛋白也会经历光依赖的降解。我们建议通过实验将这一现象作为研究蛋白质功能的通用工具。

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.