C-terminal Modification On PDR1 Resulting In Azole Hypersusceptibility In Candida Glabrata

Objective: This study aimed to analyze the mechanism of decreased expression level of C-terminally 3x FLAG tagged Pdr1,to determine if decreased transcriptional autoregulation is partially involved in reduced gene expression and protein expressiol of pdr1,to investigate if C-terminally3 x FLAG tagged Pdr1 interacts with Gal11 A efficiently,to uncover that if unbiquitin-proteasome system is overactivated in degrading C-terminally3 x FLAG tagged Pdr1,in order to provide a novel therapeutic targets for treating azole-resistant C.glabrata.Methods: In section one,we confirmed the phenotype of C-terminally3 x FLAG tagged Pdr1 with RT-q PCR experiments,spot assay,immunofluorescence,and western blotting assys.To determine if decreased m RNA and protein level of C-terminally tagged Pdr1 is caused by reduced transcriptional autoregulation of Pdr1,we constructed PDRE(in pdr1 gene promoters)knocked out strains,and analyze the phenotype of the mutants with RT-q PCR and spot assay.Further,we carried out Ch IP-q PCR to compare the promoters abundance of C-terminally tagged Pdr1 and N-terminally tagged Pdr1.In addition,with co-immunoprecipitation,we investigated if C-terminally tagged Pdr1 binds with Gal11 A,and with gal11 a knocked strains,we determined if Gal11 A efficiently facilitate C-terminally tagged Pdr1 to activate target genes.In the last section,we used MG132 and cycloheximide to stimulate mutants to understand if C-terminally tagged Pdr1 is degraded abnormally.With Co-immunoprecipitation,we tried to determine if C-terminally tagged Pdr1 binds much more ubiquitin than N-terminally tagged Pdr1.In addition,to investigate if MG132 could also rescue transcriptional activity of C-terminally tagged Pdr1,we carried out immunofluorescence assay,subcellular fractionation,spot assay and RT-q PCR assay.Results: We confirmed in the first section that C-terminally tagging Pdr1 with 3x FLAG strongly reduced pdr1 gene and protein level in either azole-resistant or azole-susceptible strains background,while C-terminally1 x FLAG tagged Pdr1 and N-terminally 3x FLAG tagged Pdr1 functions slightly hyperactive compared to untagged wild type strain.In addition,strains expressing C-terminally 3x FLAG tagged Pdr1 displayed decreased azole-resistance.C-terminally tagged Pdr1 displayed not only decreased transcriptional activity but also cytoplasmic accumulation.In the second section,we confirmed that transcriptional autoregulation of Pdr1 contributes half of m RNA expression of pdr1 gene.With Ch IP analysis,we confirmed that reduced amount of Pdr1 binding to chromation is partially involved in decreased m RNA and protein level of C-terminally tagged Pdr1.In the last section,we confirmed that inhibiting proteasome activity restores protein level of C-terminally tagged Pdr1 to 2-3 fold and it is over binding with ubiquitin via Co-immunoprecipitation experiments,indicating that over-activated ubiquitin-proteasome system participate in decreasing protein level of C-terminally tagged Pdr1.Conclusion: In this study,we found that tagging Pdr1 with 3x FLAG at C-terminus strongly affects its transcriptional autoregualtion,and coactivator Gal11 A could not efficiently facilitate Pdr1 to activating target genes.Besides abnormal function of C-terminus,C-terminally tagged Pdr1 is also over binding with ubiquitin that results in over ubiquitin-proteasome dependent degrading on C-terminally tagged Pdr1 protein.Together,this study presents us a novel therapeutic target for treating azole-resistant C.glabrata.