Angiotensin-Converting Enzyme 2 Metabolizes and Partially Inactivates Pyr-Apelin-13 and Apelin-17

Abstract

Abstract-Apelin peptides mediate beneficial effects on the cardiovascular system and are being targeted as potential new drugs. However, apelin peptides have extremely short biological half-lives, and improved understanding of apelin peptide metabolism may lead to the discovery of biologically stable analogues with therapeutic potential. We examined the ability of angiotensin-converting enzyme 2 (ACE2) to cleave and inactivate pyr-apelin 13 and apelin 17, the dominant apelin peptides. Computer-assisted modeling shows a conserved binding of pyr-apelin 13 and apelin 17 to the ACE2 catalytic site. In ACE2 knockout mice, hypotensive action of pyr-apelin 13 and apelin 17 was potentiated, with a corresponding greater elevation in plasma apelin levels. Similarly, pharmacological inhibition of ACE2 potentiated the vasodepressor action of apelin peptides. Biochemical analysis confirmed that recombinant human ACE2 can cleave pyr-apelin 13 and apelin 17 efficiently, and apelin peptides are degraded slower in ACE2-deficient plasma. The biological relevance of ACE2-mediated proteolytic processing of apelin peptides was further supported by the reduced potency of pyr-apelin 12 and apelin 16 on the activation of signaling pathways and nitric oxide production from endothelial cells. Importantly, although pyr-apelin 13 and apelin 17 rescued contractile function in a myocardial ischemia-reperfusion model, ACE2 cleavage products, pyr-apelin 12 and 16, were devoid of these cardioprotective effects. We designed and synthesized active apelin analogues that were resistant to ACE2-mediated degradation, thereby confirming that stable apelin analogues can be designed as potential drugs. We conclude that ACE2 represents a major negative regulator of apelin action in the vasculature and heart. residue and the enzymatic processes involved in its removal from the native apelin peptide remains poorly defined. Using loss-of-function and gain-of-function strategies, we here define a critical role of angiotensin-converting enzyme 2 (ACE2) in the proteolytic cleavage of the C-terminal phenylalanine residue in pyr-apelin 13 and apelin 17. Importantly, this degradative site can be modified to produce relatively stable apelin analogues as potential therapeutic agents. Methods In Silico Modeling of Apelin Peptide Binding to ACE2We selected the structure of human apo-ACE2 (PDB ID: 1R42) mainly because of its high resolution, appropriate R-factor, and errorless electron density map. 16 This structure was equilibrated in constant pressure-temperature condition (NVT, NPT) in the Groningen Machine for Chemical Simulations (GROMACS). 17 We performed knowledgebased docking using our understanding from ACE-substrate bound complex structure. For this purpose, we have used the Angiotensin II (Ang II)-bound ACE complex (PDB ID: 4APH) 18 and MLN-4760 inhibitor bound ACE2 complex (PDB ID: 1R4L). 16,[18][19][20] We modeled the ACE2-Ang II, ACE2-pyr-apelin 13, and ACE2-apelin 17 complexes using these 2 above mentioned reference structures as a template. We built th...

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“…By 2030, the prevalence of stroke will increase by more than 20% over 2012 and the direct medical costs are projected to reach $184.1 billion, which is a great challenge to human society and healthcare system (Mozaffarian et al, 2016). The situation is much worse in China, where stroke is the leading cause of death (Wang H. et al, 2016). Ischemic stroke is caused by the blockage of an artery in the brain, accounting for approximately 87% of stroke cases, which contributes to the major portion of death and post-stroke disability in patients (Mozaffarian et al, 2016).…”

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“…In summary, the studies by Wang et al 6 represent an important step forward in our understanding of the enzymatic degradation of apelin peptides and offer proof of concept for an approach to augmenting apelin agonism through the generation of synthetic long-acting analogues. It also suggests that inhibition of apelin metabolism is a worthwhile endeavor and that this approach could have major therapeutic potential especially if more apelin-specific peptidases are identified.…”

“…One major barrier to progress on both of these fronts has been a poor understanding of the post-translational processing, cleavage, and inactivation of apelin peptides. The studies by Wang et al, 6 therefore, represent an important step toward our ability to study the effects of chronic apelin agonism: a prerequisite for exploring its therapeutic potential.…”

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