Interaction Of The E3 Ubiquitin Ligase HRD1 And Renal Epithelial Sodium Channel In Mammalian Cells

The renal epithelial sodium channel(ENaC)is composed of three homologous subunits,located on the membrane of polarized cells,maintaining body salt and water homeostasis.The dysfunction of ENaC caused by it’s trafficking,degradation and regulatory hurdles can affect normal physiologic function seriously.E3 ubiquitin ligase plays an important role in protein degradation,by which involve in many of physiological process in the cell.In recent years,most of researches on ENaC demonstrated that it’s degradation rely on Endoplasmic reticulum associated protein degradation(ERAD).HMG-CoA reductase degradation protein 1(HRD1)is an E3 ubiquitin ligase that located on Endoplasmic reticulum.According to the research of Teresa M.Buck et al,the E3 ubiquitin ligase Hrd1 was identified to involve in the ubiquitin degradation of ENaC in yeast(Mol Biol Cell.2010;21:1047-1058).We will figure out whether the E3 ubiquitin ligase HRD1 plays an important role in ENaC ubiquitylation in the mammalian cells.Firstly,we used MG132 to inhibit proteasome in mCCD cells and detected the level of α-ENaC protein.We found that α-ENaC expression significantly raised in mCCD cells treated with MG132.These findings suggested that α-ENaC obviously was regulated by ubiquitin-proteasome pathway.Then,we overexpressed HRD1 andα-ENaC in HEK293T cells,overexpressed HRD1 in mCCD only and down regulated HRD1 in HepG2 with si-HRD1 RNA.And we found overexpression or downregulation of HRD1 had no any effects on the protein level of α-ENaC.These data indicated that the E3 ubiquitin ligase HRD1 may not involve in the degradation of α-ENaC.Mass spectrometry was used to screen the binding proteins of α-ENaC in HEK293T cells.The results indicated there are many proteins have contact withα-ENaC other than HRD1.Therefore,the data aboved suggested that HRD1 may be not the E3 ubiquitin ligase of a-ENaC for its ubiquitylation.Then,we did co-transfection in HEK293T cells with HRD1 and a-ENaC plasmids,and used immunofluorescence assay to figure out the sub-localization of HRD1 and α-ENaC in HEK293T cells.To our surprise,the results indicated that HRD1 maybe co-localized with a-ENaC.In order to further confirm this result,co-immunoprecipitations were performed using cell lysates from HEK293T cells with HRD1 and α-ENaC in HEK293T at the same time transfection,or mCCD cell transfected with HRD1.In the result,we found HRD1 and α-ENaC couldn’t be pulled down by each other.The experiment of Co-Immunoprecipitation demonstrated HRD1 and a-ENaC have no interaction in mammalian cells.We also finished the cycloheximide experiment to detect a-ENaC expression at different times in mCCD cells overexpressed HRD1.We found that HRD1 overexpressed couldn’t promote the degradation of a-ENaC protein,compared with control group.Thus,these findings demonstrated that HRD1 don’t participate in the ubiquitylation of a-ENaC protein in mammalian cells.In summary,there was no interaction between HRD1 and α-ENaC in mammalian cells.HRD1 may be not an E3 ubiquitin ligase which was involved inα-ENaC ubiquitylation.Our studies also indicated that the system of the biogenesis and degradation of protein in mammalian was quite different from yeast system.