Nuclear Protein STIP Mediates The Molecμlar Regμlation Of Deubiquitinating Enzyme USP7

STIP is a nuclear phosphoprotein and forms large ploymers with a rod-likeshape when expressed in mammalian cells-stiposome. STIP is expressed in mμltipleorgans of human body, such as kidney, liver, lung, and in various developmentalstages. STIP is highly homologous from C. elegans to human being. Knockdown ofSTIP by RNAi arrested development around the16-cell stage, sμggesting itsessential role in biological evolution. However, there are still plenty that we don’tknow about its function and mechanism in cells so far.USP7is an important member of Ubiquitin-specific proteases (USPs) whichbelongs to cysteine proteases. It is the first substrate-specific ubiquitinase recognizedagainst p53which can bind and deubiquinate p53directly, resμlting in p53’s stability.Meanwhile, USP7can also stabilizes MDM2in the same way which serves as E3ubiquitin ligase in negative regμlation of p53.Structural and biochemicalcharacterization of USP7reveals that the affinity between MDM2and USP7is morethan ten times higher than that of p53,which makes MDM2the preferred substrateunder non stress conditions. Furthermore, USP7also regμlate the activity andfunction of numbers of intracellμlar protein substrates, including the inhibitoryprotein, DNA repair proteins, immune response proteins, viral proteins, andepigenetic regμlators which plays an important role in the pathogenesis anddevelopment of diseases.In the previous study, we have found that STIP prolongs half-life of p53andMDM2by reducing the ubiquitination levels of them, leading toMDM2and p53’sstability, and promotes the growth of tumor cells. Since STIP has no enzyme activityand function, in order to reveal the molecμlar mechanism ofMDM2and p53’sstability mediated by STIP, firstly we analyzed binding partners on the sequence ofSTIP throμgh Bioinformatics method, we found that there are several binding sites ofUSP7on STIP, sμggesting STIP may have an intracellμlar interaction with USP7.Inorder to verify the hypothesis, we used laser scanning confocal microscope,discovered that endogenous STIP co-localized with USP7within stiposome innucleus. Further Co-IP and GST pμll-down experiments makes it clear that STIP andUSP7has a direct interaction within cells. To address the binding site of STIP onUSP7, we constructed various plasmids that contain different domains of USP7. In immunoprecipitations of cells transfected with these plasmids, STIP selectively bindsto the N terminal domain and C terminal domain of USP7.As a deubiquitinase, USP7can not only deubiquitinates and stabilizes p53but also do the same to p53’s negativeregμlator MDM2.In order to determine that STIP stabilizesMDM2and p53throμghUSP7,we utilized strategies such as overexpression of USP7plasmid, knockdown ofUSP7and overexpression of enzyme activity mutative USP7plasmid, demonstratedthat STIP stabilizesMDM2and p53depending on the deubiquitinase activity andfunction of USP7.This study will reveal a new mechanism of stabilizingMDM2andp53, and determine a new molecμlar that mediating USP7’s regμlation onMDM2andp53.This study will not only contribute to the theory of molecμlar regμlation ofMdm2-p53pathway, but will lay a foundation of further research of STIP’s functionin tumor.