MDM2-Mediated Ubiquitination of Angiotensin-Converting Enzyme 2 Contributes to the Development of Pulmonary Arterial Hypertension

Abstract

Background: Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II (Ang II), a potent vasoconstrictor, to Ang 1-7 and is also a membrane protein that enables COVID-19 infectivity. AMP-activated protein kinase (AMPK) phosphorylation of ACE2 enhances ACE2 stability. This mode of post-translational modification (PTM) of ACE2 in vascular endothelial cells (ECs) is causative of a pulmonary hypertension (PH)-protective phenotype. The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that ubiquitinates its substrates to cause their degradation. In this study, we investigated whether MDM2 is involved in the PTM of ACE2 via its ubiquitination of ACE2, and whether an AMPK and MDM2 crosstalk regulates the pathogenesis of PH. Methods: Bioinformatic analyses were used to explore E3 ligase that ubiquitinates ACE2. Cultured endothelial cells, mouse models, and specimens from patients with idiopathic pulmonary arterial hypertension were used to investigate the crosstalk between AMPK and MDM2 in regulating ACE2 phosphorylation and ubiquitination in the context of PH. Results: Levels of MDM2 were increased and those of ACE2 decreased in lung tissues or pulmonary arterial endothelial cells from patients with idiopathic pulmonary arterial hypertension and rodent models of experimental PH. MDM2 inhibition by JNJ-165 reversed the SU5416/hypoxia-induced PH in C57BL/6 mice. ACE2-S680L mice (dephosphorylation at S680) showed PH susceptibility, and ectopic expression of ACE2-S680L/K788R (deubiquitination at K788) reduced experimental PH. Moreover, ACE2-K788R overexpression in mice with endothelial cell-specific AMPKα2 knockout mitigated PH. Conclusions: Maladapted PTM (phosphorylation and ubiquitination) of ACE2 at Ser-680 and Lys-788 is involved in the pathogenesis of pulmonary arterial hypertension and experimental PH. Thus, a combined intervention of AMPK and MDM2 in the pulmonary endothelium might be therapeutically effective in PH treatment.

SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2

“…It appears that S protein in ECs increases redox stress which may lead to AMPK deactivation, MDM2 upregulation, and ultimately ACE2 destabilization. 4 Although these findings need to be confirmed with the SARS-CoV-2 virus in the future study, it seems paradoxical that ACE2 reduction by S protein would decrease the virus infectivity, thereby protecting endothelium. However, a dysregulated renin-angiotensin system due to ACE2 reduction may exacerbate endothelial dysfunction, leading to endotheliitis.…”

“…AMPK (AMP-activated protein kinase) phosphorylates ACE2 Ser-680, MDM2 (murine double minute 2) ubiquitinates ACE2 Lys-788, and crosstalk between AMPK and MDM2 determines the ACE2 level. 4 In the damaged lungs, levels of pAMPK (phospho-AMPK), pACE2 (phospho-ACE2), and ACE2 decreased but those of MDM2 increased (Figure [B], i). Furthermore, complementary increased and decreased phosphorylation of eNOS (endothelial NO synthase) Thr-494 and Ser-1176 indicated impaired eNOS activity.…”

“…Confocal images of ECs treated with S1 protein revealed increased mitochondrial fragmentation, indicating altered mitochondrial dynamics (Figure [C], i). To examine whether these mitochondrial changes were due, in part, to the decreased amount of ACE2, we overexpressed ACE2 S680D (ACE2-D, a phospho-mimetic ACE2 with increased stability) or S680L (ACE2-L, a dephospho-mimetic with decreased stability) 4 in ECs. As shown in Figure [C], ii, ECs with ACE2-L had a higher number of fragmented mitochondria when compared to those with ACE2-D.…”

“…We also compared the expressions of mitochondria- and glycolysis-related genes in lung ECs isolated from ACE2-D or ACE2-L knock-in mice. 4 Shown in Figure [E], the mRNA levels of NRF1 , HO1 , and TFAM (mitochondria biogenesis-related genes) were increased, whereas those of HK2 , PFKFB3 , and ENO2 (glycolysis-related genes) were decreased in lung ECs in ACE2-D mice, as compared to those in ACE2-L mice.…”

Lenalidomide downregulates ACE2 protein abundance to alleviate infection by SARS-CoV-2 spike protein conditioned pseudoviruses

“…The E3 ligase MDM2 was reported to target ACE2 for degradation in an AMPK phosphorylation-dependent manner. 2 Intriguingly, increased ACE2 levels were observed in lungs from patients with comorbidities associated with severe COVID-19. 3 Modulating ACE2 protein homeostasis is an attractive direction to prevent/control SARS-CoV-2 infection; however, regulatory mechanisms of ACE2 protein control remain elusive.…”