Ubiquitin-like Protein Nedd8 Regulates The Fuction Of PDX-1

PDX-1(pancreatic duodenal homobox-1),one of the most important transcriptional factors regulating the synthesis and secretion of insulin,is involved in maintaining the function of mature pancreatic β cells.The protein level of PDX-1 is modulated at both transcriptional and post-translational levels in order to maintain a high level of PDX-1 expression in mature β cells.Under oxidative stress,PDX-1 is rapidly degraded via the ubiquitin-proteasome pathway,resulting in pancreatic β cell impairment.It has been reported that the Cullin3-pcif1 complex is an E3 ubiqintin ligase of PDX-1.However,which signaling contributes to activating this complex is still far from known.Nedd8 is an ubiquitin-like protein and functions as neddylation modification of target proteins.The substrates of Nedd8 are mainly Cullin family members including Cullin3.Moreover,neddylation conjugated Cullins can be activated as the E3 ubiquitin-ligase.This study is aim to explore whether Nedd8 is the key factor leading to PDX-1 degradation induced by cullin3-pcif1 complex.Our results demonstrated that the level of neddylation enhanced significantly with palm and IL-1β treatment in INS-1 cells,whereas the protein level of PDX-1 decreased relative to that of control.Transiently transfected with Nedd8 resulted in an increase of neddylation modification and a decrease of PDX-1 protein level.The protein level of PDX-1 and the degree of Neddylation was opposite trend.Nedd8 overexpression did not affect the gene expression level of PDX-1.CHX was introduced to block the de novo protein synthesis in INS-1 cells and we found that enhanced neddylation modification by Nedd8 overexpression reduced of the protein stability of PDX-1.Using MG132 to inhibit ubiquitin-proteasome led to the recovery of PDX-1 protein level,suggesting that the PDX-1 protein degradation induced by Nedd8 was associate with ubiquitin-proteasome pathway.On the contrary,treatment with MLN4924,an inhibitor of Nedd8 activator enzyme,increased PDX-1 protein level in INS-1 cells.In order to clarify the relationship between Nedd8 and PDX-1,we used the Laser scanning confocal microscope and co-immunoprecipitation(CO-IP)technologies.The results showed that Nedd8 and PDX-1 were colocalized and interacted with each other.Luciferase reporter gene assay revealed that overexpression of Nedd8 inhibited transcriptional activity of PDX-1,and mainly reduced its binding ability to insulin promoter A3/A4 region.Quantitative RT-PCR experiments showed that enhanced neddylation modification was able to decrease the transcriptional level of insulin.Potassium-stimulated insulin secretion assay(KSIS)further showed that Nedd8 reduced the basal insulin secretion as well as insulin content.In summary,this study demonstrated that Nedd8 was capable to regulate the degree of PDX-1 protein degradation via the ubiquitin-proteasome pathway.Oxidative stress triggered Nedd8 conjugating to Cullin3-pcif1 complex which functioned as an activated E3 ubiquitin ligase to accelerate the degradation of PDX-1 protein,thereby inhibiting insulin gene transcription,reducing the basal insulin secretion and insulin content in pancreatic β cells.Simultaneously,PDX-1 protein level enhanced after inhibiting cellular neddylation level.Take together,this study makes sense to further explore the mediation of PDX-1 by Nedd8 under physiological as well as pathological condition,offering potential targets for prevention and treatment of diabetes.