The Study Of Interaction Regions Between The Small Ubiquitin Related Modifier (SUMO) Conjugating Enzyme UBE2I And The Interferon Induced Protein Nmi

Objective:UBE2I is the specific conjugating enzyme E2 in the sumo modification process, which can transfer an ubiquitin-like protein named SUMO to target proteins and cause target proteins SUMOylation; Nmi, an interferon-inducible protein, can augment IL-2 and IFN-γ dependent transcriptional activity. Our previous experiments show that the interferon induced protein Nmi can interact with the small ubiquitin related modifier(SUMO) conjugating enzyme UBE2 I and alter the nuclear localization of UBE2I;This research was to identify the key component of UBE2 I responsible for the interaction with Nmi, that will establish a foundation for exploring the mechanisms of the regulation of IFN-γ signaling pathway.Methods:1. We designed a number of truncated and deleted UBE2 I mutants, which were inserted into prokaryotic plasmid p GEX-4T1 to express mutant protein. The constructed prokaryotic expression plasmid were transformed into the competent cell of E.coli. UBE2 I mutants were induced by IPTG, and purified by affinity chromatography of GSH agarose beads. Nmi was expressed in mammalian cells.The key component of UBE2 I responsible for the interaction with Nmi was studied by means of GST-pull down. 2. The key component of UBE2 I responsible for the interaction with Nmi was confirmed by coimmunoprecipitation and immunohistochemistry experiments.Results:The 62 nd leucine of UBE2 I was discovered to be the key amino acid responsible for the interaction with Nmi by GST-pull down. Coimmunoprecipitation experiment demonstated 62 nd leucine-deleted UBE2 I mutant can not interact with Nmi. And immunohistochemistry experiment showed that Nmi can not alter the nuclear localization of 62 nd leucine-deleted UBE2 I mutant.Conclusions:Our data show that the 62 nd leucine of UBE2 I was the key amino acid responsible for the interaction with Nmi.