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

我们这个项目的目的是阐明蛋白质表达对发育的调节。我们已经分析了F-盒蛋白β-TrCP2的功能，并阐明了它在胚胎发育分化中的作用，β-TrCP1是泛素化β-连环蛋白的泛素连接酶，我们培育的β-TrCP1基因靶向小鼠κ&gt；βα仍对β-连环蛋白和L；κ&gt；βα有降解作用，提示存在其他泛素连接酶β-catenin和L；κ&gt；βα。β-TrCP1基因靶向小鼠的β-TrCP1同源基因在胚胎9.5日是胚胎死亡的。β-TrCP1基因敲除小鼠和β-TrCP2基因敲除小鼠的这种表型差异表明，尽管还没有报道其生化功能的差异，但这两种蛋白质具有显著的生物学功能。我们分析了四种基于胚胎早期和中期表达的蛋白质以及β-TrCP2识别的共同序列。然而，我们没有检测到这些蛋白与β-TrCP2结合，以及β-TrCP2泛素化和降解。我们产生了β-TrCP2条件性基因敲除小鼠。B-TrCP1基因敲除的原代胚胎成纤维细胞显示出周期2蛋白表达的异常调节，这表明维持昼夜节律需要β-TrCP1对蛋白质的降解。

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.