Search for physiological substrates and functional analysis of F-box protein β-TrCP in vivo

本研究的目的是阐明蛋白质表达对发育的调控。我们分析了F-box蛋白β-TrCP 2的功能，证实了它在胚胎发育过程中的发育和分化中的作用，β-TrCP 1是泛素连接酶，它能泛素化β-catenin和l_κ βα，我们构建的β-TrCP 1基因靶向小鼠仍表现出β-catenin和l_κ βα的降解，提示β-catenin和l_<κ>βα还存在其它泛素连接酶。β-TrCP 1同源物β-TrCP 2基因靶向小鼠在胚胎9.5天时具有胚胎致死性。本研究从β-TrCP 1基因敲除小鼠和β-TrCP 2基因敲除小鼠的表型差异中筛选出4个蛋白质，并对其在胚胎发育早期和中期的表达情况以及与β-TrCP 2识别的共有序列进行了分析。我们建立了β-TrCP 2条件性基因敲除小鼠模型，B-TrCP 1基因敲除小鼠胚胎成纤维细胞周期2蛋白表达的异常调控，提示昼夜节律的维持需要β-TrCP 1的蛋白降解。

Our purpose of this project is elucidation of the regulation of protein expression on development. We have analyzed the function of F-box proteins β-TrCP2, and demonstrated its role on development and differentiation during embryogenesis.β-TrCP1 is the ubiquitin ligase which ubiquitinates β-catenin and l_<κ>βα The gene-targeted mouse of β-TrCP1, which we have generated, still exhibited degradation of β-catenin and l_<κ>βα, suggesting existence of other ubiquitin ligases for β-catenin and l_<κ>βα.The gene-targeted mouse of β-TrCP2, the homologue of β-TrCP1 is embryonic lethal at embryonic day9.5. This difference of phenotype between β-TrCP1 knockout mouse and that of β-TrCP2, indicate that each proteins have different biological functions significantly, even though the difference of biochemical functions have not been reported.We analyzed four proteins picked up based on the expression during early and middle embryogenesis and the consensus sequences, which are recognized by β-TrCP2. However, we did not detected bindings between these proteins and β-TrCP2, and ubiquitination and degradation by β-TrCP2.We have generated β-TrCP2 conditional knockout mouse.B-TrCP1 knockout primary embryonic fibroblasts have shown the abnormal regulation of Period 2 protein expression, suggesting that maintenance of circadian rhythm requires protein degradation by β-TrCP1.