Mechanism Of Cleavage Of RIPK3 By 3C Proteases Of EV71 And CA6 As Well As Caspase-8

Background:Enteroviruses are belong to Picornaviridae and are the main pathogens that cause hand,foot and mouth disease,including novel enteroviruses,coxsackieviruses,echoviruses and polioviruses.Children are more susceptible.Mild clinical symptoms include fatigue,low-grade fever,diarrhea,and herpes on the limbs and mouth.A small number of severe patients may experience neurological and respiratory complications,such as paralysis,viral meningitis,myocardial damage,and even death.The pathogenic mechanism of enteroviruses has not been fully studied,and there are no mature and effective targeted antiviral drugs or vaccines.3C protease,which is a key factor in the replication process of enterovirus,plays an important role in inhibiting host gene expression by affecting the processing of host pre-m RNA as well as processing viral proteins to mature.It is also an important drug target.Receptor interacting protein 3(RIPK3)and cysteinyl aspartate specific proteinase family(Caspase)are key molecules involved in the programmed death of host cells.This study focused on exploring the specific regulation mechanism of 3C proteases of Enterovirus 71(EV71)and Coxsackievirus A6(CA6)on RIPK3 and Caspase,in order to provide a new idea for the development of antiviral drugs.Methods:The first part The human kidney epithelial cell line 293 T was used as host cells.In the first step,the p CMV-HA-RIPK3 plasmid(about 60 KD in full length)was transiently transfected into 293 T cells,and the apoptosis inhibitors Z-DEVD-FMK(inhibits Caspase-3 activation)and Z-IETD-FMK(inhibits Caspase-8 activation)andZ-LEHD-FMK(inhibiting Caspase-9 activation)were used to treat with 293 T cells in order to observe the expression of RIPK3 and screen the Caspase proteins that regulate RIPK3.By inhibiting common degradation pathways,proteasome inhibitor(MG132--inhibits the E3 proteasome degradation pathway)and autophagy inhibitor(3MA--inhibits the formation of autophagosomes and chloroquine--inhibits the fusion process of autophagosomes and lysosomes)were used to treat with 293 T cells to eliminate possible contributing factors.In the second step,VR1012-FLAG-RIPK3-MYC plasmid,which was reconstructed in our ladoratory was transiently transfected into 293 T cells,and then treated with apoptosis inhibitors Z-DEVD-FMK,Z-IETD-FMK and Z-LEHD-FMK,respectively.The expression of RIPK3 was detected by the FLAG and MYC tag antibodies,and the Caspase protein to be screened was further identified.In the third step,according to the amino acid sequence of RIPK3 and the usual recognition sequence of Caspase-3,-8 and-9,the possible cleavage site of Caspase on RIPK3 is searched.The mutant plasmid was constructed,and the binding of RIPK3 to Caspase was detected by co-immunoprecipitation experiment,and the Caspase protein that regulates RIPK3 was finally determined.The second part In the first step,VR1012-FLAG-RIPK3-MYC and VR1012-EV71 3C-HA were co-transfected into 293 T cells,and the expression of RIPK3 was detected by FLAG(N-terminal)and MYC(C-terminal)tag antibodies to verify whether 3C protease has a cleavage effect on RIPK3.And observing whether the cleavage of RIPK3 by Caspase-8 is affected by 3C protease.In the second step,the protease catalytic active site mutation plasmids,which are VR1012-EV71 3C(His40)-HA,VR1012-EV713C(Glu71)-HA and VR1012-EV71 3C(Cys147)-HA,respectively,were used to further verify the cleavage effect of 3C protease on RIPK3 and to verify whether through 3C enzymatic activity,3C protease could gradually weakens the ability of Caspase-8 to cleave RIPK3.The third step is to verify whether the 3C protease of Coxsackievirus A6 also has the ability to cleave RIPK3,and whether the CA6 3C protease will produce similar results to EV71 3C protease in the aspect of interfering with Caspase-8.To further confirm the interference of enterovirus 3C protease on RIPK3 and Caspase-8,VR1012-CA6 3C-MYC and VR1012-FLAG-RIPK3-MYC were co-transfected into 293 T cells,and the changes between RIPK3 bands were observed.Results and analysis:The first part1.Z-DEVD-FMK(inhibits Caspase-3 activation)and Z-IETD-FMK(inhibits Caspase-8 activation)can inhibit the down-regulation of RIPK3 in 293 T cells,Z-LEHD-FMK(inhibits Caspase-9 activation),Proteasome inhibitors(MG132)and autophagy inhibitors(3MA and chloroquine)had no effect on RIPK3 expression,suggesting that downregulation of RIPK3 is related to Caspase-3 and/or-8,independent of degradation pathways and Caspase-9.2.Transfection of VR1012-FLAG-RIPK3-MYC into 293 T cells can produce35KD(FLAG-N-terminal)and 25KD(MYC-C-terminal)cleavage bands.And Z-DEVD-FMK(inhibits Caspase-3 activation)and Z-IETD-FMK(inhibits Caspase-8activation)but not Z-LEHD-FMK(inhibits Caspase-9 activation)inhibit the production of cleavage bands,indicating that Caspase-3 and/or-8 down-regulated RIPK3 through cleavage.3.According to the amino acid sequence of RIPK3 and the usual recognition sequences of Caspase-3,-8 and-9,which are DXXD,XEXD and I/V/L---EXD,respectively,It was found that there is a site on RIPK3(TEMD)that can be a candidate site for Caspase-3,-8,-9 cleavage.And after mutating the D328 site of RIPK3 to A328,the cleavage bands of 35 KD and 25 KD disappeared.It indicated that the cleavage band produced by RIPK3 transfected 293 T cells was related to the D328 site.And it is possible that only Caspase-8 is involved in the process of cleaving RIPK3(Caspase-3 recognition sequence is DXXD,and Caspase-9 recognition sequence is I/V/L---EXD,which are different from TEMD).It was further confirmed by the experiment of co-immunoprecipitation that Caspase-8 could bind RIPK3,but Caspase-3 could not bind RIPK3.Therefore,in summary,the regulatory protein that could cleaves RIPK3 in 293 T cells is Caspase-8.The second part1.VR1012-FLAG-RIPK3-MYC and VR1012-EV71 3C-HA were co-transfected into 293 T cells,which produced two bands of about 47 KD and 13 KD.Thus the 3C protease of EV71 could cleave RIPK3.2.The mutant EV71 3C protein could inhibit the cleavage of RIPK3 by 3C protease under the condition that it loses the catalytic function but still has the binding function,but could not inhibit the cleavage of RIPK3 by Caspase-8.Therefore,the catalytic active site of 3C protease is an important site for cleaving RIPK3,and it is also an important site for interfering with the cleavage of RIPK3 by Caspase-8,while the steric hindrance of the binding activity of 3C protease could not interfere with the cleavage of RIPK3 by Caspase-8,so,after cleaving RIPK3 by 3C protease,it might interfere with the recognition of RIPK3 by Caspase-8.3.CA6 3C protease also cleaved RIPK3 to produce the bands around 47 KD and13KD,and the bands around 25 KD and 35 KD generated by Caspase-8 cleaving RIPK3 might also be down-regulated due to the action of CA6 3C protease.Conclusions:1.Overexpression of RIPK3 can activate Caspase activation in 293 T cells,and activated Caspase-8 but not Caspase-3 and-9 can cleave RIPK3 by recognizing the D328 site(TEMD)of RIPK3,and RIPK3 is not degraded by the pathways of E3 proteasome and autolysosome.2.The 3C proteases of EV71 and CA6 can cleave RIPK3.3.The 3C protease of EV71 interferes with the cleavage of RIPK3 by Caspase-8through its enzymatic catalytic activity rather than its enzymatic binding activity.4.The cleavage of RIPK3 by 3C protease may affect the recognition of RIPK3 by Caspase-8.Innovation:1.It was first discovered that the 3C proteases of EV71 and CA6 have the cleavage effect on necroptosis factor RIPK3.2.It was first discovered that the 3C proteases of EV71 and CA6 could affect the cleavage of RIPK3 by Caspase-8 after cleaving RIPK3.3.It was first discovered that the interference phenomenon of EV71 3C protease to Caspase-8 is through the catalytic activity of 3C protease rather than the binding activity of 3C protease.