New cancer therapy by degradation of specific proteins

我们提出通过修饰泛素连接酶来诱导特定蛋白的降解，最近有报道称许多蛋白的表达水平不仅受转录调控，而且受降解率调控。泛素-载体体系统因其特异性而成为调控蛋白表达水平的重要系统之一。在这个独特的点上，我们试图创造新的泛素连接酶来识别和诱导降解对我们身体有毒的蛋白质。在这个项目中，我们使用癌蛋白Myc作为模型蛋白。由于已知Myc与Max结合，我们制作了几个嵌合蛋白，Max/Nedd4, Max/b-TrCP1, Max/CHIP。ned4是一种hect型泛素连接酶。b-TrCP1是一种F-box蛋白，是scf型泛素连接酶的一个组成部分。CHIP是一种u型泛素连接酶。据推测，Myc会被这些嵌合蛋白结合并泛素化。我们在体外系统和体内免疫沉淀实验中证实了该人工蛋白Max/CHIP与Myc的结合。通过表达载体过表达Max/CHIP蛋白能最有效地降低Myc在哺乳动物细胞中的半衰期。斑块实验和集落形成实验表明，Max/CHIP嵌合蛋白的表达抑制了myc诱导的转化。现在，我们观察对整个身体的影响。我们将过表达Max/CHIP嵌合蛋白的肿瘤细胞植入裸鼠体内，观察其对肿瘤发生的影响。

We proposed to induce to degradation of specific protein by the modification of ubiquitin-ligase, Recently, it is reported that expression level of many proteins are regulated by not only transcription but degradation rate. Ubiquitin-porteasome system is one of the most useful system to regulate expression level of protein because of its specificity. On this unique point we try to create new ubiquitin-ligase to recognize and induce to degradate proteins which are toxic for our bodies.In this project, we used oncoprotein, Myc as model protein. Because Myc is known to bind to Max, we made several chimeric proteins, Max/Nedd4, Max/b-TrCP1, Max/CHIP. Nedd-4 is a HECT-type ubiquitin-ligase. b-TrCP1 is a F-box proteins which is a component of SCF-type ubiquitin-ligase. CHIP is a U-box type ubiquitin-ligase. It was hypothesized that Myc would be bound and ubiquitylated by these chmeric proteins.We confirmed that this artificial protein Max/CHIP bound to Myc in vitro system and also immunoprecipitation assay in vivo. The half-life of Myc reduced by overexpression of Max/CHIP protein in mammalian cells by expression vector most efficiently.Plaque assays and colony-formation assay shown that Myc-induced transformation is inhibited by Max/CHIP chimeric protein expression.Now, we observe the effect on whole bode. We implanted tumor cells which overexpressed Max/CHIP chimeric protein in Nude mouse to examine the effect of tumorigenesis.