Ubiquitylome Analysis Of Marek's Tumor And Identification Of CDK1 Ubiquitination

Marek's disease(MD)is avian carcinogenic lymphoproliferative disease caused by Marek's disease virus serotype 1(MDV-1).CD4~+T cells are susceptible to infection and transformation by MDV,and then forms T lymphomas in peripheral nerves and internal organs,and subsequently causes immunosuppression.It has been found that some MDV-encoded genes are involved in the pathogenesis of MDV,but the detailed tumorigenicity and immunosuppression mechanisms of MDV are still unclear.There have been some omics analyses that revealed systemic changes in MD tumor,such as transcriptomic and proteomic analyses,improved the understanding of MDV pathogenecity.Recently,we found that MDV-encoded UL36 has deubiquitination activity and is highly expressed in MDCC-MSB1 cells.However,it has been known that ubiquitination is one of the most important post-translational modification mechinery in cells.Thus,we hypothesized that MDV may manipulate the ubiquitination regulatory system in chicken T lymphocyte to promote tumorigenecity and immunosupression.In present study,we used a monoclonal antibody against ubiquitinated peptide(K-?-GG)to separate ubiquitinated peptides from CD4~+T lymphocytes and MD turmor cell line,MDCC-MSB1 cells,and then qualitatively and quantitatively analyzed the ubiquitinated peptides by mass spectrometry and bioinformatics analysis.Our study found that ubiquitination of 717 proteins in MSB cells were significantly up-regulated compared to CD4+T lymphocytes,while ubiquitination of 778 proteins were significantly down-regulated.In ubiquitination alteration,some proteins were up-regulated by MDV and other proteins in similar proportion were down-regulated by MDV.Among the significantly changed proteins,ubiquitination of the transferase,especially Serine/threonine protein kinase,is a major target of MDV.MDV primarily alters the ubiquitination in the pathways,such as signal transduction,immune system,cancer,and infectious disease pathways in MSB cells.In these pathways,the ubiquitination of CDK1,IL-18,PRKCB,ETV6 and EST1 proteins were confirmed to be significantly up-regulated or down-regulated using specific antibodies against these proteins by western blotting.According to the results of ubiquitylome analysis and western blotting,it was revealed that CDK1 protein contains multiple ubiquitination sites with significant up-regulation in which the highest one is 152 folds.As a cell cycle-dependent kinase,CDK1 plays an important role in the regulation of the cell cycle.Many viruses regulate cell cycle progression through the ubiquitin system to promote viral proliferation.Thus,mutagenicity was carried out to verify which Lys sites were up-regulated in ubiquitination by MDV.The recombinant lentivirus expressing CDK1or CDK1 mutants with Lys mutations were packedged using pCDH-EF1?-CDK1(K mutation),pGAG,pREV,pVSV-G plasmids.Post infecting MSB cells,the Strep tag II tagged CDK1 and mutants were isolated from infected MSB cells.The ubiquitinated CDK1 proteins were identified by western blotting.We found that ubiquitinated CDK1 still was detected,even all of the Lys sites on CDK1 were mutated to Arg.We can infer that the ubiquitination may occur at the other residues,such as Cys or Ser/Thr sites on CDK1.Our results from the current study could promote the understanding of MD tumorigenecity from the perspective of ubiquitination regulation.