CAGE-prox: A Unified Approach for Time-Resolved Protein Activation in Living Systems

Wang，Jie1,2; Liu，Yuan1; Liu，Yanjun1; Wang，Chu1,3; Chen，Peng R.1,3

2021-06-01

10.1002/cpz1.180

Current protocols   影响因子和分区

2691-1299

Temporal activation of proteins of interest (POIs) offers a gain-of-function approach to investigate protein functions in dynamic biological processes. Fusion of photo/chemical-switchable proteins to a POI, or site-specific blockage/decaging of catalytic residue(s) on a POI, are the most widely utilized strategies for selective protein activation. These methods, however, either lack generality (e.g., active site decaging) or would modify the POI with a bulky tag (e.g., genetic fusion). Recently, a computationally aided and genetically encoded proximal decaging strategy (CAGE-prox) has been developed for time-resolved photoactivation of a broad range of proteins in living systems. In contrast to the direct decaging of the active site of a POI, CAGE-prox relies on a unified caged amino acid that can be anchored in proximity to a protein's functional site for temporal blockage of its activity until rescued by photo/chemical decaging. In order to identify the optimal site for photo-caged unnatural amino acid insertion, which is key for the effective blockade and re-activation of the POI, a computational algorithm was developed to screen all possible positions in close proximity to the functional site that would enable turning off/on protein activity via caging/decaging operations. Here, we describe the CAGE-prox strategy, from in silico design to experimental validation, and provide various examples of its application. © 2021 Wiley Periodicals LLC Basic Protocol 1: In silico design and experimental validation of CAGE-prox Basic Protocol 2: Orthogonal activation of a POI by CAGE-prox while minimizing the activity from the endogenous protein Basic Protocol 3: CAGE-prox-enabled, time-resolved proteomics for the identification of substrates of a proteolytic enzyme Basic Protocol 4: Controlled activation of protein-based prodrugs for tumor therapy.

chemical decaging chemical proteomics computational design protein activation proximal decaging

英语

其他

2-s2.0-85110251050

Scopus

1.Synthetic and Functional Biomolecules Center,Beijing National Laboratory for Molecular Sciences,Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education,College of Chemistry and Molecular Engineering,Peking University,China
2.Department of Chemistry,Southern University of Science and Technology,Shenzhen,China
3.Peking-Tsinghua Center for Life Sciences,Peking University,China

Wang，Jie,Liu，Yuan,Liu，Yanjun,et al. CAGE-prox: A Unified Approach for Time-Resolved Protein Activation in Living Systems[J]. Current protocols,2021,1(6).

Wang，Jie,Liu，Yuan,Liu，Yanjun,Wang，Chu,&Chen，Peng R..(2021).CAGE-prox: A Unified Approach for Time-Resolved Protein Activation in Living Systems.Current protocols,1(6).

Wang，Jie,et al."CAGE-prox: A Unified Approach for Time-Resolved Protein Activation in Living Systems".Current protocols 1.6(2021).