Identification of prolyl carboxypeptidase as an alternative enzyme for processing of renal angiotensin II using mass spectrometry

Angiotensin-converting enzyme 2 (ACE2) catalyzes conversion of ANG II to ANG-(1–7). The present study uses newly established proteomic approaches and genetic mouse models to examine the contribution of alternative renal peptidases to ACE2-independent formation of ANG-(1–7). In situ and in vitro mass spectrometric characterization showed that substrate concentration and pH control renal ANG II processing. At pH ≥6, ANG-(1–7) formation was significantly reduced in ACE2 knockout (KO) mice. However, at pH <6, formation of ANG-(1–7) in ACE2 KO mice was similar to that in wild-type (WT) mice, suggesting alternative peptidases for renal ANG II processing. Furthermore, the dual prolyl carboxypeptidase (PCP)-prolyl endopeptidase (PEP) inhibitor Z-prolyl-prolinal reduced ANG-(1–7) formation in ACE2 KO mice, while the ACE2 inhibitor MLN-4760 had no effect. Unlike the ACE2 KO mice, ANG-(1–7) formation from ANG II in PEP KO mice was not different from that in WT mice at any tested pH. However, at pH 5, this reaction was significantly reduced in kidneys and urine of PCP-depleted mice. In conclusion, results suggest that ACE2 metabolizes ANG II in the kidney at neutral and basic pH, while PCP catalyzes the same reaction at acidic pH. This is the first report demonstrating that renal ANG-(1–7) formation from ANG II is independent of ACE2. Elucidation of ACE2-independent ANG-(1–7) production pathways may have clinically important implications in patients with metabolic and renal disease.

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An ACE2 Triple Decoy that neutralizes SARS-CoV-2 shows enhanced affinity for virus variants

Scientific Reports ◽

10.1038/s41598-021-91809-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shiho Tanaka ◽

Gard Nelson ◽

C. Anders Olson ◽

Oleksandr Buzko ◽

Wendy Higashide ◽

...

Keyword(s):

Md Simulation ◽

Therapeutic Option ◽

Receptor Binding Domain ◽

Converting Enzyme ◽

High Likelihood ◽

Wild Type ◽

Angiotensin Converting Enzyme 2 ◽

High Affinity ◽

Sustained Efficacy

AbstractThe SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.

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Directed evolution of angiotensin-converting enzyme 2 (ACE2) variants with enhanced peptidase activity profiles for novel therapeutic applications

10.1101/2022.01.03.474817 ◽

2022 ◽

Author(s):

Pete Heinzelman ◽

Philip A Romero

Keyword(s):

Angiotensin Converting Enzyme ◽

Directed Evolution ◽

Peptidase Activity ◽

Ang Ii ◽

Converting Enzyme ◽

Wild Type ◽

Peptide Substrate ◽

Angiotensin Converting Enzyme 2 ◽

Therapeutic Applications ◽

Target Peptide

Mutants of the Angiotensin Converting-Enzyme 2 (ACE2) carboxypeptidase possessing enhanced hydrolytic activity and specificity hold potential to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis with increased efficacy and reduced potential side effects relative to wild type ACE2. In pursuing this goal, we established a yeast display-based liquid chromatography screen that enabled use of directed evolution to identify ACE2 mutants with improved target peptide substrate, Angiotensin-II (Ang-II), activity and specificity relative to Apelin-13, an off-target peptide substrate. Screening yeast-displayed ACE2 active site residue saturation mutant libraries revealed three substitution-tolerant positions that can be mutated to enhance ACE2's activity profile. Double mutant libraries combining substitutions at these positions, M360, T371 and Y510, yielded candidate improved ACE2 mutants that were recombinantly expressed and purified at 1 mg/L yield and > 90% homogeneity. Relative to wild type, the leading mutant, T371L/Y510Ile, has seven-fold increased kcat toward Ang-II and six-fold decreased kcat/Km for Apelin-13 hydrolysis. In single substrate hydrolysis assays featuring physiologically relevant substrate concentrations T371L/Y510Ile hydrolyzes more Ang-II than wild type with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Additionally, T371L/Y510Ile hydrolyzed Ang-II at rates greater than wild type, with Apelin-13 hydrolysis reductions of up to 80 percent, in multiplex assays containing a mixture of peptides relevant to the ATR therapeutic axis. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to established and unexplored ACE2 therapeutic applications and demonstrate the feasibility of developing ACE2 variants for use in biomedical contexts unrelated to the ATR axis such as localized activation of peptide-based prodrugs.

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ACE2-targeting monoclonal antibody as potent and broad-spectrum coronavirus blocker

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00740-y ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Yuning Chen ◽

Ya-Nan Zhang ◽

Renhong Yan ◽

Guifeng Wang ◽

Yuanyuan Zhang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Broad Spectrum ◽

Management Strategy ◽

Spectrum Management ◽

Clinical Development ◽

Severe Toxicity ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Binding Residues

AbstractThe evolution of coronaviruses, such as SARS-CoV-2, makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after. Here we report a human angiotensin-converting enzyme 2 (ACE2)-targeting monoclonal antibody, 3E8, blocked the S1-subunits and pseudo-typed virus constructs from multiple coronaviruses including SARS-CoV-2, SARS-CoV-2 mutant variants (SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617.1, and P.1), SARS-CoV and HCoV-NL63, without markedly affecting the physiological activities of ACE2 or causing severe toxicity in ACE2 “knock-in” mice. 3E8 also blocked live SARS-CoV-2 infection in vitro and in a prophylactic mouse model of COVID-19. Cryo-EM and “alanine walk” studies revealed the key binding residues on ACE2 interacting with the CDR3 domain of 3E8 heavy chain. Although full evaluation of safety in non-human primates is necessary before clinical development of 3E8, we provided a potentially potent and “broad-spectrum” management strategy against all coronaviruses that utilize ACE2 as entry receptors and disclosed an anti-coronavirus epitope on human ACE2.

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Membrane potential measurements in renal afferent and efferent arterioles: actions of angiotensin II

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.2.f307 ◽

1997 ◽

Vol 273 (2) ◽

pp. F307-F314 ◽

Cited By ~ 40

Author(s):

R. Loutzenhiser ◽

L. Chilton ◽

G. Trottier

Keyword(s):

Angiotensin Ii ◽

Perfusion Pressure ◽

Rat Kidney ◽

Renal Perfusion ◽

Membrane Potentials ◽

Ang Ii ◽

Ca Channels ◽

Renal Perfusion Pressure

An adaptation of the in vitro perfused hydronephrotic rat kidney model allowing in situ measurement of arteriolar membrane potentials is described. At a renal perfusion pressure of 80 mmHg, resting membrane potentials of interlobular arteries (22 +/- 2 microns) and afferent (14 +/- 1 microns) and efferent arterioles (12 +/- 1 microns) were -40 +/- 2 (n = 8), -40 +/- 1 (n = 45), and -38 +/- 2 mV (n = 22), respectively (P = 0.75). Using a dual-pipette system to stabilize the impalement site, we measured afferent and efferent arteriolar membrane potentials during angiotensin II (ANG II)-induced vasoconstriction. ANG II (0.1 nM) reduced afferent arteriolar diameters from 13 +/- 1 to 8 +/- 1 microns (n = 8, P = 0.005) and membrane potentials from -40 +/- 2 to -29 +/- mV (P = 0.012). ANG II elicited a similar vasoconstriction in efferent arterioles, decreasing diameters from 13 +/- 1 to 8 +/- 1 microns (n = 8, P = 0.004), but failed to elicit a significant depolarization (-39 +/- 2 for control; -36 +/- 3 mV for ANG II; P = 0.27). Our findings thus indicate that resting membrane potentials of pre- and postglomerular arterioles are similar and lie near the threshold activation potential for L-type Ca channels. ANG II-induced vasoconstriction appears to be closely coupled to membrane depolarization in the afferent arteriole, whereas mechanical and electrical responses appear to be dissociated in the efferent arteriole.

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ANG II modifies cardiomyocyte function via extracardiac and intracardiac neurons: in situ and in vitro studies

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1997.272.3.r766 ◽

1997 ◽

Vol 272 (3) ◽

pp. R766-R775 ◽

Cited By ~ 6

Author(s):

M. Horackova ◽

J. A. Armour

Keyword(s):

Guinea Pig ◽

Receptor Antagonist ◽

Neuronal Activity ◽

Ang Ii ◽

Arterial Blood ◽

Cardiac Ganglia ◽

In Situ Experiments ◽

Anesthetized Dogs

To determine whether angiotensin II (ANG II) affects cardiac performance via neurons in intrathoracic cardiac ganglia, studies were performed on anesthetized dogs. To exclude possible vascular regulatory effects of ANG II, experiments were also performed using long-term cultures of adult guinea pig ventricular cardiomyocytes with or without intrathoracic neurons. 1) In in situ experiments in 10 anesthetized dogs, cardiac augmentation occurred when ANG II (10 microl or 0.1 ml; 10-100 microM) was administered into limited loci within acutely decentralized stellate or middle cervical ganglia that were neurally connected to, but not those disconnected from, the heart. In another 18 dogs, ANG II increased intrinsic cardiac neuronal activity when administered adjacent to such neurons or into their local arterial blood supply. Ventricular ionotropic effects elicited by ANG II were eliminated by timolol, whereas increases in intrinsic cardiac neuronal activity were not affected. Effects elicited by ANG II were eliminated by administration of a selective AT1 receptor antagonist (losartan) but not by a selective AT2 receptor antagonist (PD-123319). 2) In in vitro experiments, ANG II (100 nM) induced positive chronotropic effects on cultured adult guinea pig cardiomyocytes innervated with adult extrinsic or intrinsic cardiac neurons, but not those cultured without neurons. The frequency of calcium inward current (Ca(i)) transients (recorded by fura 2 fluorescence) increased in innervated cocultures but not in the noninnervated cardiomyocyte cultures; however, the amplitude of Ca(i) transients was not affected by ANG II in cultures or in freshly isolated adult guinea pig cardiomyocytes. ANG II-induced effects in cocultures were blocked by losartan but not PD-123319 or timolol. Thus 1) ANG II-sensitive neurons exist in intrathoracic extracardiac and intrinsic cardiac ganglia; 2) these neurons possess AT1 receptors; and 3) these neurons appear to act directly and indirectly via adrenergic neurons to enhance cardiomyocyte function.

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Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages of progastrin to release gastrin-34 and gastrin-17

Biochemical Journal ◽

10.1042/bj20080881 ◽

2008 ◽

Vol 415 (1) ◽

pp. 35-43 ◽

Cited By ~ 23

Author(s):

Jens F. Rehfeld ◽

Xiaorong Zhu ◽

Christina Norrbom ◽

Jens R. Bundgaard ◽

Anders H. Johnsen ◽

...

Keyword(s):

Structural Factors ◽

Cleavage Sites ◽

Wild Type ◽

Prohormone Convertases ◽

Site Specific ◽

G Cells ◽

Processing Site ◽

Vitro Effect

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and α-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg36Arg37 and Arg73Arg74 sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, α-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys53Lys54), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

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ACE2 Shedding and the Role in COVID-19

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.789180 ◽

2022 ◽

Vol 11 ◽

Author(s):

Jieqiong Wang ◽

Huiying Zhao ◽

Youzhong An

Keyword(s):

Amino Acids ◽

Viral Entry ◽

Kidney Injury ◽

Renin Angiotensin System ◽

Underlying Mechanism ◽

Ang Ii ◽

Converting Enzyme ◽

Angiotensin System ◽

Angiotensin Converting Enzyme 2 ◽

Transmembrane Glycoprotein

Angiotensin converting enzyme 2 (ACE2), a transmembrane glycoprotein, is an important part of the renin-angiotensin system (RAS). In the COVID-19 epidemic, it was found to be the receptor of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). ACE2 maintains homeostasis by inhibiting the Ang II-AT1R axis and activating the Ang I (1-7)-MasR axis, protecting against lung, heart and kidney injury. In addition, ACE2 helps transport amino acids across the membrane. ACE2 sheds from the membrane, producing soluble ACE2 (sACE2). Previous studies have pointed out that sACE2 plays a role in the pathology of the disease, but the underlying mechanism is not yet clear. Recent studies have confirmed that sACE2 can also act as the receptor of SARS-COV-2, mediating viral entry into the cell and then spreading to the infective area. Elevated concentrations of sACE2 are more related to disease. Recombinant human ACE2, an exogenous soluble ACE2, can be used to supplement endogenous ACE2. It may represent a potent COVID-19 treatment in the future. However, the specific administration concentration needs to be further investigated.

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Dysregulation of ACE (Angiotensin-Converting Enzyme)-2 and Renin-Angiotensin Peptides in SARS-CoV-2 Mediated Mortality and End-Organ Injuries

Hypertension ◽

10.1161/hypertensionaha.121.18295 ◽

2021 ◽

Author(s):

Kaiming Wang ◽

Mahmoud Gheblawi ◽

Anish Nikhanj ◽

Matt Munan ◽

Erika MacIntyre ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Tumor Necrosis Factor Receptor ◽

Renin Angiotensin System ◽

Adverse Outcomes ◽

Blood Collection ◽

Ang Ii ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Renin Angiotensin ◽

Angiotensin Peptides

ACE (angiotensin-converting enzyme)-2 as the target for SARS-CoV-2 also negatively regulates the renin-angiotensin system. Pathological activation of ADAM17 (A disintegrin and metalloproteinase-17) may potentiate inflammation and diminish ACE2-mediated tissue protection through proteolytic shedding, contributing to SARS-CoV-2 pathogenesis. We aim to examine plasma soluble ACE2 and angiotensin profiles in relation to outcomes by enrolling consecutive patients admitted for COVID-19 with baseline blood collection at admission and repeated sampling at 7 days. The primary outcome was 90-day mortality, and secondary outcomes were the incidence of end-organ injuries. Overall, 242 patients were included, the median age was 63 (52–74) years, 155 (64.0%) were men, and 57 (23.6%) patients reached the primary end point. Baseline soluble ACE2 was elevated in COVID-19 but was not associated with disease severity or mortality. In contrast, an upward trajectory of soluble ACE2 at repeat sampling was independently associated with an elevated risk of mortality and incidence of acute myocardial injury and circulatory shock. Similarly, an increase in soluble tumor necrosis factor receptor levels was also associated with adverse outcomes. Plasma Ang I, Ang 1-7 (angiotensin 1–7) levels, and the Ang 1-7/Ang II (angiotensin II) ratio were elevated during SARS-CoV-2 infection related to downregulation of ACE activity at baseline. Moreover, patients having an upward trajectory of soluble ACE2 were characterized by an imbalance in the Ang 1-7/Ang II ratio. The observed dysregulation of ACE2 and angiotensin peptides with disease progression suggest a potential role of ADAM17 inhibition and enhancing the beneficial Ang 1-7/Mas axis to improve outcomes against SARS-CoV-2 infection.

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ACE2 the Janus-faced protein – from cardiovascular protection to severe acute respiratory syndrome-coronavirus and COVID-19

Clinical Science ◽

10.1042/cs20200363 ◽

2020 ◽

Vol 134 (7) ◽

pp. 747-750 ◽

Cited By ~ 20

Author(s):

Rhian M. Touyz ◽

Hongliang Li ◽

Christian Delles

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Angiotensin Converting Enzyme ◽

Basic Science ◽

Physiological Role ◽

Ang Ii ◽

Converting Enzyme ◽

Sars Coronavirus ◽

Multifunctional Protein ◽

Angiotensin Converting Enzyme 2

Abstract Angiotensin converting enzyme 2 (ACE2) is the major enzyme responsible for conversion of Ang II into Ang-(1-7). It also acts as the receptor for severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2, which causes Coronavirus Disease (COVID)-19. In recognition of the importance of ACE2 and to celebrate 20 years since its discovery, the journal will publish a focused issue on the basic science and (patho)physiological role of this multifunctional protein.

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