Identification And Functional Study Of Apoptotic Proteins In SL2 Cell

Apoptosis is a form of strictly controlled programmed cell death in multi-cellular organisms that is responsible for eliminating of unwanted cells including damaged and virus-infected cells.Spodoptera littoralis is a generalist phytophagous insect attacking a wide range of commercially important crops and is one of the most destructive pests in the tropical and subtropical areas of the world.SL2 cell derived from Spodoptera littoralis ovary are often used in studying baculovirus infection.SL2 cell derived from Spodoptera littoralis ovary undergoes apoptosis and produces very low levels of polyhedrin in contrast to Sf9 cells when infected by Ac MNPV,which suggests that differences could be existed in the mechanism of apoptosis in SL2 cell and Sf9 cell.Only one effector caspase from Spodoptera littoralis was reported,none initiator caspase has been identified so far and a scanty few articles expounding mechanism of apoptosis in Spodoptera littoralis.Caspases play a vital role in apoptosis in both mammals and invertebrates.According to their localization in the apoptotic pathway and biological functions,caspases are classified into three groups including initiator caspases,effector caspases and inflammatory caspases.IAP is a core inhibitor of apoptosis protein,loss of IAP triggers cell death with increased caspase activity.In our study,three caspases,SlDronc,SlDredd and Sl-caspase-2,and two IAPs,SlIAP1-1 and Sl IAP1-2,were identified,two of the caspases were initiator caspases and the other one was an effector caspase.SlIAP1-1 and Sl IAP1-2 both inhibited apoptosis induced by SlDronc in SL2 cell.SlDronc was identified as a novel Spodoptera littoralis initiator caspase.Sequence analysis shows that SlDronc possesses typical structure features of initiator caspase and is highly similar to insect Dronc homologues.Recombinant SlDronc underwent autocatalytic cleavage when expressed and purified from E.coli and could cleave synthetic initiator caspase substrates.SlDronc cleaved and activated Sl-caspase-1 and Sf-caspase-1.Transient expression of SlDronc induced apoptosis in SL2 cell,and knockdown of Sldronc decreased apoptosis induced by UV irradiation in SL2 cell.The above results indicate that SlDronc is an apoptotic initiator caspase in Spodoptera littoralis.Furthermore,SlDronc lacking its prodomain was more effective at inducing cell death,and Sl IAPs inhibited apoptosis in SL2 cell induced by overexpression of SlDronc.SlDredd is also an initiator caspase.Sequence analysis shows that SlDredd possesses typical structure features of initiator caspase and is highly similar to insect Dredd homologues.Recombinant SlDredd underwent autocatalytic cleavage when expressed and purified from E.coli and could cleave synthetic initiator caspase substrates.Transient expression of SlDredd induced apoptosis in SL2 cell and activated form of SlDredd gradually increased when SL2 cells were treated by UV.These results suggest that SlDredd is an initiator caspase involved in apoptosis.Sl-caspase-2 is an effector caspase,which shares high similarity with Lep-caspase-2homologs.Recombinant Sl-caspase-2 was not processed when expressed and purified from E.coli and showed no activity to 13 synthetic caspase substrates.Transient expression of Sl-caspase-2 did not induce apoptosis in SL2 cell.Sl-caspase-2 was cleaved by SlDronc in vitro and in SL2 cell.SlIAP1-1 and SlIAP1-2 both contain two BIR domains(BIR1 and BIR2)and a RING domain.SlIAP1-1 and Sl IAP1-2 were cleaved by Sl-caspase-1 and SlDronc,SlIAP1-1and Sl IAP1-2 inhibited apoptosis in SL2 cell induced by SlDronc.Taken together,three caspases and two IAPs were identified in our study,which makes a step forward in identifying apoptotic pathway in Spodoptera littoralis.