The Molecular Mechanism Of Deubiquitylase USP11-mediated Cell Cycle Regulation Via P21 For Proliferation Of Non-small Cell Lung Cancer

USP11 is a member of the ubiquitin-specific proteases?UPS?family of deubiquitinating enzymes.The full length of USP11 contains 920 amino acids.the N-terminal contains the domain present in ubiquitin specific proteases?DUSP?and the UBL domain?UBL?,and the C-terminus is the catalytic domain,which contains the second UBL domain.In cells,USP11 deubiquitinates and stabilizes a variety of substrate proteins,such as BRCA2,ALK5,I?B?,PML,?H2AX,RanBPM,etc.,participating in cell signal transduction regulation,DNA damage repair,and affects cells proliferation and invasion.However,current research indicates that USP11functions differently in different types of tumor cells.In gliomas,USP11 inhibits proliferation,migration and invasion of glioma by deubiquitinating and stabilizing PML.In breast cancer,USP11 promotes the proliferation of breast cancer cells by stabilizing XIAP.However,the relationship between USP11 and non-small cell lung cancer?NSCLC?has not yet been elucidated.p21 is the first well-defined cyclin-dependent kinase inhibitor?CKI?that belongs to the CIP/KIP family and mediates multiple important biological processes such as cell cycle arrest,DNA replication and repair,proliferation and differentiation,senescence and apoptosis.p21 is encoded by the CDKN1A gene and contains 164amino acids.p21 plays an important role in the precise regulation of CDKs activity and in ensuring the orderly operation of the cell cycle,and its expression and protein levels are tightly regulated through transcriptional and post-transcriptional mechanisms.At the transcriptional level,p21 is normally regulated in a p53-dependent and p53-independent manner.In dividing cells,p21 is a very unstable protein with a half-life of only 20-60 minutes,mainly degraded by the ubiquitin-proteasome pathway.Current studies indicate that three E3 ubiquitin ligase complexes including SCF^SKP2,CRL4^CDT2,and APC/C^CDC20 are involved in the degradation of p21 at different cell cycles.In the G1/S phase,SCF^SKP2KP2 promotes ubiquitination of phosphorylated p21 at the Ser130 site by CDK2.In the S phase,CRL4^CDT2DT2 specifically targets p21 that binds to PCNA,resulting in ubiquitination degradation of p21.In the G2/M phase,APC/C^CDC20DC20 recognizes p21 binding to CDK1-cyclin A and CDK1-cyclin B and is responsible for p21 degradation.The SKP2,CDT2,and CDC20 proteins in the E3 ubiquitin ligase complex function as a substrate recognition subunit that acts as a bridge between p21 and other parts of the E3ubiquitin ligase complex.On the other hand,the study also found that cellular multiple proteins can stabilize p21 through different mechanisms,such as p38 and JNK,which promote p21 stabilization by phosphorylating p21;WISp39,hSSB1 and TRIM39 stabilize p21 via protein interaction;Ras stabilizes p21 via promoting the formation of the p21-cyclin D1 complex;Cabels1 promotes p21 stabilization by interfering with the binding of PSMA3 to p21,but these proteins do not have any deubiquitinating enzyme activity and function,and whether there is deubiquitinase having the ability to directly reverse the p21 ubiquitination process remains unclear in cells.In this thesis,p21 was found in the immunoprecipitate of USP11 by mass spectrometry data and database analysis,suggesting that there may be interaction between USP11 and p21.In order to further verify the mass spectrometry results,we used direct immunofluorescence staining,co-immunoprecipitation and direct in vitro protein incubation to demonstrate the direct interaction between USP11 and p21.p21is a very unstable protein that can be ubiquitinated by different E3 ubiquitin ligases and further degraded by the proteasome.Given the characteristics of USP11 as a deubiquitinating enzyme and the interaction between USP11 and p21,the effect of USP11 on the level of p21 was examined.By overexpressing USP11 in A549,HCT116 and HCT116 p53^-/-,the level of endogenous p21 was significantly increased,and inhibiting expression of USP11 can reduce the level of p21,and the regulation of p21 by USP11 was dose-dependent and dependent on the deubiquitinating enzyme activity of USP11.However,alteration of USP11 expression had no effect on p21mRNA levels,suggesting that USP11-mediated-p21 regulation occurs at post-translational modification levels.80%of the protein in the cell is mainly degraded by the proteasome pathway.In order to prove whether USP11 regulates degradation of p21 through ubiquitin-proteasome pathway,p21 ubiquitination degradation is blocked by proteasome-specific inhibitors,and it is demonstrating that p21 is affected by USP11-mediated proteasome pathway.Inhibition of nascent protein synthesis by cycloheximide CHX further revealed that knockdown of endogenous USP11 expression significantly shortened the half-life of p21,while overexpression of wild-type USP11 extended half-life of p21,indicating that USP11 enhances p21protein stability.In vivo and in vitro ubiquitination experiments further demonstrated that USP11 can directly deubiquitinate p21,mainly removing K48-linked polyubiquitin chains.Since p21 is mediated by different E3 ligases in different stages of the cell cycle,in order to clarify the role of USP11 in the cell against which E3ligase of p21.We found USP11 can resist the degradation of p21 by three E3 ligase complexes of SCF^SKP2,CRL4^CDT2DT2 and APC/C^CDC2 through ubiquitination analysis and cell cycle synchronization experiments,indicating that USP11 stabilizes p21 in a cell-cycle independent manner.As a cyclin-dependent kinase inhibitor,p21 mediates the regulation of the G1/S and G2/M phases of the cell cycle.In view of the fact that USP11 can deubiquitinate and stabilize p21,to further prove that USP11 regulates cell cycle progression through p21,we found that in cells with normal expression of p21,inhibiting USP11expression induces a significant increase in the S phase of the cell cycle,whereas in p21-deficient cells,the change in USP11 expression had no significant effect on the cell cycle via cell cycle analysis and cell cycle synchronization experiments.In the event of DNA damage,USP11 regulates cell cycle G2/M phase transition by p21.Taken together,these results reveal that USP11-mediated transition of cell cycle is in a p21-dependent manner.The main function of p21 in the nucleus is to act as a negative regulator of cell cycle,inhibiting cell cycle progression and tumor proliferation.Considering the colocalization of USP11 and p21 in the nucleus of NSCLC cell line A549,the effect of USP11 on the proliferation of NSCLC was examined.Through cell experiments and animal experiments,it was confirmed that USP11 inhibits the proliferation of non-small cell lung cancer by stabilizing p21.Further studies showed that USP11 and p21 expression levels were significantly positively correlated in clinical samples of non-small cell lung cancer.In summary,this study identified for the first time that p21 is an important downstream effector of deubiquitinating enzyme USP11.USP11 can directly interact with p21,deubiquitinate and stabilize p21.It is clarified that USP11 participates in the regulation of G1/S and G2/M phases in the cell cycle through p21,thereby mediating the molecular mechanism of NSCLC cell proliferation.This study further broadens our understanding of cell cycle regulation theory and will provide a scientific basis for targeted therapy and optimized anti-tumor treatments for NSCLC.