Engineering protein-specific proteases: targeting signaling proteins

概述：我们的目标是开发工程蛋白酶的一般原理， 可以控制细胞中的信号。这需要七个步骤：1）选择信号蛋白 （人RAS同种型和致癌突变体）; 2）动态区域的鉴定 3）在信号传导蛋白内鉴定靶序列; 4）工程化/进化针对靶序列的高特异性; 5） 工程改造由靶蛋白催化的酶原活化; 6）测试 调节E. 7）测试人细胞培养物中的信号传导控制。 我们将使用多学科方法完成这些步骤，包括 计算和体外选择为基础的蛋白质工程，酶学，多 三维核磁共振、X射线晶体学和细胞生物学。这三个具体目标是： 1)进化出靶向活性RAS的蛋白质特异性蛋白酶; 2）工程酶原 3）在细胞模型系统中测试有前景的蛋白酶。 这个项目将开发识别，折叠和催化的一般原则， 详细了解结构和机制，并利用这些知识来开发 控制细胞信号的工具。蛋白酶及其调节剂将构成蛋白酶。 酶系统，将感测活性RAS的存在，并控制 释放活性RAS特异性蛋白酶，从而调节RAS信号传导。的 在基于细胞的系统中切割RAS并由此改变RAS信号传导的能力将 代表了建立蛋白质特异性免疫的实用性的重要里程碑。 蛋白酶作为外源信号调节剂。虽然这些蛋白酶靶向活性 RAS，基本的设计原则是基本的，将适用于许多 靶蛋白。

Summary: Our objective is to develop general principles for engineering proteases that can control signaling in cells. This requires seven steps: 1) Choice of signaling proteins (Human RAS isoforms and oncogenic mutants); 2) Identification of dynamic regions within the signaling proteins; 3) Identification of a target sequence within dynamic regions; 4) Engineering/evolving high specificity against the target sequence; 5) Engineering zymogen activation that is catalyzed by the target protein; 6) Testing regulated RAS degradation in E. coli; 7) Testing control of signaling in human cell culture. We will complete these steps using a multi-disciplinary approach that includes computational and in vitro selection-based protein engineering, enzymology, multi- dimensional NMR, X-ray crystallography, and cell-biology. The three Specific Aims are: 1) Evolve protein-specific proteases that target active RAS; 2) Engineer zymogen activation catalyzed by active RAS; 3) Test promising proteases in cell model systems. This project will develop general principles of recognition, folding, and catalysis based on detailed understanding of structure and mechanism and use this knowledge to develop tools to control signaling in a cell. The protease and its regulators will comprise an enzymatic system that will sense the presence of active RAS and transduce a controlled release of the active RAS-specific protease, thereby modulating RAS signaling. The ability to cleave RAS in cell-based systems and thereby alter RAS signaling will represent a significant milestone towards establishing the utility of protein-specific proteases as exogenous signaling regulators. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.