The Effect And Mechanism Of Ubiquitination Proteasome Pathway On Centrosome Localization Of IFIT2

Background and purpose:Overexpression of Interferon-Induced protein with Tetratricopeptide repeats 2(IFIT2)leads to prolonged survival of tumor patients.Deletion of IFIT2 gene will lead to EMT transformation and promote cell migration and infiltration.Through previous studies,we found that inhibition of ubiquitination and degradation of IFIT2 protein will lead to the localization of IFIT2 in centrosome.It is speculated that the location of IFIT2 centrosome may affect the function of centrosome,and then affect cell division,apoptosis and tumor metastasis.The purpose of this study was to clarify the relationship between ubiquitination degradation of IFIT2 and its centrosome localization in tumor cells and its mechanism,and to explore the possibility of IFIT2 centrosome localization as a tumor therapeutic target.Materials and methods:1.Determine the effect of ubiquitin proteasome pathway on the localization of IFIT2 centrosome: construct IFIT2 expression plasmids p IRES-EGFP-IFIT2 and p EGFP-IFIT2,transfect them into 293 T cells,treat the cells with proteasome inhibitors and protein ubiquitin inhibitors,detect the localization of IFIT2 and γ-tubulin protein by immunofluorescence,and further observe the movement of IFIT2 in cells after inhibiting ubiquitination and protein degradation by dynamic fluorescence microscope,The ubiquitination of IFIT2 and its interaction with γ-tubulin protein were detected by protein immunoprecipitation technique.2.To determine the mechanism of ubiquitination and degradation of IFIT2:K63R mutant expression plasmid was constructed and transfected into 293 T cells to screen the ubiquitination sites of IFIT2,so as to determine its relationship with the localization of IFIT2 centrosome;Molecular biological techniques such as molecular cloning,RNA interference and overexpression were used to verify the effect of possible E3 ligase on the ubiquitination and stability of IFIT2.3.The interaction between protein and IFIT2 was identified by mass spectrometry;Five truncated IFIT2 protein mutants were constructed.The intracellular localization and molecular weight of different mutants were analyzed by WB and fluorescence,so as to study the effect of protein domain on IFIT2 interaction and function;The identification results of mass spectrometry were analyzed by Western Blot to further verify the IFIT2 interacting protein.Results:1.The role of IFIT2 centrosome localization,ubiquitination sites and mechanism1.1 After cells were treated with IFIT2 aggregation proteasome inhibitor,IFIT2 protein increased and the level of ubiquitination increased significantly,indicating that the ubiquitin proteasome pathway is involved in the degradation of IFIT family proteins.Inhibition of protein degradation pathway can lead to the increase of IFIT2 and promote the aggregation of IFIT2.1.2 Immunofluorescence analysis of the subcellular localization of IFIT2 aggregated bodies showed that a single IFIT2 body could gather around the nucleus,and two bodies were found in metaphase cells,which could be symmetrically distributed on both sides of the equatorial plate of metaphase cells.Protein immunocoprecipitation showed that IFIT2 could interact with γ-Tubulin protein in cells treated with MG132,but IFIT2 cannot interact with γ-Tubulin protein in cells without MG132 treatment.These results suggest that ubiquitin proteasome pathway is involved in the regulation of IFIT2 centrosome localization.1.3 293 T cells transfected with EGFP–IFIT2 were preincubated with different concentrations of colchicine and then retreated with MG132.The data showed that the aggregation of IFIT2 in the centrosome decreased significantly with the increase of colchicine concentration,indicating that the aggregation of IFIT2 to the centrosome depended on microtubule transport.293 T cells transfected with EGFP–IFIT2 were incubated with different doses of ciliobrevin D and then treated with MG132.The results showed that ciliobrevin D reduced the aggregation of IFIT2 mediated by proteasome inhibitors in a dose-dependent manner.It is suggested that the aggregation of IFIT2 to centrosome depends on microtubule transport system.1.4 Construct K63 R mutant,transfect k63 wild-type and k63 r mutant ubiquitin and IFIT2 into 293 T cells,add proteasome inhibitor to inhibit IFIT2 protein degradation,and analyze the effect of k63 mutation on IFIT2 localization.The results showed that IFIT2 protein was dispersed in the cytoplasm without transfection of ubiquitin molecule;When IFIT2 was co-transfected with wild-type ubiquitin,IFIT2 partially aggregated on the centrosome.When IFIT2 and K63 R mutants were cotransfected,the aggregation of IFIT2 in the centrosome increased,while the content of IFIT2 in the cytoplasm decreased significantly.Statistical analysis showed that the diameter of IFIT2 aggregate decreased significantly after K63 R mutation.These results suggested that IFIT2 centrosome aggregation depends on k63 ubiquitination.1.5 293 T cells were transfected with IFIT2,and then treated with MG132(proteasome inhibitor)and MLN4924(neddylation inhibitor)for 24 hours.The results showed that both MG132 and MLN4924 could increase IFIT2.It showed that both ubiquitination modification and neddylation were involved in the ubiquitination degradation of IFIT2.As an inhibitor of neddylation,MLN4924 can inhibit the modification of neural precursor cell-expressed developmentally downregulated 8(NEDD8).The effect of MLN4924 on the expression of IFIT2 induced by ATRA was analyzed.The results showed that inhibiting NEDD8 modification could enhance the expression of IFIT2 induced by all-trans-retinoicacid(ATRA).Based on the inhibition of protein synthesis,the effect of MLN4924 on the degradation rate of IFIT2 was observed.The results showed that the treatment of cells with Cycloheximide(CHX)alone had little effect on the NEDD8 modification of cullin1 protein,while the NEDD8 modification could not be detected after MLN4924 treatment for 2 hours.Inhibiting neddylation could significantly slow down the degradation of IFIT2.The results showed that NEDD8 modification was involved in the ubiquitination and degradation of IFIT2.1.6 Construct sh RNA expression plasmids for NEDD8,NAE1,Cullin1,etc.through the verification of its interference effect,in addition to the poor silencing effect of NAE1,it can effectively silence NEDD8,cullin1 and cullin3.The results showed that Cullin 1 and Cullin 3 had the most obvious inhibitory effect.Silencing cullin1,the results showed that inhibition of cullin1 could significantly up-regulate IFIT2 protein,suggesting that CRL-1 E3 ligase with cullin1 as scaffold may regulate the degradation of IFIT2 protein.1.7 The effect of MLN4924 on acute promyelocytic leukemia(APL)differentiation was analyzed.The results showed that MLN4924 could inhibit the modification of Cullin protein NEDD8.At the cellular level,inhibition of neddylation can inhibit the proliferation of APL cell NB4,induce apoptosis,and block the cell cycle in S phase.The effect of MLN4924 combined with ATRA on APL cell differentiation was further analyzed.The results showed that MLN4924 could induce NB4 cells to express CD11 b and morphologically enhance ATRA induced cell differentiation.At the same time,at the molecular level,MLN4924 can up-regulate the expression of death inducing protein kinase 1(DAPK1),promote the expression and phosphorylation of cell autophagy molecule Beclin1 protein,and then lead to the shear of LC3,which leads to autophagy and finally lead to the differentiation of APL cells.2.Analysis of IFIT2 interacting protein and its involved signal pathway2.1 The lentivirus expression plasmid vector containing SFB tag was successfully constructed,N-SFB-PLVX-IRES-z SGreen and C-SFB-PLVX-IRESz SGreen were constructed,and the polyclonal restriction sites of the plasmid were guaranteed.2.2 Double immunoprecipitation system analysis of IFIT2 interacting proteins:using the method of twice immunoprecipitation,the proteins that specifically interact with IFIT2 were obtained by mass spectrometry,and then 76 interacting proteins were obtained.Through the analysis of their interacting proteins,it was found that IFIT2 interacting proteins are mainly involved in myeloid cell activation,COP9 signal complex,m RNA interacting proteins,interferon regulatory network,etc.These results suggested that IFIT2 can participate in antitumor effects in a variety of ways.2.3 Interaction between IFIT2 and HSP70: Five truncated IFIT2 protein mutants were constructed.The intracellular localization and molecular weight of different mutants were analyzed by Western Blot and fluorescence.Western Blot results showed that all five mutants could express proteins in cells;The results of cell localization showed that all IFIT2 mutants were located in the cytoplasm except the mutants with 3-9TPR deletion,which suggested that the 3-4TPR domain determined the cytoplasmic localization of IFIT2.The identification results of mass spectrometry were analyzed by Western Blot.The results showed that IFIT2 interacted with HSPA1 B,but the interaction between IFIT2 and HSP90 a and HSP90 b was not detected.At the same time,the interaction between HSPA1 B and different IFIT2 mutants was analyzed.The results showed that except for the mutant IFIT2-150 with3-9TPR deletion,other mutants could interact with HSPA1,indicating that the cytoplasmic localization of IFIT2 is very important for the interaction between IFIT2 and HSPA1B.Conclusion:1.Dynein mediated microtubule transport system is involved in protein ubiquitination mediated IFIT2 centrosome aggregation.2.Ubiquitin k63 modification of IFIT2 is an important molecular basis leading to the aggregation of IFIT2 centrosomes.3.Neddylation promotes the ubiquitination and degradation of IFIT2 protein by regulating the activity of ubiquitin-E3 linked enzyme Cullin1.Inhibiting neddylation activity can inhibit the degradation of IFIT2 protein and increase the anti-leukemia effect of IFIT2.4.IFIT2 plays a biological role by interacting with COP9 signal complex,m RNA interacting protein and interferon regulatory network.