Engineering programmable enzymes for proteome editing

项目摘要/摘要 用于基因组编辑和重新编程的CRISPR/Cas9技术的开发是 过去十年中最重要的发现。将指令传递到特定DNA基因座的能力使 对广泛物种的遗传功能进行系统的询问。与基因组研究相比，我们的 操纵蛋白质组的能力远远落后，蛋白质组是细胞的功能组成部分。天然蛋白质修饰 酶，如蛋白水解酶和激动酶，通常缺乏执行有针对性的活动的精致专一性。 定向到特定的蛋白质。一种实现用户控制的、依赖于目标的酶激活的策略 将为询问、调节和利用内源性蛋白质功能提供新的机会。 在这项工作中，我们设计了自动抑制酶，这种酶可以对高度特异的抗原做出反应而被激活- 抗体相互作用。这些新的酶结构被设计成具有内置的切换能力 在靶标非结合的酶非活性构象和靶标结合的酶活性构象之间。我们 提出了一项研究计划，概述了靶向激活的蛋白酶、荧光素酶和激酶的发展， 以及随后用于控制蛋白质降解的工程酶，构建 生物传感器，以及重写细胞信号通路。这些合成酶采用了调节机制。 与内生的对应物完全不同，使基础研究和 人工合成生物学应用。 1

Project Summary/Abstract The development of CRISPR/Cas9 technology for genome editing and reprogramming was one of the most important discoveries in the past decade. The ability to deliver instructions to a specific DNA locus enables systematic interrogation of genetic functions in a broad range of species. In contrast to genomic research, our ability to manipulate the proteome, the functional components of a cell, lags far behind. Natural protein-modifying enzymes, such as proteases and kinases, generally lack the exquisite specificity to perform targeted activities directed to a particular protein. A strategy that enables user-controlled, target-dependent enzyme activation would enable new opportunities for interrogating, modulating, and harnessing endogenous protein functions. In this work, we design autoinhibited enzymes that are activated in response to a highly specific antigen- antibody interaction. These novel enzyme architectures are engineered to have a built-in capability to toggle between a target-unbound, enzyme-inactive conformation and a target-bound, enzyme-active conformation. We present a research plan that outlines the development of a target-activated protease, luciferase, and a kinase, and the subsequent application of the engineered enzymes for controlling protein degradation, constructing biosensors, and re-writing cell signaling pathways. These synthetic enzymes adopt regulatory mechanisms entirely different from the endogenous counterparts, enabling wide possibilities for both basic research and synthetic biological applications. 1