CAGE-prox: A Unified Approach for Time-Resolved Protein Activation in Living Systems
Jie Wang1,2, Yuan Liu1, Yanjun Liu1
1Synthetic 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, Beijing, China.
Abstract:
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.
Insights
A new method called CAGE-prox enables precise temporal control over protein activity in living systems. This computationally designed approach uses caged amino acids to reversibly block protein function until photoactivated.
Area of Science:
- Molecular Biology
- Biochemistry
- Chemical Biology
Background:
- Temporal activation of proteins of interest (POIs) is crucial for studying dynamic biological processes using gain-of-function approaches.
- Existing methods like protein fusion or active site decaging have limitations in generality or introduce bulky tags.
- There is a need for versatile and non-disruptive methods for time-resolved protein activation.
Purpose of the Study:
- To introduce and validate the computationally aided and genetically encoded proximal decaging (CAGE-prox) strategy.
- To demonstrate CAGE-prox's utility for time-resolved photoactivation of a broad range of proteins.
- To provide a computational framework for optimizing caged amino acid insertion sites.
Main Methods:
- Development of a computational algorithm to identify optimal sites for caged unnatural amino acid insertion near a protein's functional site.
- Genetic encoding of caged amino acids for proximity-based protein activity blockade.
- Photo/chemical decaging for temporal rescue and activation of protein function.
- Experimental validation of the CAGE-prox strategy across various applications.
Main Results:
- The CAGE-prox strategy enables effective temporal blockade and photo-induced reactivation of protein activity.
- Computational screening successfully identified optimal insertion sites for caged amino acids.
- Demonstrated orthogonal activation of POIs, minimizing interference from endogenous proteins.
- Applied CAGE-prox to time-resolved proteomics and controlled activation of protein-based prodrugs.
Conclusions:
- CAGE-prox offers a general, non-disruptive, and computationally guided approach for precise temporal control of protein function in living systems.
- This strategy facilitates gain-of-function studies and novel therapeutic applications.
- CAGE-prox represents a significant advancement in optogenetic and chemogenetic tools for biological research.
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