UBR1 Promotes Sex-Dependent ACE2 Ubiquitination in Hypertension

Abstract

BACKGROUND: Ang-II (angiotensin II) impairs the function of the antihypertensive enzyme ACE2 (angiotensin-converting enzyme 2) by promoting its internalization, ubiquitination, and degradation, thus contributing to hypertension. However, few ACE2 ubiquitination partners have been identified, and their role in hypertension remains unknown. METHODS: Proteomics and bioinformatic analyses were used to identify ACE2 ubiquitination partners in the brain, heart, and kidney of hypertensive C57BL6/J mice of both sexes. The interaction between UBR1 (ubiquitin protein ligase E3 component N-recognin) and ACE2 was validated in cells. Central and peripheral UBR1 knockdown was then performed in male mice to investigate its role in the maintenance of hypertension. RESULTS: Proteomics analysis of the hypothalamus identified UBR1 as a potential E3 (ubiquitin protein ligase) ligase promoting ACE2 ubiquitination. Enhanced UBR1 expression, associated with ACE2 reduction, was confirmed in various tissues from hypertensive male mice and human samples. Treatment of endothelial and smooth muscle cells with testosterone, but not 17β-estradiol, confirmed a sex-specific regulation of UBR1. In vivo silencing of UBR1 using chronic administration of small interference RNA resulted in the restoration of ACE2 levels in hypertensive male mice. A transient decrease in blood pressure after intracerebroventricular, but not systemic, infusion was also observed. Interestingly, UBR1 knockdown increased brain activation of Nedd4-2 (neural precursor cell expressed developmentally downregulated protein 4), an E3 ligase promoting ACE2 ubiquitination, and reduced expression of serum and glucocorticoid-regulated kinase 1, the kinase that inactivates Nedd4-2. CONCLUSIONS: These data demonstrate that UBR1 is a novel E3 ubiquitin ligase targeting ACE2 in hypertension. UBR1 and Nedd4-2 appear to work synergistically to ubiquitinate ACE2. Targeting these ubiquitin ligases may represent a novel strategy to restore ACE2 compensatory activity in hypertension.