The molecular mechanisms of interactions between bioactive peptides and angiotensin-converting enzyme

The ability of milk protein derived Ile-Pro-Ala (IPA), Phe-Pro (FP) and Gly-Lys-Pro (GKP) peptides to inhibit angiotensin I-converting enzyme (ACE), a protein with an important role in blood-pressure regulation, were verified in vitro and in vivo. This work elucidates the modes and molecular mechanisms of the interaction of IPA, FP and GKP with ACE, including mechanisms that bind the peptides to the cofactor Zn²⁺. It was observed that the best docking poses obtained for IPA, FP and GKP were at the ACE catalytic site with very similar modes of interaction, including the interaction with Zn²⁺. The interactions, including H-bonds, hydrophobic, hydrophilic, and electrostatic interactions, as well as the interaction with Zn²⁺, were responsible for the binding between the bioactive peptides and ACE.     

Effects of Lactobacillus helveticus fermented milk on bone cells in vitro

Milk fermented with Lactobacillus helveticus (L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3-1.4 times with the 1 × 10⁻⁵, 1 × 10⁻⁴ and 1 × 10⁻³ solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.     

The antihypertensive effect of peptides: a novel alternative to drugs?

Many types of bioactive peptides that inhibit angiotensin I, angiotensin I converting enzyme (ACE) and Ang II type 1 receptor (AT1) in the cardiovascular system contribute to the prevention and treatment of hypertension. These inhibitory peptides are derived from many food proteins or artificial synthetic products. Further research examining the bioavailability of ACE inhibitory peptides will lead to the development of more effective ACE inhibitory peptides and foods. Our research also demonstrates that ACE inhibitory peptide LAP may lower blood pressure with no adverse effects.     

Influence of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) on the release of endothelin-1 by endothelial cells

Milk protein-derived peptides with angiotensin-converting enzyme (ACE) inhibitory activity can reduce blood pressure in hypertensive subjects. The lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) is an ACE-inhibitory peptide released by tryptic digestion from the milk protein beta-lactoglobulin. Its ACE-inhibitory activity is 100 times lower than that of captopril. The latter is known to inhibit the release of the vasoconstrictor endothelin-1 (ET-1) by endothelial cells. The effects of ALPMHIR on the endothelium are currently unknown. In this study, the influence of ALPMHIR on release of ET-1 by endothelial cells was investigated. The basal ET-1 release of the cells was reduced by 29% (p<0.01) in the presence of 1 mM ALPMHIR, compared to 42% (p<0.01) for 0.1 mM captopril. Addition of 10 U/ml thrombin to the incubation medium increased the release of ET-1 by 66% (p<0.01). Co-incubation of 10 U/ml thrombin with 1 microM captopril or with 0.1 mM ALPMHIR inhibited the stimulated ET-1 release by 45% (p<0.01) and by 32% (p<0.01), respectively. These data indicate that dietary peptides, such as ALPMHIR, can modulate ET-1 release by endothelial cells. These effects, among other mechanisms, may play a role in the anti-hypertensive effect of milk protein-derived peptides.     

Solid-phase synthesis and conformational properties of angiotensin converting enzyme catalytic-site peptides: the basis for a structural study on the enzyme-substrate interaction

The solution NMR conformational properties of two angiotensin converting enzyme (ACE) Zn catalytic-site 36-residue peptides, with the general sequence HEMGHX23EAIGDX3, synthesized through solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, is reported. The 1H resonance assignment of Zn-bound peptides is presented and the characteristic features of the NMR solution models of the two ACE Zn(II)-bound peptides are reported. The solid-state and solution structures of the ACE C-domain catalytic site are compared while biologically important structural similarities and differences of the N- and C-terminal catalytic sites are discussed. Additionally, the structural features of the ACE substrate, the angiotensin I (AI) decapeptide, are studied using NMR spectroscopy, in order to set the structural basis for the ACE-substrate interaction in solution.     

Milk-Derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) Promote Adipocyte Differentiation and Inhibit Inflammation in 3T3-F442A Cells

Milk derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) have shown promise as anti-hypertensive agents due to their inhibitory effects on angiotensin converting enzyme (ACE). Due to the key inter-related roles of hypertension, chronic inflammation and insulin resistance in the pathogenesis of metabolic syndrome, there is growing interest in investigating established anti-hypertensive agents for their effects on insulin sensitivity and inflammation. In this study, we examined the effects of IPP and VPP on 3T3-F442A murine pre-adipocytes, a widely used model for studying metabolic diseases. We found that both IPP and VPP induced beneficial adipogenic differentiation as manifested by intracellular lipid accumulation, upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) and secretion of the protective lipid hormone adiponectin by these cells. The observed effects were similar to those induced by insulin, suggesting potential benefits in the presence of insulin resistance. IPP and VPP also inhibited cytokine induced pro-inflammatory changes such as reduction in adipokine levels and activation of the nuclear factor kappa B (NF-κB) pathway. Taken together, our findings suggest that IPP and VPP exert insulin-mimetic adipogenic effects and prevent inflammatory changes in adipocytes, which may offer protection against metabolic disease.     

Prediction of off-target effects on angiotensin-converting enzyme 2

The authors describe a structure-based strategy to identify therapeutically beneficial off-target effects by screening a chemical library of Food and Drug Administration (FDA)-approved small-molecule drugs matching pharmacophores defined for specific target proteins. They applied this strategy to angiotensin-converting enzyme 2 (ACE2), an enzyme that generates vasodilatory peptides and promotes protection from hypertension-associated cardiovascular disease. The conformation-based structural selection method by molecular docking using DOCK allowed them to identify a series of FDA-approved drugs that enhance catalytic efficiency of ACE2 in vitro. These data demonstrate that libraries of approved drugs can be rapidly screened to identify potential side effects due to interactions with specific proteins other than the intended targets.     

Production of angiotensin-I-converting enzyme inhibitory peptides from β-lactoglobulin- and casein-derived peptides: an integrative approach

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin- and casein-derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N "Amano" in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50=285 μg/mL. In this hydrolysate we identified the well-known potent ACE-inhibitor and antihypertensive tripeptide Ile-Pro-Pro (IPP) and another novel octapeptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=28 μg/mL. This hydrolysate contained a tetrapeptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.     

In vitro and in silico studies of novel synthetic ACE-inhibitory peptides derived from Saccharomyces cerevisiae protein hydrolysate

The structure-function relation of YR-10 (YGKPVAVPAR) was investigated by synthesizing four structural analogs of that including YHR-10 (YGKHVAVHAR), GA-8 (GKPVAVPA), GHA-8 (GKHVAVHA), and PAR-3 (PAR). GA-8 (GKPVAVPA) was synthesized on the basis of simulated enzymatic gastrointestinal digestion performed by bioinformatics tools (expasy-peptide cutter). This study explains the molecular mechanisms for the interaction of synthetic peptides with ACE. The IC₅₀ values of each were 139.554 ± 2.3, 61.91 ± 1.2, 463.230 ± 3.56, 135.135 ± 2.1, 514.024 ± 5.86 µM, respectively. Results indicated that Pro replacement with His in YR-10 and GA-8 increased ACE inhibitory activity respectively, by 55.63% and 70.82%. Removal of Tyr and Arg from respectively N and C terminal positions of YR-10, following in silico simulated gastrointestinal digestion caused the 3.31 fold decrease in ACE inhibitory activity. YHR-10 showed the best docking poses, and GHA-8 exhibited interaction with Zn²⁺. Lineweaver–Burk plots of most active peptides suggest that they act as noncompetitive inhibitors against ACE.     

Pharmacokinetics of proline-rich tripeptides in the pig

Tripeptides may possess bioactive properties. For instance, blood pressure lowering is attributed to the proline-rich tripeptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP), and Val-Pro-Pro (VPP). However, little is known about their absorption, distribution, and elimination characteristics. The aim of this study was to characterize the pharmacokinetic behavior of IPP, LPP, and VPP in a conscious pig model. Synthetic IPP, LPP, and VPP were administered intravenously or intragastrically (4.0 mg kg⁻¹ BW in saline) to 10 piglets (approximately 25 kg body weight) in the postabsorptive state. After intravenous dosing, the elimination half-life for IPP was significantly higher (P < 0.001) than for LPP and VPP (2.5 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 min, respectively). After intragastric dosing, however, the elimination half-lives were not significantly different between the peptides (9 ± 1, 15 ± 4, and 12 ± 6 min, respectively). Maximum plasma concentrations were about 10 nmol l⁻¹ for the three tripeptides. The fraction dose absorbed was 0.077 ± 0.010, 0.059 ± 0.009, and 0.073 ± 0.015%, for IPP, LPP, and VPP, respectively. Proline-rich tripeptides reach the blood circulation intact, with an absolute bioavailability of about 0.1% when administered via a saline solution. Because half-lives of absorption and elimination were maximally about 5 and 15 min, respectively, this suggests that under these conditions a bioactive effect of these tripeptides would be rather acute.     

Bisphenol-A Can Inhibit the Enzymatic Activity of Human Superoxide Dismutase

Bisphenol-A (BPA), a synthetic xenoestrogen, is currently being used to produce a wide variety of consumer products. Humans as well as animals are exposed to this ubiquitous compound through ingestion, inhalation, and dermal exposure. The effect of this compound on superoxide dismutase (SOD), an antioxidant enzyme, isolated from human blood was studied using an enzyme inhibition assay. The mode of interaction of BPA on SOD was investigated using modeling and docking studies. Purified human SOD from erythrocytes was used to study the enzyme inhibition assay of BPA. Molecular level interactions of BPA on SOD were also analyzed by modeling and docking studies. Our study demonstrates that BPA has an inhibitory effect on SOD. The docking results showed that it could bind to the active site residues of SOD and could interfere with the catalytic activity of the enzyme. Our study reveals for the first time that BPA can directly inhibit the enzymatic activity of human SOD and thus impairs the free radical scavenging mechanism.     

Enhanced Lacto-Tri-Peptide Bio-Availability by Co-Ingestion of Macronutrients

Some food-derived peptides possess bioactive properties, and may affect health positively. For example, the C-terminal lacto-tri-peptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP) and Val-Pro-Pro (VPP) (together named here XPP) are described to lower blood pressure. The bioactivity depends on their availability at the site of action. Quantitative trans-organ availability/kinetic measurements will provide more insight in C-terminal tri-peptides behavior in the body. We hypothesize that the composition of the meal will modify their systemic availability. We studied trans-organ XPP fluxes in catheterized pigs (25 kg; n=10) to determine systemic and portal availability, as well as renal and hepatic uptake of a water-based single dose of synthetic XPP and a XPP containing protein matrix (casein hydrolyte, CasH). In a second experiment (n=10), we compared the CasH-containing protein matrix with a CasH-containing meal matrix and the modifying effects of macronutrients in a meal on the availability (high carbohydrates, low quality protein, high fat, and fiber). Portal availability of synthetic XPP was 0.08 ± 0.01% of intake and increased when a protein matrix was present (respectively 3.1, 1.8 and 83 times for IPP, LPP and VPP). Difference between individual XPP was probably due to release from longer peptides. CasH prolonged portal bioavailability with 18 min (absorption half-life, synthetic XPP: 15 ± 2 min, CasH: 33 ± 3 min, p<0.0001) and increased systemic elimination with 20 min (synthetic XPP: 12 ± 2 min; CasH: 32 ± 3 min, p<0.0001). Subsequent renal and hepatic uptake is about 75% of the portal release. A meal containing CasH, increased portal 1.8 and systemic bioavailability 1.2 times. Low protein quality and fiber increased XPP systemic bioavailability further (respectively 1.5 and 1.4 times). We conclude that the amount and quality of the protein, and the presence of fiber in a meal, are the main factors that increase the systemic bioavailability of food-derived XPP.     

Inhibition mechanism and model of an angiotensin I-converting enzyme (ACE)-inhibitory hexapeptide from yeast (Saccharomyces cerevisiae)

Angiotensin I-converting enzyme (ACE) has an important function in blood pressure regulation. ACE-inhibitory peptides can lower blood pressure by inhibiting ACE activity. Based on the sequence of an ACE-inhibitory hexapeptide (TPTQQS) purified from yeast, enzyme kinetics experiments, isothermal titration calorimetry (ITC), and a docking simulation were performed. The hexapeptide was found to inhibit ACE in a non-competitive manner, as supported by the structural model. The hexapeptide bound to ACE via interactions of the N-terminal Thr1, Thr3, and Gln4 residues with the residues on the lid structure of ACE, and the C-terminal Ser6 attracted the zinc ion, which is vital for ACE catalysis. The displacement of the zinc ion from the active site resulted in the inhibition of ACE activity. The structural model based on the docking simulation was supported by experiments in which the peptide was modified. This study provides a new inhibitory mechanism of ACE by a peptide which broads our knowledge for drug designing against enzyme targets.     

Structure and physiological importance of angiotensin converting enzyme domains

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), exhibiting different biochemical properties. The catalytically active ACE isoforms consisted of just one domain have been also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when their crystal structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control of different physiological functions. The C-terminal catalytic domain plays an important role in the regulation of blood pressure: it catalyzes angiotensin I cleavage in vivo. The N-domain contributes to the processing of other bioactive peptides for which it exhibits high affinity. The role of the N-domain is not ultimately associated with functioning of the rennin-angiotensin system and it contributes processing of other bioactive peptides for which it exhibits high affinity (goralatide, luliberin, enkephalin heptapeptide, beta-amyloid peptide). Domain-selective inhibitors selectively blocking either the N- or C-domain of ACE have been developed.     

Effect of an Excess Intake of Casein Hydrolysate Containing Val-Pro-Pro and Ile-Pro-Pro in Subjects with Normal Blood Pressure,High-Normal Blood Pressure,or Mild Hypertension

A double-blind, placebo-controlled, randomized clinical trial was conducted to evaluate the effects of ingesting an excess of tablets containing casein hydrolysate, incorporating angiotensin I-converting enzyme (ACE) inhibitory peptides such as Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in subjects with blood pressure ranging from normal to mild hypertension. A total of 48 subjects were given either 5 times more than the effective amount of casein hydrolysate or a placebo in tablet form for 4 weeks. In the active group, systolic blood pressure (SBP) decreased significantly as compared with the placebo group. In stratified analysis, however, this antihypertensive effect was not found in normotensive subjects. In addition, neither an acute or nor an excessive reduction in blood pressure nor clinically important adverse events were observed in this study. These findings suggest that intake of a 5-fold excess of tablets containing casein hydrolysate can lead to a mild improvement in hypertension without side effects.     

Effect of an excess intake of casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro in subjects with normal blood pressure, high-normal blood pressure, or mild hypertension

A double-blind, placebo-controlled, randomized clinical trial was conducted to evaluate the effects of ingesting an excess of tablets containing casein hydrolysate, incorporating angiotensin I-converting enzyme (ACE) inhibitory peptides such as Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in subjects with blood pressure ranging from normal to mild hypertension. A total of 48 subjects were given either 5 times more than the effective amount of casein hydrolysate or a placebo in tablet form for 4 weeks. In the active group, systolic blood pressure (SBP) decreased significantly as compared with the placebo group. In stratified analysis, however, this antihypertensive effect was not found in normotensive subjects. In addition, neither an acute or nor an excessive reduction in blood pressure nor clinically important adverse events were observed in this study. These findings suggest that intake of a 5-fold excess of tablets containing casein hydrolysate can lead to a mild improvement in hypertension without side effects.     

Influence of the Lactotripeptides Isoleucine–Proline–Proline and Valine–Proline–Proline on Systolic Blood Pressure in Japanese Subjects: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Background

The lactotripeptides isoleucine–proline–proline (IPP) and valine–proline–proline (VPP) have been shown to decrease systolic blood pressure (SBP) in several populations, but the size of the effect varies among studies. We performed a meta-analysis including all published studies to evaluate the SBP-lowering effect of IPP/VPP in Japanese subjects more comprehensively.

Methods and Findings

Eligible randomized controlled trials were searched for within four bibliographic databases, including two Japanese ones. Eighteen studies (including a total of 1194 subjects) were included in the meta-analysis. A random effect model using the restricted maximum likelihood (REML) estimator was used for the analysis. The analysis showed that consumption of IPP/VPP induced a significant reduction in SBP as compared with placebo in Japanese subjects, with an estimated effect of -5.63 mm Hg (95% CI, -6.87 to -4.39, P<0.0001) and no evidence of publication bias. A significant heterogeneity between series was evident, which could be explained by a significant influence of the baseline blood pressure status of the subjects, the effect of IPP/VPP on SBP being stronger in hypertensive subjects (-8.35 mm Hg, P<0.0001) than in non-hypertensive subjects (-3.42mm Hg, P<0.0001). Furthermore, the effect of IPP/VPP on SBP remained significant when limiting the analysis to series that tested the usual doses of IPP/VPP consumed daily (below 5 mg/d), with estimated effects of -6.01 mm Hg in the overall population and -3.32 mm Hg in non-hypertensive subjects.

Conclusions

Results from this meta-analysis show that IPP/VPP lactotripeptides can significantly reduce office SBP in Japanese subjects with or without overt hypertension, and for doses that can potentially be consumed as an everyday supplement. This suggests that these peptides could play a role in controlling blood pressure in Japanese subjects. The systematic review protocol was published on the PROSPERO register (CRD42014014322).     

血管紧张素转化酶抑制肽的研究进展

血管紧张素转化酶 (angiotensin I convertingenzyme ,ACE)在血压调节方面起着重要的作用 ,当其受到抑制时血压就会降低。许多合成的ACE抑制剂被广泛地应用于临床 ,但会造成多种副作用。近年来 ,对天然ACE抑制肽的研究表明 ,一些来源于蛋白酶解产生的活性肽可以对ACE起到有效的抑制作用。综述了血管紧张素转化酶的抑制肽的降压原理 ,种类和来源以及结构特点等的研究进展 ,并对其应用前景进行了展望。     

In silico identification of novel ACE and DPP-IV inhibitory peptides derived from buffalo milk proteins and evaluation of their inhibitory mechanisms

The capacity of buffalo milk proteins to release bioactive peptides was evaluated and novel bioactive peptides were identified. The sequential similarity between buffalo milk proteins and their cow counterparts was analysed. Buffalo milk proteins were simulated to yield theoretical peptides via in silico proteolysis. The potential of selected proteins to release specific bioactive peptides was evaluated by the A value obtained from the BIOPEP–UWM database (Minkiewicz et al. in Int J Mol Sci 20(23):5978, 2019). Buffalo milk protein is a suitable precursor to produce bioactive peptides, particularly dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory peptides. Two novel ACE inhibitory peptides (KPW and RGP) and four potential DPP-IV inhibitory peptides (RGP, KPW, FPK and KFTW) derived from in silico proteolysis of buffalo milk proteins were screened using different integrated bioinformatic approaches (PeptideRanker, Innovagen, peptide-cutter and molecular docking). The Lineweaver–Burk plots showed that KPW (IC₅₀?=?136.28?±?10.77 μM) and RGP (104.72?±?8.37 μM) acted as a competitive inhibitor against ACE. Similarly, KFTW (IC₅₀?=?873.92?±?32.89 μM) was also a competitive inhibitor of DPP-IV, while KPW and FPK (82.52?±?10.37 and 126.57?±?8.45 μM, respectively) were mixed-type inhibitors. It should be emphasized that this study does not involve any clinical trial.