Establishment Of Screening Method For Small Molecule Inhibitors Of USP7-MDM2 Interaction And Mechanism Studies Of USP7 On LSD1

Deubiquitination is the process that DUBs hydrolyze the thioester bond between ubiquitin and protein and then remove the ubiquitin molecules from substrates,so as to protect substrate from proteasome hydrolysis or regulate subcellular localization or activity of substrate.USP7 is a member of the ubiquitin specific protease family,which is a cysteine protease.USP 7 can regulate the expression and activity of many substrates in cells and plays an important role in the development of tumors.However,the mechanism of USP7 in gastric cancer is not clear and needs further study.MDM2 is one of the substrates of USP7,and it is also a carcinogenic protein and E3 ligase,which promotes the degradation of tumor suppressor p53.Therefore,the first part of this project aims to establish an inhibitor screening method based on the interaction between USP7 and MDM2,in order to screen inhibitors that can block the interaction between USP7 and MDM2,so as to reduce the expression of MDM2 protein,and then increase the level of p53 in cells.Finally,tumor growth can be inhibited.In addition,as an important tumor target,LSD 1 is also one of the substrates of USP7.USP7 can stabilize LSD1.Therefore,the second part of this project focuses on the regulatory mechanism of USP7 on LSD1.The main research contents of this project include:(1)Discovery of USP7 related proteins based on tissue microarray technology and related databasesIn this study,the expression of USP7 in cancer tissues was significantly higher than that in adjacent normal tissues was found through analysis of tissue microarray combined with immunohistochemistry results.Furthermore,through the Kaplan Meier survival analysis website,we found that the overall survival of gastric cancer patients with high expression of USP7 or MDM2 was significantly shorter than that of patients with low expression.In addition,there was a significant positive correlation between the expression of USP7 and LSD1 in gastric cancer(r=0.302,P<0.0001).At last,we found that USP7 and LSD1 can interact with each other based on BioGRID 3.5 database.(2)Establishment of screening method for small molecule inhibitors of USP7-MDM2 interactionThe N-terminal of USP7 and MDM2 prokaryotic expression vectors were constructed respectively,and the proteins were purified by affinity chromatography.The recombinant protein of USP7 and MDM2 with high purity were obtained.HTRF technology was used to successfully establish the screening method for inhibitors of the interaction between USP7 and MDM2.(3)Study on the regulation mechanism of USP7 on LSD 1In this study,we found that the expression of USP7 was positively correlated with the expression of LSD1 in tissues with high AR expression(r=0.394,P=0.0001).At the cellular level,USP7 can interact with the Tower and AOL domains of LSD1,and maintain the stability of LSD 1 in a concentration dependent manner in gastric cancer cells.Further mechanism studies show that USP7 can significantly prolong the half-life of LSD 1 by removing polyubiquitin of LSD 1.Finally,it was confirmed that USP7 could remove LSD1 ubiquitination in an AR dependent manner and maintain the stability of LSD 1 in gastric cancer cells.In conclusion,the screening method for inhibitors of USP7-MDM2 interaction based on HTRF technology was established for the first time.It was found that USP7 could regulate LSD1 and maintain the stability of LSD 1 in an AR dependent manner.The results are of great significance for the future clinical treatment of gastric cancer patients.