Function And Mechanism Of The Modification Of Ubiquitin Ligase Smurf2 By Ubiquitin-Like Protein Nedd8

Nedd8 is a ubiquitin-like protein and neddylation is a posttranslational modification play an important role in the life-cycle.Nedd8 is highly homologous to ubiquitin in amino acid sequence and protein structure.The early study of Nedd8 found Nedd8 was highest in the early mouse embryo and was developmentally down-regulated in the mouse embryo of brain.In current study,Nedd8 is positively correlative with tumorigenesis.Unlike ubiquitin which was equally expression in the nucleus and cytosol,Nedd8 is highly enrich in the nuclear and is weaker in the cytosol.Neddylation regulate ubiquitination though dual direction regulation.When the Cullin-RING is in the neddylation stage,the structure of Cullin-RING will change to facilitate recruitment of E2.This indicate that neddylation is essential for Cullin-RING ligases and promotes ubiquitination.However,when the neddylation site compete with Ubiquitination site in certain substrates,neddylation will inhibit the ubiquitin congection.Currently,all reported Nedd8 E3 enzymes can also function as ubiquitin E3 enzymes.The best-studed Nedd8 ligase are RBXs,a component of the complex of Ubiquitin ligase Culin-RING E3.In recent study,the most reported non Cullin-RING Nedd8 E3 enzymes include MDM2,Smurf1,ITCH and NEDL2.Smurf2 is highly homologous to Smurf1,they are one of the member of HECT Nedd4-like ubiquitin ligases.There are some difference in crystal structure between Smurf1 and Smurf2.Smurf1 have two WW domains,and Smurf2 have three WW domains.They have functional redundancy and substrate specificity,because of the difference and similarity between Smurf1 and Smurf2.For example,Smurf1 promotes tumor progression,while Smurf2 inhibits tumor development in early-stage,while promotes tumor progression in later-stage.The neddylation of Smurf1 gives us a clue that Smurf2 may be a Nedd8 E3 ligase.Neddylation is a new modification of Smurf2.We find that Smurf2 could be modified by Nedd8 in HCT116 cell line which highly expresses Nedd8.We use MLN4924 and deneddylation enzyme which could inhabit Neddylation to conform the result.Although both Smurf1 and Smurf2 could be modified by Nedd8,Our study find that Smurf2 is different from Smurf1 in the Nedd8-modification.We can not find active site of Nedd8 ligase in HECT domain.Multiple sequence alignment of the HECT family indicates that Nedd8-ligase active site is non-conserve in the family.All Smurf2’s domains could be modified by Nedd8,when Nedd8 was over expressed in cell.Previous study have demonstrated that c2 domain could interact with HECT domain.We find Smurf1 and Smurf2 directly interact in vitro.The interaction between Smurf1 and Smurf2 may enhance Smurf2 neddylation.Because of Smurf2 neddylation,the protein level is downregulated and the Smurf2 ubiquitination is upregulated.We also find that Nedd8 promote degredation of Smad3 and RNF20 in protein levels.The study show some similarity and also difference between Smurf1 and Smurf2.A new modification of Smurf2 could be enhanced by its homologous protein Smurf1.This will help us to understand homologous protein in modification level.When design a target drug of Smurf1 or Smurf2,we should think about the drug target validation and identification of secondary drug target effects.