Designing an apoptosis reporter by mutagenesis-based insertion of caspase-3 cleavage motif into green fluorescence protein

Introduction

Apoptosis is an essential process for organisms and animal development, and its dysregulation is related to the progression of several diseases. Therefore, methods to detect apoptosis are necessary for mechanistic research and drug development. To overcome the disadvantages of traditional methods for detecting apoptosis, a variety of imaging strategies based on enzyme-mediated fluorescence activation have been developed.

Objectives

This study aims to design a novel strategy for apoptosis fluorescent reporters which are inactivated by caspase-3.

Methods

Four candidate EGFP mutations containing DEVD-similar sequences were selected by their structural positions and hydrophilicities, and an EGFP mutant was chosen by investigating fluorescence expression. This EGFP mutant was stably expressed in various cells using the safe harbor locus. To verify our apoptosis reporter system, protein levels, colocalization, and intensity were investigated in apoptosis-induced EGFP mutant-expressing cells.

Results

The fluorescence intensity of the mutant EGFP was decreased in a time- and concentration-dependent manner by staurosporine and H₂O₂, which induce apoptosis. Furthermore, compared to a dark-to-bright reporter of apoptosis (caspase-activatable GFP), our bright-to-dark system showed greater sensitivity for apoptosis. Our system is useful in various models, including other species.

Conclusion

Our method does not require additional peptides, which makes it easily adaptable to other systems. And, our apoptosis reporter may be useful in a variety of research fields as well as drug screening.