基于正交脱笼策略的蛋白质在体激活技术系列进展

Recent Progress on Bioorthogonal Decaging-Enabled Protein Activation in Living Systems

在活细胞等生理环境下开展蛋白质功能的原位研究具有重要的科学意义。陈鹏课题组长期致力于发展蛋白质的原位激活技术,希望为活细胞内的每一个蛋白质安装“调控开关”。课题组先前提出的“化学脱笼”策略,可通过对蛋白质关键残基的化学保护和脱保护反应,实现对其活性的“关-开”调控。然而,受可供脱笼的氨基酸种类的限制,该方法无法适用于所有蛋白质。为了解决这一瓶颈问题,陈鹏课题组与王初课题组合作提出了基于“邻近脱笼”策略的新方法——CAGEprox,极大地扩展了脱笼策略的适用范围。该方法通过在目标蛋白活性中心邻近位点定点引入侧链保护的非天然氨基酸,来实现对其活性的抑制和激活。在该方法中,王初课题组发挥自身在蛋白质功能位点计算方面的经验,建立了一个虚拟筛选合适邻近激活位点的计算模型和流程,避免了繁琐的人为实验操作,极大地提高了CAGE-prox方法的成功率和普适性。两个课题组合作分别在一系列不同类型的蛋白上展示了CAGE-prox方法在活体环境下瞬时激活目标蛋白的普适性。其中,结合CAGE-prox和王初课题组在定量化学蛋白质组学领域的技术优势,两个课题组合作开发了具有时间分辨率的定量蛋白质组学技术,对细胞凋亡这一动态生物学过程中蛋白质水解的底物进行了捕捉和鉴定,为理解凋亡动态的过程提供了有力的支撑。

In situ study of protein function in physiological conditions,such as living cells,is highly important and desired.Chen Peng’s group focuses on developing the methods of in situ manipulation of proteins,and dedicate themselves to install“switches”for any protein of interest in living cells.Chemical decaging,which was first developed by Chen Peng’s group,has emerged as an attractive strategy for gain-of-function study of proteins by chemical protection and deprotection of key residues of proteins.However,this strategy was limited because current bioorthogonal rescue strategies are largely centered on liberating the side-chain of lysine or tyrosine residue on a protein of interest.In order to overcome this bottleneck,Chen Peng’s group and Wang Chu’s group proposed a new strategy—“proximal decaging”,which greatly expanded the scope of application.The universal“proximal cage”can be introduced in close proximity with a protein’s functional site for temporal blockage of its activity until being rescued by photo-decaging.As Wang Chu’s group mainly focuses on the chemoproteomics profiling and computational design of functional sites of proteins,they developed here a computational model to virtually screen adjacent activation sites to insert the proximal cage,which significantly reduces the experimental efforts with cumbersome human operations.Subsequently,the two research groups demonstrate that,the so-called“CAGE-prox”method can be applied to block the activity of a wide range of proteins regardless of the existence and/or chemical identity of the catalytic residues.Noteworthy,they co-developed a time-resolved proteomics technique,which could be applied to capture and identify substrates for protein hydrolysis in cell apoptosis,providing valuable resources for understanding the dynamic biological process.