A Study On The Deubiquitination And Stabilization Of PD-L1 By Deubiquitinase USP22 In Cancer Cells

Research background and purposeIn recent years,the incidence and mortality rate of cancer in China have been increasing year by year,which has become a social problem that can not be ignored.Tumor immunotherapy offers hope to many cancer patients by activating the body's own immune system to kill tumor cells.In the process of tumor development,tumor cells tend to tune the expression of specific proteins on the surface of the plasma membrane,which bind to immunocyte plasma membrane proteins,inhibit the destruction of tumors by immune cells,and promote immune escape.These proteins,which inhibit immune system function,are called immune checkpoints and are important targets for tumor immunotherapy.Inhibitors of immune checkpoint PD-1 and its ligand PD-L1 have been used in the treatment of many cancers with great success.However,the effectiveness of PDL1/PD-1 therapy is only 10%-40%.Among them,the response rate of lung cancer is only about 20%.How to further improve the response rate of PD-L1/PD-1 therapy,so that more patients benefit from it,is currently facing a major challenge.PD-L1 is highly expressed in tumor cells,dentritic cells,macrophages,fibroblasts and many other cells.High expression of PD-L1 in tumor cells is associated with poor prognosis.The high expression of PD-L1 in tumor cells is an influence factor affecting the effect of PD-L1/PD-1 inhibitors,understanding the molecular mechanism of PDL1 expression regulation can help improve the effectiveness of PD-L1/PD-1 therapy.The regulation of PD-L1 can occur at the gene level,transcription level,posttranscription level,post-translation level and so on.In the aspect of post-translation modification,it has been reported that there are many modifications such as glycosylation,phosphorylation,ubiquitinization,palmylization,acetylation,etc.,which can affect the protein level and positioning of PD-L1.At present,the regulatory mechanism of PD-L1 is not clear enough.Therefore,it is necessary to further study and improve its regulatory network.The ubiquitin-proteasome pathway is one of the main ways of protein degradation in cells.Ubiquitin ligase catalize substrates ubiquitination and promote substrate degradation through proteasome.Deubiquitinating enzyme reduces ubiquitination of the substrate,inhibits its degradation and improves the stability of the protein.USP22 belongs to the USP family of deubiquitinase,which was initially found to act as a component of transcriptional co-activation complex SAGA,regulating the mono ubiquitinization of histones,thereby affecting gene expression.It was later found that USP22 could remove polyubiquitination of nuclear and cytoplastic proteins alone and affect the stability of the protein.USP22 played an important role in cell cycle,telomere stabilization,apoptosis,signal transduction,etc.,and was considered as an oncogene.Studies have shown that high expression of USP22 is associated with poor prognosis and tumor metastasis in patients.The COP9 Signalosome(CSN)is a highly conservative protein complex in eukaryote such as animals and plants.The COP9 Signalosome consists of 8 components,CSN1-CSN8.CSN5 contains an MPN domain that contains JAMM motif with isopeptide enzyme activity that removes the neddylation modification of the Cullin E3 Ubiquitin ligase,thereby inhibiting the activity of the Cullin E3 Ubiquitin ligase.At the same time,CSN5 can also hydrolysate the ubibin modification of the substrate,the substrate deubiquitination enhances the stability of the protein.It has been reported that PD-L1 is the substrate protein of CSN5,and CSN5 can reduce the polyubiquitination of PD-L1,stabilize PD-L1 protein,and affect the anti-tumor immune response.CSN5 has a number of binding proteins that stabilize some proteins and make some proteins unstable.Therefore,the role of CSN5 may be more complex and need further study.Scientific problems to be solved(1)Molecular mechanism of USP22 regulating PD-L1(2)The relationship between USP22 and CSN5 in the regulation of PD-L1(3)Does the regulation of USP22 on PD-L1 affect the anti-tumor immune response of immune systemResearch contentsThere is an interaction between USP22 and PD-L1 in tumor cells.Database showed that USP22 may be an interactive protein of PD-L1.The coimmunoprecipitation experiment verified that USP22 can bind to PD-L1.At the same time,the segmented domain experiment shows that USP22 interacted with PD-L1 intracellular domain,while PD-L1 interacted with catalytic domain of USP22.Immunofluorescence experiments have shown that USP22 and PD-L1 co-localized in cytoplasm.USP22 regulates protein levels of PD-L1 in tumor cells.USP22 knock-down does not affect mRNA levels of PD-L1.Immunoblotting experiments show that USP22 knock-down decreases PD-L1 protein level while USP22 overexpression increases PDL1 protein level.Further studies have found that USP22 regulates the stability of the PD-L1 protein.Moreover,USP22 knock-down causing PD-L1 degradation through protease pathways.USP22 can regulate the degradation of PD-L1 proteasome pathway by regulating the protein stability of PD-L1.In tumor cells,USP22 regulates the ubiquitination of PD-L1 intracellular domain through its deubiquitinase activity,which in turn regulates the protein level of PD-L1.Co-immunoprecipitation experiments showed that USP22 overexpression reduced polyubiquitination of PD-L1,and USP22 knock-down enhanced polyubiquitination of PD-L1.The USP22 deubiquitinase activity dead mutat lost the ability to reduce PD-L1 polyubiquitination.USP22 reduces PD-L1 K6,K11,K27,K29,K33,K63-linked ubiquitin chain,and does not remove K48-linked ubiquitin chain.There are 5 lysines in the intracellular domain of PD-L1,all of which mutate into arginine,and the mutant plasmid is used to study the effect of USP22 on PD-L1 deubiquitination.The results showed that USP22 knock-down could not regulate the protein level of the mutant PDL1.USP22 may regulate endoplasmic reticulum PD-L1 in tumor cells.The E3 ubiquitin ligase HRD1,which has been reported in the endoplasmic reticulum-related degradation(ERAD)process,cause PD-L1 ubiquitiantion and affects its degradation.Experiments in this paper show that USP22 inhibits the increase of PD-L1 ubiquitination caused by HRD1.At the same time,USP22 alleviates the decline in PDL1 protein levels caused by HRD1.Glycosylation of PD-L1 does not affect the deubiquitination of PD-L1 by USP22 in tumor cells.It has been reported that the glycosylation modification of PD-L1 inhibits the ubiquitination of PD-L1 and stabilizes PD-L1.The unglycosylated form of PD-L1 can be obtained by mutation of the glycosylation site of PD-L1.Experiments in this paper show that USP22 reduces ubiquitinization of both glycosylated and unglycosylated form of PD-L1.USP22 decreases the polyubiquitination of CSN5 and stabilizes CSN5 protein in tumor cells.It has been reported that the deubiquitinase CSN5 can reduce PD-L1 ubiquitination.This paper proves that USP22 interacts and co-localizes with CSN5 through co-immunoprecipitation experiments and immunofluorescence experiments.USP22 does not affect the mRNA level of CSN5,affects the protein level of CSN5,regulates the stability of CSN5 protein and the degradation of proteasome pathways.Wild-type USP22 instead of catalytic dead USP22 mutant reduces polyubiquitination of CSN5.USP22 remove CSN5 K6,K11,K27,K29,K33,K63-linked ubiquitin chains and does not affect K48-1 inked ubiquitin chains.USP22 and CSN5 cooperatively regulate protein levels of PD-L1 in tumor cells.Experiments in this paper show that USP22 promotes the interaction between PD-L1 and CSN5,while CSN5 promotes the interaction between PD-L1 and USP22.USP22 and CSN5 may enhance the regulatory role of PD-L1 by recruiting each other.Experiments in this paper show that USP22 and CSN5 double knock-down further decrease PD-L1 protein level compared to USP22 or CSN5 sigle knock-down.USP22 affects the PD-L1/PD-1 signaling pathway,alters the activity of T cells,and ultimately regulates the anti-tumor immune response by regulating the protein level of PD-L1 in tumor cells.Flow cytometry experiments showed that USP22 knock-down led to a decrease in the content of PD-L1 in the plasma membrane.Subcutaneous tumor injection experiments showed that USP22 knock-down inhibited tumor growth.immunohistochemical and immunofluorescence experiments revealed that CSN5 and PD-L1 protein level were downregulated,while Tumor infiltrating CD8-T cells was upregulated in USP22 knock-down tumor tissue.T-cell killing experiments show that USP22 knock-down promoted T-cell killing ability towards tumor cells,PD-L1 overexpression can restore T-cell killing ability.The TCGA database shows that USP22 low expression is associated with better prognosis.In order to explore the pathological relationship between USP22 and PD-L1,this paper analyzes the expression of USP22 and PD-L1 in different tumor cell line and patient samples.The protein levels of USP22 and PD-L1 are positively related in tumor cell line and patient samples,providing pathological evidence for USP22 as the regulatory protein of PD-L1.Conclusion(1)USP22 interacts with PD-L1,which can directly regulate the de ubiquitination of PD-L1 and stabilize the protein level of PD-Ll.(2)USP22 interacts with CSN5,a known deubiquitinase of PD-L1,which regulates the deubiquitination of CSN5,stabilizes CSN5 protein and indirectly stabilizes PD-L1 protein level.(3)In USP22 knockdown tumor tissue,PD-L1 protein level was down regulated,and CD8+ T cells were increased,which showed higher anti-tumor immune response.(4)USP22 knockdown promotes T cell killing ability and actrivates T cell immune response.(5)The protein levels of USP22 and PD-L1 were positively correlated in tumor tissues.Research SignificanceThis paper identifies USP22 as a new regulatory protein of PD-L1,reveals the molecular mechanism of USP22 regulating PD-L1,and explores the relationship between USP22 and CSN5.In mice and in mouse tumor cell line,the regulation of USP22 on PD-L1 affects PD-L1-mediated immunosuppression.In patient tumor tissue,USP22 was positively associated with PD-L1 protein levels.In summary,USP22 can be used as a potential target for tumor immunotherapy,and USP22 inhibitors may be able to improve the effectiveness of PD-L1/PD-1 inhibitor.