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 stud...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 m Neon Green2-based molecular biosensor.m Neon Green2 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 Dna E intein to abolish the fluorescence(inactive state).Caspase-3-catalyzed cleavage of DEVD linearizes m Neon Green2 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 He La and MCF-7 tumor cells and inhibition of Zika virus invasion.展开更多
基金supported by the National Natural Science Foundation of China(21890743)National Key Research and Development Program of China(2017YFA0205500,2018YFA0902702)the Strategic Priority Research Program of the Chinese Academy of Sciences,China(XDB29050100)。
文摘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 m Neon Green2-based molecular biosensor.m Neon Green2 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 Dna E intein to abolish the fluorescence(inactive state).Caspase-3-catalyzed cleavage of DEVD linearizes m Neon Green2 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 He La and MCF-7 tumor cells and inhibition of Zika virus invasion.
