The Mechanism Through Which Neddylation-mediated Nedd4-2 Activation Regulates NBCe1 Ubiquitination In Kidney

Introduction:Solute Carrier?SLC,Solute Carrier?family is the body's largest transporter family.SLC4 is the most important subfamily of SLC,which is in charge of bicarbonate transport in the body.NBCe1 is an important member of SLC4 family,which is encoded by SLC4A4 gene and mainly expressed in the basolateral membrane of renal proximal tubules.NBCe1 plays a vital role in the reabsorption of HCO3^–in renal tubules.The abnormal function of NBCe1 can lead to severe HCO3^–reabsorption disorder and cause renal tubular acidosis,which is characterized by low concentration of HCO3^–in the blood and decreased pH value.It has been reported that the NBCe1 mutation not only causes low blood pH value in patients,which is manifested as severe acidosis,but also presents abnormalities such as short stature,cataracts,migraines and mental retardation.Other studies have shown that NBCe1 knockout mice also exhibit very severe renal tubular acidosis and other abnormalities.It can be seen that NBCe1 has very important physiological functions,and its abnormality seriously affects human health.Therefore,it is of great significance to study the regulation of expression of NBCe1.But at present,there are few reports about it.Ubiquitination is an important post-translational modification of proteins.Proteins undergoing ubiquitination modification can be recognized and degraded by 26S proteasomes,indicating that ubiquitination modification plays an important role in regulating protein expression.Ubiquitin E3 ligase plays a key role in the process of ubiquitination by specific recognition of target proteins.As an important member of the Nedd4 family,Nedd4-2 is an important ubiquitin E3 ligase.Studies have confirmed that the key ion channel proteins in the kidney,such as ENaC,NCC and NKCC2,are all the target proteins of Nedd4-2,among which ENaC is the most clearly studied.Nedd4-2 plays a key role in the regulation of water electrolyte balance by mediating ubiquitination modification of the above proteins and affecting their protein expression.But as an important ubiquitin E3 ligase,Nedd4-2 does much more than that.Therefore,identification of the new Nedd4-2 target protein will help to discover the new function of Nedd4-2 in the kidney.Through bioinformatics prediction,we found that there were motifs in the protein sequence of NBCe1 that could interact with Nedd4-2.At the same time,we searched the UbiBrowser site for the ubiquitin E3 ligase of NBCe1,and the highest ranked and confidence one was Nedd4-2.Based on these evidences,we speculate that Nedd4-2 may interact with NBCe1 and mediate its ubiquitination and degradation as its E3 ligase.It is also noteworthy that as a ubiquitin E3 ligase,the regulation of activity and expression for Nedd4-2 itself will have an important impact on its function,and the mechanism of its regulation is still unclear.Neddylation is a kind of ubiquitin-like modification mediated by a cascade of three nedd8-specific enzymes.Studies have shown that the ubiquitin E3 ligases of the HECT family,Smurf1 and Smurf2,will undergo Neddylation modification,and Neddylation modification may affect the activity of the ubiquitin E3 ligases,and then affect the ubiquitination level of their downstream target proteins.Since Nedd4-2 also contains the HECT domain,we speculated that Nedd4-2 may also undergo Neddylation modification.The interaction between Nedd4-2 and Nedd8 was found in our preliminary experiment,which supported our above hypothesis.Therefore,we speculated that neddylation modification could activate Nedd4-2,and then affect the ubiquitination modification of target protein NBCe1 and its protein expression level.In order to verify the above hypothesis,we will first study the regulation of NBCe1 by Nedd4-2 and the underlying mechanisms.Then we will further study whether neddylation might regulate the protein level of NBCe1 via Nedd4-2.This study would find a new regulatory pathway:activation of Nedd4-2 mediated by neddylation modification would increase the ubiquitination level of NBCe1 and lead to the reduction of the protein level.The confirmation of this pathway will provide an important scientific basis for the in-depth study of the regulation of acid-base balance in the kidney,also provide new targets for drug development of related diseases.Materials and methods:1.Materials:Mouse kidney M1 cell line,recombinant protein GST-Nedd4-2,His-NBCe1,Conditional gene knockout mice line Nedd4-2fl/fl;Ksp1.3?C57B6J?,in vitr o ubiquitination kit,in vitro neddylation kit,expression vectors of NBCe1,Nedd4-2 and Nedd8,Nedd4-2 shRNA vector,related antibodies,etc.2.Methods:cell culture,transient gene transfection method,Real-time quantitative PCR assay,Western Blot assay,Co-immunoprecipitation experiment,Protein purification experiment,GST pull-down assay,in vitro ubiquitination assay,in vitro neddylation assay and statistical analysis.Results:1.The results of Co-IP experiments showed that Nedd4-2 could interact with NBCe1,and NBCe1 could interact with ubiquitin as well.The result of GST-Pulldown experiment showed that there is a direct interaction between Nedd4-2 and NBCe1.In vivo and in vitro experiments suggested that Nedd4-2 could interact with NBCe1 and lead to ubiquitination of NBCe1.2.M1 cells were treated with proteasome inhibitor MG132.Western Blot results showed that NBCe1 expression was up-regulated?P<0.05?,indicating that NBCe1 may be regulated by ubiquitination.The result of in vitro ubiquitination assay further proved that Nedd4-2 can mediate the ubiquitination modification of NBCe1 as its ubiquitin E3 ligase.3.When Nedd4-2 was overexpressed in mouse kidney M1 cells,Western Blot results showed that compared with the control group,the expression level of NBCe1 is significantly reduced?P<0.05?.MG132 treatment could restore the decreased expression of NBCe1 caused by Nedd4-2 overexpression?P<0.05?;Knockdown of Nedd4-2significantly increased NBCe1 expression?P<0.05?.4.When Nedd4-2 was overexpressed in M1 cells,Western Blot results showed that compared with the control group,the abundance of NBCe1 protein in the membrane of M1 cells decreased significantly?P<0.05?,while the abundance of cytosolic NBCe1 protein increased significantly?P<0.05?.5.Total protein of kidneys in Nedd4-2 knockout mice and control mice were extracted and subjected to Western Blot experiments.The results showed that compared with the control group of Nedd4-2^fl/fl mice,the expression of NBCe1 was significantly up-regulated in Nedd4-2^fl/fl;Ksp1.3 mice?P<0.05?,which is consistent with the results obtained at the cellular level after knocking down Nedd4-2.6.The ubiquitination level of NBCe1 was detected by Co-IP experiments,and the results showed that compared with control group Nedd4-2^fl/+;Ksp1.3 and Ksp1.3-Cre mice,the ubiquitination level of NBCe1 was downregulated in kidneys of Nedd4-2^fl/fl;Ksp1.3 mice.7.The total protein of mouse kidney M1 cells was extracted for Co-IP experiments,and the results showed that Nedd4-2 could interact with Nedd8.The results of in vitro neddylation assay showed that Nedd8 could interact with Nedd4-2 directly;compared with the control group,the binding capacity of Nedd8 and Nedd4-2 was enhanced obviously.8.The results of real-time PCR showed that mRNA level of NBCe1 was not significantly changed when Nedd8 was overexpressed in mouse kidney M1 cells,compared with the control group.9.The results of Western Blot experiments showed that after overexpressing Nedd8,the protein level of NBCe1 was significantly down-regulated?P<0.05?,and the expression level of ENaC as a positive control was also significantly down-regulated?P<0.05?.When M1 cells were co-transfected with Nedd8 expression vector and Nedd4-2 interference vector,the expression level of NBCe1 and ENaC were significantly up-regulated?P<0.05?.10.The results of Western Blot showed that the protein level of NBCe1 in M1 cells was up-regulated after stimulation by the neddylation E1?NAE?inhibitor MLN4924?P<0.05?,the expression of ENaC was also significantly up-regulated?P<0.05?.Conclusions:1.Nedd4-2 is the ubiquitin E3 ligase of NBCe1,resulting in its ubiquitination and degradation.2.The expression level of NBCe1 was significantly up-regulated,while the ubiquitination level of NBCe1 was down-regulated in the kidney of Nedd4-2^fl/fl;Ksp1.3 knockout mice.3.The ubiquitin E3 ligase Nedd4-2 can be modified by neddylation,and neddylation modification could activate Nedd4-2 to regulate the degradation of NBCe1.