UBQLN2 Regulates TBK1 To Promote IFN1 Expression

Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease but the pathogenesis is unclear.Both Ubiquilin 2(UBQLN2)gene and Tank-binding kinase 1(TBK1)gene are pathogenic genes of ALS.Mutations in either UBQLN2 or TBK1 can impair the autophagy system,leading to an ALS-like phenotype in animals.However,the interplay of TBK1 and UBQLN2 has not been reported.Phosphorylation of interferon regulatory factor 3(IRF3)by activated TBK1 is known to promote the production of IFN1 and pro-inflammatory cytokines.Neuroinflammation plays an important role in the development of ALS,whether UBQLN2 interacts with TBK1 to promote the expression of IFN1 and proinflammatory factors by regulating IRF3 remains unclear.Objective: To explore the relationship between UBQLN2 and TBK1 and whether it regulates the expression of IFN1 via the TBK1-IRF3 signaling pathway.Methods:(1)The Ubqln2 and mutant(P497H,P506T)plasmids,TBK1 and mutant(R47H)plasmids were constructed and transfected into HEK-293 T cells respectively,and the relationship between UBQLN2 and TBK1 was detected by Co-IP and GST pull down.(2)The effects of overexpression of UBQLN2 or UBQLN2 mutations on the expression of P-TBK1,P-IRF3,IFN1 and related pro-inflammatory factors were detected by WB,RT-q PCR,dual-luciferase reporter assay.The IRF3 plasmid,p62 plasmid and OPTN plasmid were constructed and transfected into HEK-293 T cells respectively.Co-IP was used to detect the effect of UBQLN2 mutations on the interaction between TBK1 with its partners,including IRF3,p62 and OPTN.(3)To examine whether UBQLN2 enhanced the IFN1 expression via TBK1-IRF3 or TBK1-IRF7,we generated CRISPR-Cas9-mediated IRF3 and IRF7 knockout(KO)HEK-293 T cells.Results:(1)TBK1 interacted with UBQLN2 primarily via the TBK1CCD1,CCD2 and KD domains.UBQLN2 interacted with TBK1 mainly via the UBA domain.(2)Over-expression of UBQLN2 promoted the phosphorylation of TBK1 and IRF3,the level of P-TBK1 and P-IRF3 decreased after UBQLN2 mutations.(3)Overexpression of UBQLN2 promoted the level of IFN1 m RNA and related pro-inflammatory factors m RNA,and UBQLN2 mutations reduced the expression of them.(4)UBQLN2 mutations significantly decreased the binding affinity of TBK1 for IRF3,p62 and OPTN.(5)In IRF3-KO cells: overexpression of UBQLN2 promoted the phosphorylation of TBK1,while overexpression of UBQLN2 or UBQLN2 mutantions had no effect on the level of IFN1 m RNA and related proinflammatory factor m RNA.(6)In IRF7-KO cells:overexpression of UBQLN2 promoted the phosphorylation of TBK1 and the expression of IFN1 m RNA and related pro-inflammatory factors m RNA.Overexpression of UBQLN2 mutations decreased the level of PTBK1 and the m RNA expression of IFN1 and related pro-inflammatory factors.Conclusion: UBQLN2 interacted with TBK1 in HEK-293 T cells and regulated the expression of IFN1 via the TBK1-IRF3 signaling pathway.