ACE2 Internalization and Degradation is Controlled by Ubiquitin Ligase NEDD4

Abstract

Angiotensin converting enzyme 2 (ACE2) catalyzes the conversion of Angiotensin (Ang)‐II to Ang‐(1–7), attenuating the deleterious effects of angiotensin type 1 receptor (AT1R) activation on the cardiovascular system. We previously demonstrated that increased Ang‐II levels enhance the interactions between ACE2 and AT1R followed by enzyme degradation into lysosomes, leading to a diminished compensatory activity against an overactive renin‐angiotensin system (RAS). A major mechanism known to mediate plasma membrane protein internalization is ubiquitination which involves the addition of ubiquitin moiety to lysine residues. We have shown that Ang‐II treatment increases the levels of ACE2 ubiquitination and that reduction of the enzyme activity by Ang‐II is abolished in an ACE2 mutant lacking intracellular lysine residues. Ubiquitin is attached under the sequential action of three types of enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). The function of these enzymes in cardiovascular diseases, particularly in hypertension, were not studied until now.To gain insight in the mechanisms regulating ACE2 ubiquitination, we performed a quantitative discovery‐based proteomic experiment in hypothalami from control and DOCA‐salt‐treated hypertensive mice using Tandem Mass Tags (TMTs). We found that the expression of several E3 ligases was significantly augmented in hypertensive animals. These ligases included NEDD4 (Neural precursor cell‐Expressed Developmentally Downregulated gene 4) and HECW2 (C2 and WW domain containing E3 ubiquitin protein ligase 2). To assess the clinical significance of these findings, we tested the effects of NEDD4 overexpression on ACE2 expression and activity in HEK293T cells. Increased NEDD4 cellular levels significantly reduced basal ACE2 activity to 56 ± 7.5 % from control (n=12). Interestingly, in NEDD4 overexpressing cells, Ang‐II treatment (100 nM, 4h) failed to further reduce ACE2 activity, indicating that NEDD4‐mediated ACE2 ubiquitination can be the primary mechanism mobilized by the overactive RAS to reduce ACE2 compensatory activity. In contrast, no changes in ACE2 activity in basal conditions or after Ang‐II treatment were observed in HEK293T cells transfected with a catalytically inactive NEDD4 dominant negative mutant (125 ± 5.25 %, n=12). Supporting these results, western blot analysis demonstrated a significant decrease of ACE2 cellular levels in NEDD4 overexpressing cells (48.97 ± 2.45 % of control).These results are the first demonstration of a role of E3 ligases, particularly NEDD4, on ACE2 ubiquitination and provide a new target to preserve ACE2 compensatory activity during the development of hypertension.Support or Funding InformationSupported by Veterans Affairs (Merit Award 1I01 BX004294‐01 to EL and CMF), NHLBI (HL093178 to EL), NCRR (RR018766‐09 to JJG) and NIGMS (GM103514 and GM106392 to EL and JJG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.