Nedd4L Mediates Ubiquitination Of CL^-/HCO₃^- Transporter Pendrin To Induce Pendrin Degradation

Objective:Pre-ubiquitination is a post-translational modification process of a protein widely existing in cells.The ubiquitination process contains a variety of related functional proteins,among which three major proteins are ubiquitin activator E1,ubiquitin-binding enzyme E2 and ubiquitin ligase E3.These three enzymes constitute specific cascade reactions,which link ubiquitin labels to specific substrate proteins and bind to the base of ubiquitin labels.Proteins are recognized by proteasomes or lysosomes and further hydrolyzed into peptide segments or changes in activity.Combined with our research on Nedd4L and bioinformatics website,Nedd4L needs to recognize specific amino acid sequence,which is called PY protein sequence.The sequence consists of PPXY/LPXY,where X represents any amino acid.We search for a number of interested proteins and find out whether there are PY motifs in the amino acid sequence,mainly for renal ion channel proteins.In this study,we validated the ubiquitination of Pendrin mediated by Nedd4L at the molecular level,and constructed the eukaryotic expression vector of Nedd4L.The ubiquitination of Pendrin by Nedd4L was detected in 293T cells,which ultimately led to the change of Pendrin.Research methods:1.Cell culture.293T cells were cultured in DMEM high sugar medium at 37 C and 5.0%CO2.2.Detection of Nedd4L expression and Endrin expression in kidney tissue of mice by WesternBlot3.Detection of intracellular Nedd4L binding with Prin by Co-IP4.Construction of Nedd4L-EGFP eukatyotic expression vector5.Cell transfection.Protein was extracted 72 hours after transfection.The expression of Nedd4L and Pendrin was detected by Western Blot.6.The expression ofNedd4L and Pendrin membrane after transfection were detected by immunofluorescence.7.Detection of in vitro binding by GST-Pull down8.Changes of Pendrin ubiquitmation after Co-IP verification transfectionResult:1.Nedd4L was well exptessed in mouse kidneys atnd 293T cell,and Pendrin was also highly expressed in mouse kidney and 293T cells.2.Co-IP experiments confirm that Nedd4L bbds to Pekirin b vivo3.Petvdrin expression decreased 72 hours after Nedd4L72 transfection.4.Immunofluorescence detection revealed changes of Pendrin membrane location after transfection5.After transfection,CoIP experiment and Western Blot experiment showed that Pendrin's ubiquitbatian increased with the increase of Nedd4L expression.CONCLUSION:(1)Pendrin interacts with Nedd4L in 293T cells(2)Nedd4L can make Pendrin ubiquitinate.(3)Nedd4L can increase Pedrin ubiquitinate expression and decrease Pendrin expression.(4)Nedd4L mediated Pendrin ubiquitination d decrease Pendrin expression.(4)Nedd4L mediated Pendrin ubiquitination odification can change the.