| Object iveArtemisitene(ATT)activates the nuclear factor(erythroid-derived 2)-like 2(Nrf2)by increasing its stabilization and reducing ubiquitination.The cysteine(Cys)residues of the cytosolic Nrf2 repressor Kelch-like ECH-associated protein-1(Keapl)function as redox sensors,and may be crucial in activating Nrf2.Methods1.Cells were treated with different concentrations of artemisitene(0μM,1μM,4μM,7μM,10μM)for 16 hours with Keap 1-deficient cell lines and tBHQ 50μM as positive control.The protein levels of Nrf2 were detected by Western blotting.The Keapl genotype was knocked out in MDA-MB231 cell line.The wild type of MDA-MB231 cell was used as negative control.After treatment with 5μM artemisinin for 16 hours,the levels of Nrf2 and HO-1 were detected by Western blotting.2.Keap1 was overexpressed in Cos-1 cells.Artemisine at different concentrations(0μM,1μM,4μM,7μM,10μM)was used to treat the cells for 16 hours.Protein was collected from lysed cells and the level of Keapl was detected by Western blotting.Then,we constructed the Keapl vector of unit point mutation,and sequenced and compared the cysteine(-77,-151,-257,-273,-288 and-297)of Keapl mutation,then expanded the culture and extracted the plasmid.After drug treatment,lysed cells were detected for the next step.3.Cells expressing wild-type or cys151 ser-keapl were treated with artemisitene,cycloheximide was added,and cells were harvested at different time points for immunoblotting to determine the half-life of endogenous Nrf2 protein.MDA-MB-231 cells were co-transfected with Keapl-C151S/Keap1,Nrf2 and hemagglutinin ubiquitin,treat with artemisitene.Cell lysates were collected for co-immunoprecipitation to detect ubiquitination levels.4.After 16 hours of artemisitene treatment,the supernatant of lysed cells was collected and the expression of Nrf2 and HO-1 was detected by Western blot.The transcriptional activity of Nrf2 was detected by Luciferase.Results1.Using tBHQ as positive control,A549 cells with keap1 deletion and MB231 cells with keapl knockout treated with artemisitene at different concentrations showed no significant difference in Nrf2 protein level in A549 cells,and MB231 cells with keapl knockout treated with artemisitene also failed to enhance Nrf2 level and the transcription level of downstream genes.Artemisitene activate the Nrf2 pathway dependent on Keap1.2.When Cos-1 cells were treated with artemisitene,the higher the concentration was,the higher the gray value was,at 130kD,and the lower the gray value was,at 68kD.Six homocysteine sites were detected on Keap1 by site-directed mutagenesis.After artemisitene treatment,only 68kD bands were found in cells transfected with cysteine 151,but no 130kD bands were found.Artemisinin activates the Nrf2 pathway in a concentration-dependent manner and is associated with Keap1 151 cysteine.3.The wild type group without artemisitene treatment and transfected keapl-cys151 treated with artemisitene did not extend the half-life of Nrf2 protein.The immunco-precipitation results showed that the wild-type Keapl non-drug treated group was ubiquitinated,while the wild-type cells treated with artemisitene were not ubiquitinated with Nrf2,artemisitene had no effect on the ubiquitination status of Nrf2 in the keap1-c151s histone lysate treated with or without artemisitene.Artemisinin reduces the ubiquitin stability of nrf2-dependent Keap1 151cysteine.4.COS-1 cells were co-transfected with WT/C 151s-Keapl,Nrf2,renilla and luciferase plasmids and treated with artemisitene.Compared with cells expressing wt-keapl,cells expressing keapl-c151s showed reduced nrf2-dependent transcription activity,indicating that the mutant keap1-c151 lost its activated phenotype.artemisitene activates the Nrf2 signaling pathway through keap1-cys151.ConclusionOnly the Cys151Ser mutant prevented ATT-mediated activation of Nrf2.It is indicating that the Cys151 residue of Keapl likely interacts with ATT,and is essential in Nrf2 stabilization and the transcription of downstream genes. |
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