Modulation of Angiotensin Converting Enzyme 2 Activity by Ubiquitination

Abstract

The major roles on the regulation of cardio‐vascular activity of angiotensin II (angII), the main effector hormone of the renin angiotensin system (RAS), were firmly established. These effects are mediated in large part by activation of angiotensin type 1 receptor (AT1R). Angiotensin converting enzyme 2 (ACE2) catalyzes the conversion of angII to angiotensin1‐7 (ang1‐7), which exerts opposite effects to AT1R activation, presumably through MAS1 receptor. Our group previously demonstrated that angII enhanced AT1R/ACE2 interactions which leads to enzyme ubiquitination followed by its degradation into lysosomes. The levels of protein ubiquitination are controlled by an enzymatic cascade involving ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). Ubiquitination can be reversed under the actions of deubiquitinating enzymes (DUBs). In this study, we aimed to characterize the mechanisms modulating ACE2 ubiquitination. Treatment with angII (100 nM for 4h) of ACE2‐transfected HEK293T cells induced a significant decrease in the enzymatic activity (72.21±12.9 % from control n=8), which was reflected in a decrease of its expression levels by 64.45±7.3 (n=3) after 18h. Pretreatment with the effective concentration of PYR‐41, a cell permeable inhibitor of E1 (50 μM for 2h), fully reversed the effects of angII (100.67±23.2% from control, n=9). In contrast, pretreatment with PR‐619, a broad‐spectrum reversible inhibitor of cysteine‐reactive DUBs (20μM, 6h), did not change the effects of angII on ACE2 activity and expression levels (60.93±9.21% from control, n=9). Furthermore, our preliminary proteomic experiments indicated that the expression levels of several E3 ligases were significantly augmented in hypothalami from DOCA‐salt hypertensive mice, including NEDD4 (Neural precursor cell‐Expressed Developmentally Downregulated gene 4). Accordingly, overexpression of NEDD4 in ACE2‐transfected HEK293T cells significantly reduced basal ACE2 activity to 56±7.5 % from control (n=12) and its expression levels (48.97±2.45 %, n=2). Remarkably, angII effects on ACE2 activity and expression levels were eliminated in cells transfected with a catalytically inactive NEDD4 dominant negative mutant (125 ± 5.25%, n=12, and 94.51±15.56, n=2, respectively from control), indicating that angII requires NEDD4 activity to attenuate ACE2 actions. Similarly, angII treatment was unable to change the activity of an ACE2 ubiquitination‐deficient mutant. This mutant also displayed enhanced ACE2 activity in basal conditions (195±4.5%, n=12). In summary, NEDD4 modulates the levels of ACE2 ubiquitination, whereas PYR‐41 prevented the effects of angII on ACE2, indicating that ubiquitination may represent a novel target for the treatment of cardio‐vascular diseases.Support or Funding InformationThis work was supported in part by a Bridge Grant from Howard University to CMF, National Institute of General Medical Sciences P30 GM106392 to JJG and Veterans Affairs Merit Award (BX004294) and LSUHSC‐NO Research Enhancement Program to EL.