Characterization Of Specificity And Activity Of Chicken ChUSP1/chUAF1Deubiquitinase Complex

Post-translational modification of proteins and peptides by ubiquitin has emergedas a major regulatory mechanism in various cellular activities. This system isimportant for nearly every aspect of cellular physiology. The ubiquitin specificprocessing proteases (USPs) is known to be the largest family which sharing the mostdiverse structure in deubiquitinating enzymes(DUBs) family. USPs are able to removeubiquitin or ubiquitin chains from target proteins to eliminate or reverse the biologicalfunctions of these ubiquitinated proteins.USP1is a critical regulator of the DNA repair pathways by deubiquitinatingPCNA-ub and FANCD2-Ub (Fanconi Anemia D2) in human cells. UAF1(USP1-associated factor1) was found in cells as immune protein initially, and itsmolecular is about80kDa having highly conservation during evolution. UAF1forms acomplex with USP1(USP1/UAF1) and then increases USP1’s activity. USP1has twonuclear localization signals (NLSs) that mediate the nuclear import of theUSP1/UAF1complex.The amino acid sequence identity between human USP1and chicken USP1is upto72%. USPs harbored two highly conserved active-site motifs, the Cys and Hisboxes, which contain the catalytic cysteine and histidine residues. We have alreadyidentified that chUSP1/chUAF1complex was able to cleave substrates Di-Ub WT（K63）and Di-Ub WT（K48）efficiently. However, the activity and specificity ofchUSP1/chUAF1complex have not been illustrated yet.In current study,we tested the cleavage specificity of purified WT-chUSP1/chUAF1. Under our reaction condition, it was able to efficiently cleave substratesDi-Ub WT（K6）,Di-Ub WT（K11）, Di-Ub WT（K27）, Di-Ub WT（K33）,Di-UbWT（K48）and Di-Ub WT（K63）,but not Di-Ub WT（K29）and Ub-like substrates FAT10,ISG15,Ufm1.The ability to cleave substrates NEDD8was very low.Using Ub-AMC as substrate, kinetic analysis revealed that the activity ofchUSP1GG680,681AA/chUAF1is ten times of the chUSP1CGG91,680,681SAA/chUAF1,thirty-three times of the chUSP1GGD680,681,758AAA/chUAF1, seven times of thechUSP1HGG603,680,681AAA/chUAF1. chUSP1HGG594,680,681AAA/chUAF1and chUSP1CHGG91,594,680,681SAAA/chUAF1showed no deubiquitinating activity.These results suggested the USP1/UAF1complex could efficiently hydrolysis theDi-Ub-chains substrates except for Di-UbWT(K29). The residues onchUSP1,C91,D680,H594,are essential for its deubiquitinating activity. chUAF1could binding to chUSP1in reaction buffer as well as during intracellularcoexpression, and then enhence chUSP1deubiquitinating activivty.