Mapping of SUMOylated Proteins in Renal Cortical Collecting Duct (CCD) and Distal Convoluted Tubule (DCT) Cells

Abstract

Kidney epithelial cells are continuously subjected to various forms of cellular stress, including osmotic, oxidative and mechanical stress. To respond and survive during stress, renal cells adapt using various defence mechanisms. One of these mechanisms is proposed to be protein post‐translational modification with the small‐ubiquitin‐related‐modifier (SUMO), which modulates protein and cellular function to counterbalance the subjected stress. In mammals, five SUMO isoforms (SUMO1‐5) are expressed, but their role in kidney function has never been addressed. The aim of this study was to profile SUMO1 and SUMO2 modified proteins in cortical collecting duct (mpkCCD) and distal convoluted tubule (mpkDCT) cells as a precursor to targeted approaches to assess SUMO function. A modified form of SUMO1 or SUMO2, with a histidine tag and a T‐K mutation preceding the carboxyl‐terminal GG motif (position where SUMO attaches to substrates) was expressed in mpkCCD or mpkDCT cells. SUMO levels in cells following heat shock, treatment with deSUMOylation inhibitors, or aldosterone were assessed. Following cell lysis under denaturing conditions, SUMO‐conjugated proteins were purified using the histidine tag, and after cleavage with the endoproteinase Lys‐C, a GG‐specific antibody was used to enrich SUMOylated peptides. Peptides were identified using LC‐MS/MS and analysed using Proteome Discoverer and MaxQuant. 1428 SUMO1 and 1957 SUMO2 modified sites were identified in mpkCCD cells, corresponding to 741 SUMO1 and 971 SUMO2 proteins, respectively. In mpkDCT cells, 248 SUMO1 and 258 SUMO2 modified sites corresponding to 173 SUMO1 and 174 SUMO2 proteins, respectively, were identified. 588 proteins for mpkCCD and 74 proteins for mpkDCT were SUMOylated by both SUMO1 and SUMO2 at the same site. A well established SUMOylation KxE motif and reversed KxD/E‐motif were identified. Although several classes of SUMOylated proteins were highlighted by gene ontology, the majority of SUMOylated proteins in mpkCCD/DCT cells were related to transcription. Furthermore, we determined that the levels of SUMOylation at a specific site on the proton and oligopeptide/antibiotic cotransporter protein, PEPT2, were greatly increased after aldosterone stimulation, suggesting that SUMOylation of proteins in the CCD or DCT is under physiological regulation. In conclusion, we assessed proteome‐wide SUMOylation in renal epithelial cells for the first time. These studies lay the foundation for ongoing work assessing the role of individual SUMOylation sites in protein and kidney function.Support or Funding InformationFunding is provided by the Novo Nordisk Foundation, the Lundbeck Foundation, the Danish Medical Research Council and the Foundation Leducq (to R.A.F). Q.W. is supported by the European Union Horizon 2020 Marie Skłodowska‐Curie Individual Fellowship (Project No. 705682) and the Danish Medical Research Council (Ref. No. 6110‐00118B).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.