Screening Of Porcine ISG15-Modified Targets And Functional Identification Of ISGylation E3 Ligase HERC6

Interferon-stimulated genes(ISGs)are a general term for a large class of proteins that are up-regulated after cells are stimulated by interferon(IFN)during virion invasion.Among them,interferon-stimulated gene 15(ISG15)is one of the earliest discovered ISGs.The protein product of ISG15 belongs to ubiquitin-like protein,which is composed of two domains with high homology to ubiquitin in tandem,and is closely related to ubiquitin.Mature ISG15 has the same C-terminal motifs(LRLRGG)with ubiquitin and therefore has biological functions similar to ubiquitin.Form binding to achieve post-translational modification of the target protein,a process called protein ISG modification(ISGylation).At present,it is believed that ISGylation can not only act on viral proteins,but also modify the host’s own proteins to regulate the biological functions of proteins.However,since the ISG15 gene has only 50%homology among different mammals,the function of ISG modification of porcine host proteins and the protein targets of ISGylation have not yet been studied.In this study,ISGylated proteins were enriched in immortalized porcine alveolar macrophages CRL-2843 stimulated with interferon by using a porcine ISG15-specific monoclonal antibody,and then ISG on host proteins was obtained by mass spectrometry and proteomic analysis.Specific modification targets,and further selected the key E3 ligase HERC6(HECT and RLD domain containing E3 ubiquitin protein ligase family member 6)in the ISGylation enzyme-linked system as the research object.The amino acid residues were used to identify the ISGylation targets of HERC6,and their biological functions were also studied.The main work and results of this study are as follows:1.Proteomic analysis of ISGylated proteins: First,we used the porcine ISG15-specific monoclonal antibody to capture the ISGylated proteins in CRL-2843 cells which stimulated by enriched interferon.Then enzyme digestion and mass spectrometry were performed to determine Potential ISGylation target proteins were analyzed to obtain a total of 190 potential ISGylation sites distributed on 98 host proteins.Through Gene Ontology(GO)annotation and enrichment analysis and protein-protein interaction(PPI)network analysis of these proteins,the results show that ISG modifications occurring in pig cells mainly affect host metabolic pathways,especially the nucleotide metabolism pathway.In addition,some proteins encoded by interferon-stimulated genes and related proteins may also be target proteins of ISGylation.These data suggest that the antiviral function of porcine ISG15 may depend on the regulation of host metabolism and antiviral regulatory mechanisms mediated by certain interferon-stimulated genes.2.Determine the key protein E3 ligase HERC6 in the porcine ISGylation system as an interferon-stimulated gene: Using the interferon-stimulated CRL-2843 cell c DNA as a template,the porcine HERC6 gene was successfully cloned.After sequencing,DNAStar was used for multiple sequence alignment,found that porcine HERC6 was significantly different from human HERC6 and mouse HERC6.Subsequently,a recombinant protein containing 300 amino acids at the C-terminal end of porcine HERC6 was expressed,and BALB/c mice were immunized to obtain a polyclonal antibody against porcine HERC6.After stimulating cells with IFN-α and IFN-λ,intracellular porcine HERC6 was up-regulated at both m RNA and protein levels,confirming that HERC6 is an interferon-stimulated gene.3.The mechanism of ISGylation mediated by porcine HERC6: After co-transfection of the encoded porcine HERC6 and porcine ISG15 into HEK-293 T cells,obvious ISG modification was observed without IFN treatment.It is suggested that porcine HERC6 can act as an E3 ligase of ISG modification system to mediate the modification of porcine ISG15 and this modification depends on the presence of the specific E3 ligase HERC6 independent of interferon stimulation.When porcine ISG15 plasmid was transfected into human HEK-293 T cells alone,exogenous interferon stimulation could not mediate the coupling of porcine ISG15 to the target protein for ISGylation.It is shown that,unlike the ubiquitin modification system,the occurrence of host protein ISGylation depends on the paired presence of species-specific ISG15 and E3 ligase HERC6.4.Identification and functional identification of porcine HERC6’s own ISGylation sites: Two ISG-modified lysine residues in porcine HERC6 were identified according to proteomic analysis,and recombinant plasmids with these two site mutations were constructed by overlapping PCR,after the mutant and wild-type HERC6 were co-transfected with ISG15 in HEK-293 T cells,the HERC6 protein was enriched by co-immunoprecipitation to detect the ISGylation and the total intracellular protein ISGylation level was detected on the whole cells.The results showed that the transfection of HERC6 Compared with wild-type HERC6,the degree of cellular ISGylation of the mutant was decreased,which proved that the ISGylation of HERC6 itself can promote its activity as an E3 ligase and enhance the intracellular ISGylation.