基于光引发生物相容反应的蛋白质降解调控

Methods for spatiotemporal control the degradation of specific proteins are desirable tools for probing complex biological systems. However, few effective methods have been reported up to now. We are seeking to develop a general method to reversible and spatiotemporal control of protein degradation by light-induced biocompatible reactions, evolving the commercial protein tag named SNAP-tag as a protein degradation regulation tag, dubbed Par-tag, on account of the degradation after self-alkylation of hAGT, which is the prototype of SNAP-tag. Firstly, based on light-induced biocompatible chemical reactions we previously reported, small molecular probes are designed, synthesized and exploited as a switch with light to turn hAGT activity “off” and “on”. With this, the subtle balance between degradability and stability of hAGT as well as protein fused with hAGT are achieved. Finally, the structure of hAGT is optimized by directed evolution to generate Par-tag and used in regulation of protein stability and abundance. Par-tag will be a new chemical genetics tool with both high efficiency and wide practicability as SNAP-tag. Our researches will provide new idea in precise control of protein stability and bring new methods for probing the spatial and temporal distribution of protein and the function in complex biological systems.

具有时空分辨率的蛋白质降解调控对探索生命奥秘具有重要价值，但目前尚缺乏有效的技术手段。本项目拟基于光引发生物相容反应及商业化蛋白标签SNAP-tag原型蛋白人O6-烷基鸟嘌呤-DNA烷基转移酶（hAGT）自烷基化降解机制，发展一种可逆的具有时空分辨率的蛋白降解调控技术。首先，基于课题组开发的光引发的生物相容反应设计合成小分子探针，用于hAGT的共价标记及活性调控，建立基于小分子和光响应的hAGT活性可逆调控开关；随后利用这一开关调控hAGT及其融合蛋白的降解；最后，通过蛋白质工程手段优化hAGT的结构，建立蛋白降解调控标签Par-tag，并将其用于调控胞内蛋白质的稳定性及丰度。Par-tag将成为一种兼具SNAP-tag高效性与实用性的化学遗传学研究新工具。本研究将为精准调控胞内蛋白质的稳定性研究提供新思路，为揭示复杂生物体系中蛋白质及其功能的时空分布规律提供新方法。

具有时空分辨率的蛋白降解调控对探索生命奥秘具有重要价值，但目前尚缺乏有效的技术手段。本项目拟基于光引发生物相容反应设计合成小分子探针调控人O6-烷基鸟嘌呤-DNA烷基转移酶（hAGT）活性，结合hAGT自烷基化降解机制，发展具有时空分辨率的蛋白降解调控技术。本项目在hAGT蛋白功能的光调控及可见光引发生物相容反应开发方面均取得突破性进展。建立了hAGT活性快速分析检测手段，设计并合成了四种光响应的小分子探针，确认其中一种探针分子可用于hAGT活性的快速加笼及紫外光去笼，最终在纯蛋白体系，大肠杆菌体系以及癌细胞体系实现hAGT活性的光调控；为减少紫外光反应可能带来的光毒性，发展了可见光促进的硼酸酮酸加成反应，无需添加额外试剂可见光照射下高效合成乳酸类化合物。反应具有良好的生物兼容性，可用于hAGT快速烷化修饰。本研究为揭示hAGT生物功能提供了新方法，为精准调控内源蛋白功能及选择性靶向治疗以及未来精准治疗提供新思路。本项目资助工作在国际学术期刊已发表论文2篇，待发表论文1篇。