The Mechanism Of The Promotion Of Oncolytic Virus-triggered Pyroptosis By ISG12a

ISG12a belongs to the interferon-inducible protein 27(IFI27)family,which contains a conservative 80 amino acid motif called the ISG12 motif.ISG12 a is distributed on mitochondria,endoplasmic reticulum and nuclear membrane,and its variable distribution may be the reason why it plays a role in different biological processes.The location of ISG12 a in mitochondria is closely related to its function of promoting apoptosis.ISG12 a alone or together with other transmembrane proteins can oligomerize to form pores in the mitochondria,leading to the rupture of the outer mitochondrial membrane and the release of cytochrome C and other apoptotic factors.When ISG12 a is located in the nuclear membrane,it seems to control the shuttle between the cytoplasm and the nucleus.ISG12 a has been reported to resist viral infections,promote cell apoptosis,and is closely related to the occurrence and development of many types of tumors.Cell pyroptosis is a rapid process that triggers inflammation.Members of the Gasdermins family are effectors of cell pyrosis.In humans,the gasdermin family consists of six members: GSDMA,GSDMB,GSDMC,GSDMD,GSDME(also known as DFNA5)and DFNB59.Except DFNB59,the N-terminal domain of gasdermin protein can perforate the cell membrane.Some proteases can cleave in the middle domain of gasdermin family proteins,result in releasing the N-terminal domain.Subsequently,the N-terminal domain shifts to the cell membrane and oligomerizes and punches holes in the membrane,which eventually causes changes in membrane permeability,leading to cell pyrolysis.Surprisingly,emerging studies have indicated that the active gasdermin proteins in tumour cells can trigger strong anti-tumour immunity.However,only a few studies have investigated the role of gasdermin protein-dependent pyroptosis in virus-mediated tumour cell killing.Therefore,we conducted the following researchs.The chapter 2 determines that viral infection can induce cell pyroptosis in liver cancer cells and lung cancer cells.In order to explore whether viral infection can induce pyroptosis of tumor cells,NDV and VSV were used to infect liver cancer cells.Through observing cell morphology,we found that tumor cells would blow bubbles after virus infection.This phenomenon indicates that tumor cells may undergo cell pyroptosis after virus infection.Through conducting detection of LDH release,Hoechst and PI co-staining and western blotting,we determined that the viral infection induced caspase-3-GSDME-mediated pyroptosis of cancer cells.We previously reported on the role of ISG12 a in promoting virus-induced cell apoptosis.We speculated that ISG12 a may regulate cell pyroptosis.In chapter 3,ISG12 a was found to promote virus-induced pyroptosis.As reported in our previous study,ISG12 a silencing reduced PARP activation in virus-infected cells.ISG12 a silencing attenuated pyroptosis in a time-dependent manner as indicated by GSDME and caspase-3 cleavage.These findings suggested that the interferon-inducible gene ISG12 a contributes virus-induced cell pyroptosis.The role of ISGs in cell pyroptosis has not been reported.So we will further explore the mechanism of ISG12 a regulating cell pyroptosis.In chapter 4,TRIM21 was identified as the E3 ligase of ISG12 a.TRIM21 was found as the potential interacting protein of ISG12 a by mass spectrometry.Immunoprecipitation experiment and immunofluorescence assays confirmed that TRIM21 binds ISG12 a.Through bioinformatics analysis and immunoprecipitation experiments,it was further confirmed that the Domain II segment of ISG12 a binds to the PRY/SPRY segment of TRIM21.Considering that TRIM21 is an E3 ligase,we suspected that TRIM21 may ubiquitinate ISG12 a.Through ubiquitination experiments,TRIM21 ubiquitination modified ISG12 a was confirmed.Using point mutation methods and ubiquitination experiments,it was further determined that TRIM21 targets the K69 lysine of ISG12 a,and modified ISG12 a by K6 type ubiquitination.Based on the above experimental results,we believed that TRIM21 is the E3 ligase of ISG12 a.In view of the fact that virus infection can induce the expression of TRIM21,we suggested that TRIM21 also be involved in the regulation of cell pyroptosis.In chapter 5,we determined that TRIM21 can also promote virus-induced pyroptosis of tumor cells and ISG12 a is required for TRIM21-mediated pyroptosis.By detecting the release of LDH and the cleavage of GSDME,we found that overexpression of TRIM21 promoted virus-induced pyroptosis,and silencing TRIM21 inhibited pyroptosis.Considering that the function of E3 ligase depends on its ubiquitination modification on the substrate,TRIM21 was overexpressed in tumor cells and then ISG12 a was silenced.We found that silencing ISG12 a reversed the promotion of the release of LDH and the cleavage of GSDME by overexpression of TRIM21.These data shown that ISG12 a is required for TRIM21-mediated pyroptosis.In view of this result,the functional regulation of ISG12 a by TRIM21 through ubiquitination may be the mechanism of ISG12 a regulates pyroptosis.In chapter 6,We revealed that TRIM21 ubiquitination modified ISG12 a promoted its mitochondrial translocation and promoted cell pyroptosis.The mitochondrial localization of ISG12 a is necessary to promote the release of cytochrome c and activate caspase-3.Cytoplasmic mitochondrial extraction and immunofluorescence assays shown that viral infection promoted the mitochondrial translocation of ISG12 a,and silencing TRIM21 inhibited the mitochondrial translocation of ISG12 a.By overexpressing the K69 R mutant of ISG12 a,we found that the lysine mutation at position K69 of ISG12 a deprived its mitochondrial translocation and promote pyroptosis.In view of these results,it is shown that TRIM21 ubiquitination modifies ISG12 a,which promotes the translocation of mitochondria and promotes pyroptosis.Virus induces tumor cell pyroptosis,so oncolytic virus combined with cell pyroptosis may become a new anti-tumor strategy.In chapter 7,we determined that GSDME-mediated pyroptosis enhances the anti-tumor ability of oncolytic viruses.Through animal experiments,we found that silencing GSDME can reverse the inhibitory effect of NDV on tumors.These data shown that pyroptosis promotes the killing of tumor cells and resists the development of tumors.In summary,on the one hand,this work reveals the new function of ISG12 a in regulating cell pyroptosis.On the other hand,we firstly revealed that pyroptosis as a method of cell death can also eliminate tumor cells.This study proposes a new strategy of oncolytic virus combined with pyroptosis for the elimination of tumor cells.