Infections of many viruses induce caspase activation to regulate multiple cellular pathways,including programmed cell death,immune signaling and etc.Characterizations of caspase cleavage sites and substrates are impor...Infections of many viruses induce caspase activation to regulate multiple cellular pathways,including programmed cell death,immune signaling and etc.Characterizations of caspase cleavage sites and substrates are important for understanding the regulation mechanisms of caspase activation.Here,we identified and analyzed a novel caspase cleavage motif AEAD,and confirmed its caspase dependent cleavage activity in natural substrate,such as nitric oxide-associated protein 1(NOA1).Fusing the enhanced green fluorescent protein(EGFP)with the mitochondrial marker protein Tom20 through the AEAD motif peptide localized EGFP to the mitochondria.Upon the activation of caspase triggered by Sendai virus(SeV)or herpes simplex virus type 1(HSV-1)infection,EGFP diffusely localized to the cell due to the caspase-mediated cleavage,thus allowing visual detection of the virusinduced caspase activation.An AEAD peptide-derived inhibitor Z-AEAD-FMK were developed,which significantly inhibited the activities of caspases-1,-3,-6,-7,-8 and-9,exhibiting a broad caspase inhibition effect.The inhibitor further prevented caspases-mediated cleavage of downstream substrates,including BID,PARP1,LMNA,pro-IL-1β,pro-IL-18,GSDMD and GSDME,protecting cells from virus-induced apoptotic and pyroptotic cell death.Together,our findings provide a new perspective for the identification of novel caspase cleavage motifs and the development of new caspase inhibitors and anti-inflammatory drugs.展开更多
基金supported by the National Key R&D Program of China(2021YFC2300700)National Science and Technology Major Project(No.2018ZX10101004001005)National Natural Science Foundation of China(numbers 32070179).We thank Dr.Qinxue Hu(Wuhan Institute of Virology)and Dr.Yuchen Xia(Wuhan University)for help with materials.We thank Ding Gao from Center for Instrumental Analysis and Metrology at Wuhan Institute of Virology for his help with the Leica confocal microscope and the Operetta.
文摘Infections of many viruses induce caspase activation to regulate multiple cellular pathways,including programmed cell death,immune signaling and etc.Characterizations of caspase cleavage sites and substrates are important for understanding the regulation mechanisms of caspase activation.Here,we identified and analyzed a novel caspase cleavage motif AEAD,and confirmed its caspase dependent cleavage activity in natural substrate,such as nitric oxide-associated protein 1(NOA1).Fusing the enhanced green fluorescent protein(EGFP)with the mitochondrial marker protein Tom20 through the AEAD motif peptide localized EGFP to the mitochondria.Upon the activation of caspase triggered by Sendai virus(SeV)or herpes simplex virus type 1(HSV-1)infection,EGFP diffusely localized to the cell due to the caspase-mediated cleavage,thus allowing visual detection of the virusinduced caspase activation.An AEAD peptide-derived inhibitor Z-AEAD-FMK were developed,which significantly inhibited the activities of caspases-1,-3,-6,-7,-8 and-9,exhibiting a broad caspase inhibition effect.The inhibitor further prevented caspases-mediated cleavage of downstream substrates,including BID,PARP1,LMNA,pro-IL-1β,pro-IL-18,GSDMD and GSDME,protecting cells from virus-induced apoptotic and pyroptotic cell death.Together,our findings provide a new perspective for the identification of novel caspase cleavage motifs and the development of new caspase inhibitors and anti-inflammatory drugs.
