MECHANISMS OF LIGHT-MEDIATED PROTEIN DEGRADATION

描述（由申请人提供）：隐色素是植物和动物中作为蓝光光感受器的黄蛋白家族。隐花色素作为一种脑深部、非眼的光感受器，用于昼夜节律的光诱导。在体内，隐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，它也会在光线照射后引发自身的降解。我们建立了一种在细胞培养中研究光依赖性隐花色素降解的体外实验，通过将生物发光酶荧光素酶融合到全长隐花色素上。超过80%的荧光素酶活性在光照一小时内丧失。将光触发的降解赋予与隐花色素融合的功能蛋白质是研究细胞中特定蛋白质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性降解所需的最小隐花色素片段，并确定通过与隐花色素融合可以使其光不稳定的蛋白质范围。我们将把这种方法应用到整个果蝇的蛋白质研究中。我们将鉴定光介导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。

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.