Modular Peroxidase-Based Reporters for Detecting Protease Activity and Protein Interactions with Temporal Gating

Abstract

Enzymatic reporters have been widely applied to study various biological processes because they can amplify signal through enzymatic reactions and provide good sensitivity. However, there is still a need for modular motifs for designing a series of enzymatic reporters. Here, we report a modular peroxidase-based motif, named CLAPon, that features acid–base coil-caged enhanced ascorbate peroxidase (APEX). We demonstrate the modularity of CLAPon by designing a series of reporters for detecting protease activity and protein–protein interactions (PPIs). CLAPon for protease activity showed a 390-fold fluorescent signal increase upon tobacco etch virus protease cleavage. CLAPon for PPI detection (PPI-CLAPon) has two variants, PPI-CLAPon1.0 and 1.1. PPI-CLAPon1.0 showed a signal-to-noise ratio (SNR) of up to 107 for high-affinity PPI pairs and enabled imaging with sub-cellular spatial resolution. However, the more sensitive PPI-CLAPon1.1 is required for detecting low-affinity PPI pairs. PPI-CLAPon1.0 was further engineered to a reporter with light-dependent temporal gating, called LiPPI-CLAPon1.0, which can detect a 3-min calcium-dependent PPI with an SNR of 17. LiPPI-CLAPon enables PPI detection within a specific time window with rapid APEX activation and diverse readout. Lastly, PPI-CLAPon1.0 was designed to have chemical gating, providing more versatility to complement the LiPPI-CLAPon. These CLAPon-based reporter designs can be broadly applied to study various signaling processes that involve protease activity and PPIs and provide a versatile platform to design various genetically encoded reporters.

Protein-Based Tools for Studying Neuromodulation

“…Protease cleavage releases the steric hindrance so that within minutes split APEX reconstitutes, allowing for further fluorescent labeling and analysis in live or fixed tissues. 5 LiPPI-CLAPon achieves light-and PPI-dependence similar to the SPARK design. 32 As shown in Figure 3A, the tobacco etch virus protease (TEVp) cleavage site (TEVcs) is sterically blocked by the light, oxygen, and voltage sensing domain (LOV) in the dark.…”

“…To address the limitations of transcriptional reporters, we designed a new class of integrator, a light-and PPI-dependent enzymatic reporter, called LiPPI-CLAPon (Light-and PPIdependent Cleavage of Leucine zipper-caged APEX for Protease detectiON). 5 The key in LiPPI-CLAPon is a new protease cleavage-dependent enzymatic sensor motif we designed called CLAPon. In CLAPon, the reconstitution of the split APEX2 (enhanced ascorbate peroxidase) is sterically blocked by acid− base coils (Figure 3A).…”

“…In CLAPon, the reconstitution of the split APEX2 (enhanced ascorbate peroxidase) is sterically blocked by acid–base coils (Figure A). Protease cleavage releases the steric hindrance so that within minutes split APEX reconstitutes, allowing for further fluorescent labeling and analysis in live or fixed tissues . LiPPI-CLAPon achieves light- and PPI-dependence similar to the SPARK design .…”

“…Therefore, there is a need for further development of integrators for detecting neuromodulators. My lab has so far designed two new classes of molecular integrators, which have the potential to be applicable for large brain volume detection in live animals, followed by post-mortem analysis. ,,,,− …”

“…We demonstrated LiPPI-CLAPon’s application in detecting dopamine using the dopamine receptor D1 (DRD1)–arrestin interaction pair in HEK293T cells (Figure B,C) . Screening of different TEVcs shows that the ENLYFQA peptide sequence gives the best SBR for LiPPI-CLAPon (Figure B), while CLAPon for detecting PPI without a light control (PPI-CLAPon) gives the best SBR with the TEVcs ENLYFQN . Therefore, it is always good to re-evaluate the TEVcs and TEVp pair used in the TEVp-based proximity cleavage reporters.…”