New cancer therapy by degradation of specific proteins

我们提出通过泛素连接酶的修饰来诱导特定蛋白质的降解。最近的研究表明，许多蛋白质的表达水平不仅受转录水平的调控，而且受降解速率的调控。泛素-转运体系统因其特异性而成为调节蛋白质表达水平最有用的系统之一。本课题正是基于这一独特的观点，试图创造一种新的泛素连接酶，以识别并诱导降解对人体有害的蛋白质。由于已知Myc与Max结合，我们制备了几种嵌合蛋白，Max/Nedd 4，Max/b-TrCP 1，Max/CHIP。Nedd-4是一种HECT型泛素连接酶。b-TrCP 1是SCF型泛素连接酶的一个组成部分，是一种F盒蛋白。CHIP是一种U-box型泛素连接酶。本研究通过体外实验和体内免疫沉淀实验证实了人工合成的Max/CHIP蛋白与Myc蛋白的结合。Max/CHIP嵌合蛋白在哺乳动物细胞中的过表达能有效地降低Myc的半衰期，空斑实验和克隆形成实验表明Max/CHIP嵌合蛋白的表达能抑制Myc诱导的转化，我们观察其对整个细胞的影响。我们将过表达Max/CHIP嵌合蛋白的肿瘤细胞植入裸鼠体内，以检查肿瘤发生的效果。

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.