The Deubiquitination Of IL-33 Modulates Its Stability And DNA Binding To The IL13 Gene Locus

Interleukin-33(IL-33)is a member of the IL-1 family of cytokines with dual function which either activates target cells as a classical cytokine via binding to its receptor complex or regulates gene transcription as a nuclesr factor in the nucleus.However,its nuclear targets and the relevant regulatory mechanisms of its nuclear function remain unclear.Recent studies have revealed that the nuclear IL-33 participates in inflammatory reactions through binding to NF-k B p65 subunit or p65 promoter region.Initiating and promoting airway Th2 inflammation,IL-33 plays a crucial role in the pathogenesis of asthma as a cytokine.However,the pathophysiological roles of IL-33 as a nuclear factor remain unclear.Based on the importance of the nuclear function of IL-33,our study aimed to explore the specific function of IL-33,especially its downstream target genes.Since ubiquitin modification plays crucial roles in modulating protein stability and activity,we then attempted to figure out whether the ubiquitination and deubiquitination could affect the stability or nuclear function of IL-33.Expression plasmids encoding Flag-tagged IL-33 and His-tagged ubiquitin were transfected into human embryonic kidney(HEK)293T cells.Then ubiquitin pull-down assay was applied to isolate the ubiquitinated proteins with Ni-NTA beads.The polyubiquitination of IL-33 was observed by immune blot.The association between IL-33 and ubiquitin was confirmed in the immunoprecipitation assay.Immunoprecipitation was then performed to see which deubiquitinase(DUB)associats with IL-33.Among several DUBs,USP17 and USP21 displayed the interaction with IL-33.IL-33 colocalized with USP17 or USP21 in the cytoplasm and uncleus.We found that both USP17 and USP21 could deubiquitinate IL-33 and their enzymatically inactive mutants(USP17C89S and USP21C221A)could not deubiquitinate IL-33 efficiently.Moreover,USP17 could mediate both K48-and K63-linked deubiquitination of IL-33.We detected the protein levels of IL-33 in the presence of increasing doses of USP17 or USP21.As expected,both USP17 and USP21 dose-dependently stabilized the protein level of IL-33.Furthermore,the protein levels of IL-33 decreased upon CHX treatment and USP17 could reverse it but not its mutant C89 S.And USP21 also had this ability to stabilize IL-33.The increased mRNA level of IL-13 was found in TAP-IL-33 stable cell line via quantitative PCR(q PCR).There existed a conserved non-coding sequence(CNS)before the translation initiation site of human IL13 gene by comparing with other different species such as mouse,dog and cattle.Then we performed the chromatin immunoprecipitation(Ch IP)assay followed by q PCR and found that IL-33 could directly bind to this CNS of IL13 gene which could be down-regulated by USP17 while USP17 mutant could reverse this down-regulation.In summary,our findings identify a role of IL-33 in the regulation of IL13 gene transcription and a new regulatory mechanism of IL-33 via interacting with the deubiquitinase USP17 and USP21.Since IL-13 is an indispensable factor for the development of asthma,our finding deepens the immunological pathogenesis of asthma.It also provides new understanding for the function of IL-33 as a nuclear factor and the role of ubiquitin modification in modulating gene transcription.