EVIDENCE FOR ACE‐2 UBIQUITINATION AND PROTEASOMAL PROCESSING IN LUNG EPITHELIUM

Background and Aim The angiotensin converting enzyme 2 (ACE‐2) was shown to play a protective role in the fibrogenesis and inflammation of many organs including liver and lung. Previous studies from our lab showed that ACE‐2 is downregulated in fibrotic conditions of the adult and neonatal human lung. The exact mechanisms of ACE‐2 downregulation and protein degradation in lung cells still remain unknown. This study aims to investigate whether ACE‐2 is regulated by ubiquitination/proteasome degradation pathways in the lung. Methods Human lung epithelial cell line (A549) and mouse lung epithelial cell line (MLE12) were cultured and cell lysates were used for immunoprecipitation using ACE‐2 antibodies. The immunoprecipitate was used to detect ubiquitination. Several proteasome inhibitors (Lactacystin, clasto‐Lactacystin β‐lactone, MG132, HMB‐Val‐Ser‐Leu‐VE, and Epoxomicin) were used on MLE12 cell culture to detect the effects on ACE‐2 protein bands by western blotting and to immunoprecipitate ACE‐2 and detect the effect on the ubiquitination of ACE‐2. Several dosing of the proteasome inhibitor MG132 on MLE12 cells and of clasto‐Lactacystin β‐lactone on A549 cells were used to detect changes in ACE‐2 bands by western blotting. Results Immunoprecipitation of ACE‐2 revealed ubiquitination of 3 major bands (150, 45, 40 kDa) detected by anti‐ubiquitin antibodies in MLE12 cells, while ubiquitination of ACE2 was markedly noted in 50 kDa band of A549 cells. On MLE12 cells, all proteasome inhibitors resulted in various degrees of increased total ubiquitinated protein levels. Lactacystin and clasto‐Lactacystin β‐lactone showed increased ACE‐2 isoform band at 70 kDa and were associated with the most increase in total ubiquitinated protein levels, more noted in clasto‐Lactacystin β‐lactone treatment than Lactacystin. Additionally, increasing doses of MG132 treatment on MLE12 cells resulted in a dose dependent increase of the 70 kDa ACE‐2 band with a corresponding decrease in 40 kDa band. Similarly, treatment of A549 cells with increasing doses of clasto‐Lactacystin β‐lactone resulted in a dose dependent decrease in the 50 kDa band. Conclusion These data present the first evidence suggesting ubiquitination and proteasome dependent degradation of ACE‐2 in lung cells in a species‐dependent pattern. Future work will be directed to detect the roles of this ubiquitination/proteasome degradation in fibrotic lung diseases. Support or Funding Information PHS HL‐45136; Department of Pediatrics, Michigan State University