A switch-on molecular biosensor for detection of caspase-3 and imaging of apoptosis of cells

Abstract

Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability. Disordered apoptosis can cause a variety of diseases; therefore, sensing apoptosis can provide help in study of mechanism of the relevant diseases and drug development. It is known that caspase-3 is a key enzyme involved in apoptosis and the expression of its activity is an indication of apoptosis. Here, we present a genetically encoded switch-on mNeonGreen2-based molecular biosensor. mNeonGreen2 is the brightest monomeric green fluorescent protein. The substrate of caspase-3, DEVD amino acid residues, is inserted in it, while cyclized by insertion of Nostoc punctiforme DnaE intein to abolish the fluorescence (inactive state). Caspase-3-catalyzed cleavage of DEVD linearizes mNeonGreen2 and rebuilds the natural barrel structure to restore the fluorescence (activated state). The characterization exhibited that the Caspase-3 biosensor has shortened response time, higher sensitivity, and prolonged functional shelf life in detection of caspase-3 amongst the existing counterparts. We also used the Caspase-3 biosensor to evaluate the effect of several drugs on the induction of apoptosis of HeLa and MCF-7 tumor cells and inhibition of Zika virus invasion. Supporting Information The supporting information is available online at 10.1007/s11427-021-1986-7. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

Toxic effects of sodium dodecyl sulfate on planarian Dugesia japonica

“…Apoptosis is a strictly controlled cell suicide characterized by nuclear condensation, cell shrinkage, membrane blebbing, and DNA fragmentation ( Bertheloot, Latz & Franklin, 2021 ; Majtnerová & Roušar, 2018 ). Caspase3 is a member of the cysteine family and plays a vital role in the progress of apoptosis ( Gong et al, 2022 ; Lei et al, 2022 ). Studies have shown that aspirin could reduce the level of caspase3 protein and inhibit cell apoptosis, microplastic could induce the expression of caspase3 gene and promote cell apoptosis in planarians ( Gao et al, 2022 ; Liang et al, 2022 ).…”

Centenarian-Sourced Lactobacillus casei Combined with Dietary Fiber Complex Ameliorates Brain and Gut Function in Aged Mice

“…In recent years, caspases were found to be highly associated with cell apoptosis. Among these, caspase-3 is a common downstream effector of apoptotic pathways and a core protease that mediates cell apoptosis [54,55]. The mRNA expression of caspase-3 in the brain was significantly downregulated in mice administrated with DFC, Lactobacillus casei LTL1361, and their combination for 12 weeks, but the mRNA expression of antiapoptotic protein Bcl-2 was significantly upregulated (Figure 2).…”

A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity

“…To create a fluorogenic GZMB-responsive reporter, we took advantage of self-splicing split inteins, which bears a similarity to the elegant design of a fluorogenic caspase-3 reporter by Gong et al [13]. The C-terminal intein of Nostoc punctiforme DNA polymerase III (Npu DnaEc) was coupled to the C-terminal half of mNeonGreen2 (K159-D229) and separated by a recognition sequence for GZMB (IEPD|SG; [14]) from the N-terminal half of mNeonGreen2 (E7-D158) and the N-terminal intein (Npu DnaEn).…”

“…The GZMB-inducible biosensor described in this paper is loosely based on the construct of Gong et al [13]. The construct was designed in silico and synthesised de novo as a human-codon-optimised fragment.…”

“…By taking advantage of proteolytic specificities of certain endogenous proteases and the ability of FPs to change their conformation from a non-fluorescent to a fluorescent state, several biosensors for protease activity have been developed [11,12]. Our own construct shares some similarities with the design of Gong et al [13], whose reporter indicates the activity of the endogenous enzyme caspase-3. Caspase-3 cleavage activity results in apoptosis of the cell and is also activated downstream during T cell mediated cytotoxicity; however, additional extrinsic apoptotic pathways as well as intrinsic pathways can trigger the activation of caspase-3.…”