| Aim: To investigate the quality control of both WT-and mutant Optineurin(OPTN)in the cells.The results of this study will help us to elucidate the underling mechanisms of mutant OPTN in different disease by cell biology methods.Methods: HEK293 cells were transfected with GFP-WT-OPTN,GFP-E50K-OPTN and GFP-E478G-OPTN respectively.Twenty-four h later,the cells were treated with 125?g/ml cycloheximide(CHX)for 20 h.The cell lysates were collected at different time periods.And then cells lysates were processed for Western blot analysis with monoclonal mouse anti GFP and GAPDH antibodies.HEK293 cells were transfected as described before.Twenty-four h later,the cells were treated with 10 ?M MG132 or 100 nM Bafilomycin A1(Bafi)for 12 h.The cell lysates were collected and subjected to Western blot using antibodies against GFP and GAPDH.HEK293 cells were transfected as described as before.Twenty-four h after transfection,the cells were incubated with CHX and 10 ?M MG132 or CHX and 100 nM Baf A1(Bafi)for 14 h.The cell lysates were collected at different time periods.Cell lysates were used for Western blot analysis with monoclonal mouse anti GFP and GAPDH antibodies.HEK 293 cells were co-transfected with GFP-WT-OPTN and FLAG or FLAG-Hrd1 or myc-VCP or myc-trim5 or FLAG-RNF6 or FLAG-trim8 or FLAG-trim26.The cell lysates were collected for Western blot analysis with monoclonal mouse against GFP and GAPDH antibodies 24 h after transfection.HEK 293 cells were co-transfected with GFP-WT-OPTN or GFP-E50K-OPTN or GFP-E478G-OPTN and FLAG-Hrd1.Twenty-four h later,the cell lysates were gathered for Western blot analysis using antibodies against GFP and GAPDH.Endogenous Hrd1 were knockdown by using siRNA in HEK 293 cells.Then the cells were transfected with GFP-WT-OPTN or GFP-E50K-OPTN 48 h after knockdown.Twenty-four h later,the cells were added with or without MG132 for 12 h.Finally,the cell lysates were collected for Western blot analysis using antibodies against GFP and GAPDH.HEK 293 cells were co-transfected with GFP-WT-OPTN or GFP-E50K-OPTN or GFP-E478G-OPTN and FLAG or FLAG-Hrd1 or FLAG-Hrd1? ring finger.Twenty-four h later,the cells were fixed.Monoclonal mouse anti FLAG antibody,Alexa Flour-594-conjugated Affinipure Donkey Anti-mouse IgG were used for immunofluorescence.HEK 293 cells were co-transfected with GFP-WT-OPTN and FLAG or FLAG-Hrd1 or FLAG-Hrd1 with GFP-N3.After 24 h,the cell lysates were collected and subjected to immunoprecipitation(IP)and immunoblotted(IB)using antibodies against FLAG,GFP and Ub.HEK 293 cells were co-transfected with GFP-WT-OPTN or GFP-E50K-OPTN or GFP-E478G-OPTN and FLAG-Hrd1 for 24 h.Monoclonal mouse anti ?-tubulin was used for immunofluorescence.HEK 293 cells were transfected with GFP-WT-OPTN.Twenty-four h later,MG132 was added to the cells for 12 h or 10 MG132 plus 1 ?M Nocodazole for 12 h or MG132 for 12 h plus 400 n M TSA for 4h or MG132 for 12 h plus 1 mM EHNA for 4h.Mouse monoclonal anti ?-tubulin antibody,Alexa Flour-594-conjugated Affinipure Donkey Anti-mouse IgG were used for immunofluorescence.The cells were visualized using fluorescence microscopy.HEK 293 cells were transfected with control siRNA or Hrd1 siRNA for 48 h,and then the cells were transfected with GFP-WT-OPTN.Twenty-four h later,cells were incubated with or without MG132 for 12 h.Monoclonal mouse anti ?-tubulin antibody,Alexa Flour-594-conjugated Affinipure Donkey Anti-mouse IgG were used for immunofluorescence.The cells were visualized using fluorescence microscopy.Results: In our observations,the degradation of E50 K was most rapidly degraded among all three of them,while E478 G was the slowest(stable).With the administration of MG132,the protein expression levels were significantly increased in GFP-WT-OPTN and GFP-E50K-OPTN,but not in GFP-E478G-OPTN.In contrast MG132 treatment,a lysosome inhibitor Bafi showed no influence on OPTN degradation.Moreover,the protein degradation rate was all decreased when the cells were treated with MG132 in combination with CHX.Again,Bafi was noneffective.The expression of GFP-WT-OPTN was significantly decreased in the cells co-transfected with E3 ligase FLAG-Hrd1.Hrd1 can promote degradation of GFP-WT-OPTN and GFP-E50K-OPTN,but not GFP-E478G-OPTN.On the contrary,knockdown of Hrd1 may lead to the blockage of OPTN degradation.Hrd1 can co-localized with GFP-WT-OPTN or GFP-E50K-OPTN or GFP-E478G-OPTN.While the mutants of Hrd1 failed to regualte OPTN.Further,co-immunoprecipitation experiments also showeded an association between GFP-WT-OPTN and FLAG-Hrd1 and an enhancement of ubiquitination.Again,FLAG--Hrd1 ?ring-finger lost the ability.In the experiments,we noticed aggresome-like structures in the cells co-transfected with FLAG-Hrd1 and GFP-WT-OPTN or GFP-E478G-OPTN.Preaggresome-like structures appeared in the cells co-transfected with FLAG-Hrd1 and GFP-E50K-OPTN.Meanwhile,the structures were all co-localized with ?-tubulin in cells.HEK293 cells that were co-transfected with GFP-WT-OPTN and FLAG-Hrd1 and incubated with MG132,or MG132 plus Nocodazole(a microtubular depolymerizing agent),or MG132 plus TSA(a potent and specific inhibitor of histone deacetylase),or MG132 plus EHNA,which is a known dynein motor inhibitor showed fewer MG132-induced OPTN aggresome-like structures.In HEK293 cells,WT-OPTN was not co-localized with ?-tubulin either in the cells with knockdown of E3 ligase Hrd1 or not.MG132 treatment led to mature aggresome-like structures in control cells,which was co-localized with ?-tubulin.However,cells with siRNA knockdown of E3 ligase Hrd1 again failed to form a mature aggresome-like structure.Conclusions: WT-OPTN and E50K-OPTN proteins are degraded by UPS(but not autophagy).E3 ligase Hrd1 play an important role in both degradation of WT-OPTN or E50K-OPTN and formation of aggresome-like structures or preaggresome-like structures.While,mutant E478 G proteins are stable and can not be degraded by either UPS or lysosome.Furthermore,E3 ligase Hrd1 may promote E478 G to form a small aggresome-like structures. |
PDF Full Text Request