Southeast Louisiana Veterans Health Care System, 2400 Canal Street, New Orleans, LA 70119, USA.
Department of Pharmacology & Experimental Therapeutics, Louisiana State University Health Sciences Center, 1900 Perdido Street New Orleans, LA 70112, USA.
Cardiovasc Res. 2023 Sep 5;119(11):2130-2141. doi: 10.1093/cvr/cvad070.
Angiotensin-converting enzyme 2 (ACE2) is a critical component of the compensatory renin-angiotensin system that is down-regulated during the development of hypertension, possibly via ubiquitination. However, little is known about the mechanisms involved in ACE2 ubiquitination in neurogenic hypertension. This study aimed at identifying ACE2 ubiquitination partners, establishing causal relationships and clinical relevance, and testing a gene therapy strategy to mitigate ACE2 ubiquitination in neurogenic hypertension.
Bioinformatics and proteomics were combined to identify E3 ubiquitin ligases associated with ACE2 ubiquitination in chronically hypertensive mice. In vitro gain/loss of function experiments assessed ACE2 expression and activity to validate the interaction between ACE2 and the identified E3 ligase. Mutation experiments were further used to generate a ubiquitination-resistant ACE2 mutant (ACE2-5R). Optogenetics, blood pressure telemetry, pharmacological blockade of GABAA receptors in mice expressing ACE2-5R in the bed nucleus of the stria terminalis (BNST), and capillary western analysis were used to assess the role of ACE2 ubiquitination in neurogenic hypertension. Ubiquitination was first validated as leading to ACE2 down-regulation, and Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) was identified as a E3 ligase up-regulated in hypertension and promoting ACE2 ubiquitination. Mutation of lysine residues in the C-terminal of ACE2 was associated with increased activity and resistance to angiotensin (Ang)-II-mediated degradation. Mice transfected with ACE2-5R in the BNST exhibited enhanced GABAergic input to the paraventricular nucleus (PVN) and a reduction in hypertension. ACE2-5R expression was associated with reduced Nedd4-2 levels in the BNST.
Our data identify Nedd4-2 as the first E3 ubiquitin ligase involved in ACE2 ubiquitination in Ang-II-mediated hypertension. We demonstrate the pivotal role of ACE2 on GABAergic neurons in the maintenance of an inhibitory tone to the PVN and the regulation of pre-sympathetic activity. These findings provide a new working model where Nedd4-2 could contribute to ACE2 ubiquitination, leading to the development of neurogenic hypertension and highlighting potential novel therapeutic strategies.
血管紧张素转换酶 2（ACE2）是补偿性肾素-血管紧张素系统的关键组成部分，在高血压的发展过程中其表达下调，这种下调可能是通过泛素化实现的。然而，关于神经源性高血压中 ACE2 泛素化的机制知之甚少。本研究旨在鉴定 ACE2 泛素化的伴侣，确定因果关系和临床相关性，并测试一种基因治疗策略来减轻神经源性高血压中的 ACE2 泛素化。
生物信息学和蛋白质组学相结合，鉴定了慢性高血压小鼠中与 ACE2 泛素化相关的 E3 泛素连接酶。体外获得/丧失功能实验评估了 ACE2 的表达和活性，以验证 ACE2 与鉴定的 E3 连接酶之间的相互作用。进一步使用突变实验生成了一个泛素化抗性 ACE2 突变体（ACE2-5R）。光遗传学、血压遥测、在表达 ACE2-5R 的终纹床核（BNST）中小鼠中应用 GABAA 受体药理学阻断、毛细血管西部分析用于评估 ACE2 泛素化在神经源性高血压中的作用。首先验证了泛素化导致 ACE2 下调，并且鉴定出神经前体细胞表达的发育下调蛋白 4-2（Nedd4-2）是高血压上调并促进 ACE2 泛素化的 E3 连接酶。ACE2 羧基末端赖氨酸残基的突变与活性增加和对血管紧张素（Ang）-II 介导的降解的抗性有关。在 BNST 中转染 ACE2-5R 的小鼠表现出对室旁核（PVN）的 GABA 能传入增强和高血压降低。ACE2-5R 表达与 BNST 中 Nedd4-2 水平降低相关。
我们的数据将 Nedd4-2 鉴定为 ACE2 在 Ang-II 介导的高血压中泛素化的第一个 E3 泛素连接酶。我们证明了 ACE2 在 GABA 能神经元中的作用对于维持对 PVN 的抑制性张力和调节交感前活动至关重要。这些发现提供了一个新的工作模型，其中 Nedd4-2 可能有助于 ACE2 泛素化，导致神经源性高血压的发展，并强调了潜在的新的治疗策略。
