Interferon Induced Protein Nmi Alters The Nuclear Localization Of The Small Ubiquitin Related Modifier (SUMO) Conjugating Enzyme UBE2I

Nmi is an interferon-inducible protein. Previous studies have revealed that in stimulant withIFN-γ, Nmi can enhance the transcripts of STAT1, then augmentthe biological responds of IFN-γ,suggesting that Nmi might play an important role in IFN-γ signalling pathway. Base on thoseclues, we screened the interacting partners of Nmi via yeast two-hybrid assay to elucidate thepossible mechanism of Nmi mediate the IFN-γ signaling pathway.We used Nmi asthe bait protein, constructed the bait plasmid pGBKT7-Nmi andperformedthe autonomous activation assay to comfire that Nmi had noautonomousactivation activity. Then,we performed yeast two-hybrid assay by co-transforming bait plasmid pGBKT7-Nmi and thehuman embryo kidney cDNA library plasmid into yeast AH109strain. After selection bySD/-Ade/-His/-Leu/-Trpplate and β-galactosidase filter assays, the plasmids of positive cloneswere isolated and transformed into E.coli DH5α cell, the unique positive clones were deliveredfor sequenceing. The sequencing results were compared in GenBank database by blast program,acquired31potential interaction partners of Nmi at last. UBE2I is one of the candidates. UBE2Iis ubiquitin-conjugating enzyme2I, which can transferan ubiquitin-like protein named SUMO totarget proteins and cause target proteins SUMOylation. STAT1can be SUMOylated, and thetranscription activity of STAT1is inhibited by SUMO conjugation.For further investigate Nmi effects on SUMOlytaion of STAT1, UBE2I ORF fragmentobtained by PCR was subsequently inserted into vector pGADT7and co-transformed into yeastAH109strain with bait plasmid pGBKT7-Nmi again. The yeast two-hybrid result indicates thatNmi could interact with full length of UBE2I protein in yeast cells. Then, we constructedpGEX4T1-Nmi、pCDNA3.1(+)-UBE2I-3×Flag and pCDNA3.1(+)-Myc-Nmi plasmids for GSTPull-down assay and Co-immnoprecipitation assay, both of which confirmed the interactionbetween Nmi and UBE2I. And identified that1-199amino acid domain of Nmi interact withUBE2I.The results of immunofluorescence assay indicated that Nmi altered the nuclear localizationof UBE2I. SUMOylation of proteins usually occurs in nucleus, the change of nuclearlocalization of UBE2I probably affect SUMOylation of nuclear proteins. These evidencessuggesting that Nmi might down-regulation of STAT1SUMOylation through interacted with UBE2I, to enhance the activity of STAT1mediated IFN-γ signaling pathway.