Quantitative Proteomics Study Of Protein Ubiquitination In Antiviral Innate Immunity Pathway

Ubiquitination is an important post-translational modification of proteins in biological process,which can target protein degradation and activate protein regulatory signaling pathways.According to the different exogenous vrial nucleic acids stress,host anti-viral innate immune pathways can be divided into CGAS-STING-IFN?1 pathway against DNA viruses and RIG-I-c GAS-IFN?1 / NF?B pathway against RNA viruses.The activation of key proteins in the antiviral innate immune pathway relies on K63 ubiquitination,therefore in-depth characterization and understandings of protein ubiquitination are essential to resolve innate immune pathways.To date,no studies have systematically compared the protein ubiquitination in the two pathways.Given its highthroughput and accuracy,mass spectrometry-based proteomics has become the mainstream systematic analytical tool in post-translational modification studies.In this study,we performed a quantitative and comparative analysis of host protein ubiquitination upon DNA virus HSV and RNA virus Se V infection of THP1 cells,by combining metabolic isotope-labeling technology and ubiquitination enrichment technology prior to high-resolution LC-MS analysis.In total,we quantified 3843 proteins and 4147 ubiquitination sites.In the HSV experimental group,123 proteins were significantly up-regulated and 71 were downregulated,while 300 ubiquitination sites were significantly up-regulated and 92 were down-regulated.In the Se V experimental group,99 proteins were significantly upregulated and 68 were down-regulated proteins,whereas 144 ubiquitination sites were significantly up-regulated and 63 were down-regulated.We found that the correlation between ubiquitination levels was strong as compared to the weak correlation between ubiquitination and protein levels,indicating that most changes in ubiquitination were not affected by changes in protein amount.Motif analysis showed that ubiquitination sites were significantly enriched with neighbouring acidic amino acids or uncharged aliphatic amino acids,and reduced in basic amino acids and cysteine.Around 400 significantly regulated ubiquitination sites were found under HSV and Se V infection.The down-regulated ubiquitinated proteins were similar under two conditions but upregulated proteins were highly different.Gene Ontology analysis revealed enriched c GAS pathway in response to HSV infection and enriched RIG-I pathway upon Se V infection.Such differences were within expectations,approving the screening power of proteomics and providing resources for the subsequent studies on the key role of different ubiquitinated proteins in the two types of innate immunity pathways.Through protein-protein interaction analysis,we found that IMPDH2 highly interacted with proteins in the "innate immunity" and "VRNP assembly" pathways only in response to Sev infection.The K124 ubiquitination on IMPDH2 was significantly upregulated while no change was detected on protein level.We verified that IMPDH2 negatively regulated the NF?B pathway by inhibiting degradation of I?B? and had specific interaction with TAK1.This study compared the similarities and differences in protein ubiquitination of host proteins responsive to RNA and DNA viruses infection,and provided new concepts and resources for inspecting the key roles of protein ubiquitination in the innate immunity.