A combined in vitro and in silico study of the inhibitory mechanism of angiotensin-converting enzyme with peanut peptides

3.0 科研～小助 2025-09-01 5 4 8.19MB 13 页 1知币

侵权投诉

International Journal of Biological Macromolecules 268 (2024) 131901

Available online 26 April 2024

A combined in vitro and in silico study of the inhibitory mechanism of

angiotensin-converting enzyme with peanut peptides

Jiale Liu

, Wentian Song

, Xue Gao

, Jiaoyan Sun

, Chunlei Liu

, Li Fang

, Ji Wang

Junhua Shi

, Yue Leng

, Xiaoting Liu

, Weihong Min

College of Food Science and Engineering, National Engineering Laboratory of Wheat and Corn Deep Processing, Jilin Agricultural University, Changchun 130118, Jilin,

China

College of Food and Health, Zhejiang A&F University, Hangzhou 311300, China

ARTICLE INFO

Keywords:

Angiotensin-converting enzyme

Inhibitor

Peanut peptides

Molecular dynamics simulation

HUVEC cell

ABSTRACT

Food-derived peptides with low molecular weight, high bioavailability, and good absorptivity have been

exploited as angiotensin-converting enzyme (ACE) inhibitors. In the present study, in-vitro inhibition kinetics of

peanut peptides, in silico screening, validation of ACE inhibitory activity, molecular dynamics (MD) simulations,

and HUVEC cells were performed to systematically identify the inhibitory mechanism of ACE interacting with

peanut peptides. The results indicate that FPHPP, FPHY, and FPHFD peptides have good thermal, pH, and

digestive stability. MD trajectories elucidate the dynamic correlation between peptides and ACE and verify the

specic binding interaction. Noteworthily, FPHPP is the best inhibitor with a strongest binding afnity and

signicantly increases NO, SOD production, and AT2R expression, and decreases ROS, MDA, ET-1 levels, ACE,

and AT1R accumulation in Ang II-injury HUVEC cells.

1. Introduction

Hypertension is a dramatic risk factor in cardiovascular diseases,

including strokes and coronary heart diseases [1], and the renin-

angiotensin-aldosterone system (RAAS) is the main regulator of blood

pressure [2]. In the system, angiotensin-converting enzyme (ACE) is a

zinc dipeptidyl carboxypeptidase, which plays a pivotal role in regu-

lating blood pressure and electrolyte homeostasis through RAAS [3].

Additionally, ACE catalyzes the cleavage of the C-terminal histidine-

leucine dipeptide from angiotensin I (Ang I) to produce angiotensin II

(Ang II, a potent vasoconstrictor octapeptide) [4]. Therefore, inhibiting

ACE activity is a successful strategy for preventing and remedying high

blood pressure.

Currently, a range of ACE-inhibiting synthetic drugs usually with a

sulfhydryl (captopril), carboxyl (perindoprilat/lisinopril/enalaprilat),

or ketone (keto-ACE) blocks for coordination of the zinc ion have been

routinely applied for hypertension treatment [5]. However, after long-

term treatment, these drugs often provoke various side effects, such as

fetopathy, angioedema, hyperkalemia, persistent dry cough, and kidney

problems [6–8]. Thus, a growing attention has been drawn in identifying

food-derived ACE inhibitory (ACEI) peptides which could control blood

pressure in a safe way. Up to now, ACEI peptides have been isolated from

various kinds of food, such as seafood [9–11], plants [1,12–15], and

dairy products [16–18].

It has been demonstrated that famous peptides Ile-Trp (IW), Val-Pro-

Pro (VPP), and Ile-Pro-Pro (IPP) from milk [19], Ile-Arg-Trp (IRW) [16]

and Arg-Val-Pro-Ser-Leu (RVPSL) from eggs [20], IAVPTGVA and LPYP

from soybeans [15], YLVR from hazelnuts [21] which are dissociated by

enzymatic hydrolysis of the parent protein, microbial fermentation, and

production of recombinant peptides [22] reduced activity of ACE and

decreased blood pressure. These innovative and conventional experi-

ment approaches induce many problems, such as extensive time and

cost, and irrecoverable function. Accordingly, computational techniques

including molecular docking and molecular dynamics (MD) simulations

[23] have been performed to screen ACEI peptides, characterize inhib-

itory activities, and modify original peptides for seeking better in-

hibitors in recent years. Although a number of ACEI compounds which

assimilate easily, from the intestinal tract into blood circulation pro-

moting antihypertensive effects [24] have been produced, there is very

limited information on the relationships between structure and activity

* Corresponding author.

** Correspondence to: W. Min, College of Food and Health, Zhejiang A&F University, Hangzhou 311300, China.

E-mail addresses: liuxiaoting@jlau.edu.cn (X. Liu), minwh2000@zafu.edu.cn (W. Min).

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

https://doi.org/10.1016/j.ijbiomac.2024.131901

Received 8 January 2024; Received in revised form 6 April 2024; Accepted 24 April 2024

International Journal of Biological Macromolecules 268 (2024) 131901

(SAR) and even the specic inhibition mechanism between ACEI ligands

and the protein receptor [25–27]. Therefore, the development of stra-

tegies quantitatively characterizing the functional mechanisms of ACE is

of paramount importance, which is prot for the discovery of novel

inhibitors with simplied production process and excellent

bioavailability.

In this study, we obtained peanut-derived ACEI peptides by database

screening, molecular docking, validation of inhibitory activity, MD

simulations, as well as HUVEC cells. In addition, employing in-vitro in-

hibition kinetics of ACEI peptides by high-performance liquid chroma-

tography (HPLC) and network modeling approaches, this work

systematically examined the inhibitory mechanism of ACE interacting

with peanut peptides. Our study also offers a useful perspective of

computational biology on the screening ACEI peptides and revealing in-

depth interaction by analyzing dynamic networks and the atomic level

interaction including cation-

interaction, metal coordination, van der

Waals, and hydrogen bonds between peptides and ACE residues.

2. Materials and methods

2.1. Materials

ACE from rabbit lung and N-Hippuryl-His-Leu hydrate (HHL) and 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

were purchased from Sigma-Inc (St. Louis, MO, USA). Pepsin (3000 U/g)

and trypsin (250 U/g) was bought from Beijing Dingguo Biotechnology

Co., Ltd. (Beijing, China). Acetonitrile (HPLC grade) and triuoroacetic

acid (LC-MS grade) were obtained from Fisher Scientic (Pittsburgh, PA,

USA). Angiopoietin-2 Protein, Huma (10 mg) were from MedChemEx-

press (Shanghai, China). Human umbilical vein endothelial cells

(HUVEC) were procured from Zhongqiao Xinzhou Biotechnology Co.,

Ltd. (Shanghai, China). Fetal bovine serum, streptomycin and penicillin

were obtained from Gibco (New York, USA). NO and ET-1 ELISA kits

were bought from Biyuntian Reagent Company. Reactive oxygen species

(ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and BCA

protein concentration kits were obtained from Nanjing Jian Cheng

Bioengineering Research Institute. Antibodies against ACE, AT1R,

AT2R, and β-Actin were from Abcam (Wuhan, China). Other reagents

were of analytical grade and commercially available.

2.2. In-silico screen of peanut ACEI peptides and selected peptide

synthesis

According to the structural features and characteristic amino acids of

ACE-inhibiting peptides, some peptides were screened from the peanut

peptide sequences previously prepared and characterized in our labo-

ratory. The potential biological activity of these peptide fragments was

scored on the Peptide Ranker website (http://distilldeep.ucd.ie/Pepti

deRanker/) [28]. Moreover, ToxinPred was a unique in-silico approach

to predict toxicity of ACE inhibitory peptides [29]. The screening pep-

tides were further screened for free binding energy by molecular dock-

ing using AutoDockZn [30]. The crystal structure for ACE was taken

from the protein databank (PDB code: 4AA1) [31]. All ligands and water

molecules were removed. The peptide with the most favorable binding

energy (increasingly negative value) was considered as a potential

inhibitor.

The peanut peptides chosen were synthesized by Jiangsu Jitai Pep-

tide Industry Technology Co., Ltd. (Yancheng, China) using standard

solid-phase peptide synthesis and their purities and molecular weights

were identied by HPLC and LC-MS analysis separately.

2.3. ACE inhibition assay

ACEI activities were measured based on the method [32]. Briey, 50

L of sample solution (samples were placed in 50 mM borate buffer

solution containing 0.3 M NaCl, pH 8.3) was incubated with 50

L of

ACE (0.25 U/mL) at 37 ◦C for 10 min. The mixture was subsequently

incubated with 150

L of HHL (4.15 mM HHL in 50 mM borate buffer)

for 1 h at 37 ◦C. The reaction was terminated by adding 250

L of 1 M

HCl to the experimental samples, and the blank control group was added

with HCl and incubated for 1 h before adding the substrate. Then, add

500

L of ethyl acetate, shake for 15 s to extract hippuric acid, centrifuge

at 3000 r/min for 10 min, then collect 200

L of supernatant, and

evaporate in an oven at 110 ◦C. The residue was dissolved in 1.0 mL

distilled water, and the absorbance of the solution was measured at 228

nm. ACE inhibitory activity was calculated by the following equation:

ACE inhibition activity (%) = [(C−A)/(C−B) ] × 100

where C is the absorbance without the addition of the sample solution, A

is the absorbance of the presence of ACE with the sample solution, and B

is the absorbance of the blank group (HCl was added to inhibit reaction

before adding HHL substrate).

2.4. ACE inhibitory pattern with different peptides

To explore the inhibition mechanisms, we monitored the effects of

different concentrations of HHL substrate (0.83, 1.38, 2.75, 4.15 mM)

and used different concentrations (0, 1.0, 2.5

M) of peanut peptides.

The reaction mixture also contained different concentrations of peptides

and 0.125 U/g ACE in 50 mM borate buffer. The mixture was incubated

at 37 ◦C for 10 min, and absorbance was monitored at 228 nm. The

pattern of ACE inhibition was determined by Lineweaver-Burk in Origin.

The inhibition constants (K

) were investigated by interpretation of the

Dixon plots, in which the value of the x axis infers -K

[21].

2.5. Stability of synthetic peptides

The stability of peptides was validated by the previously described

method [33] with minor modications. The synthetic peptides were

prepared as solution with pH of 7.0 and concentration of 100

M, be-

sides as a blank control. The samples were water-bathed for 2 h at

different temperatures and pH to determine thermal and pH stability.

100

M peptide solution at pH 2 was added with a certain mass of

pepsin (1:3000) and in a water bath at 37 ◦C for 1 h. After sampling, pH

was adjusted to 7. Then an appropriate amount of trypsin (1:250) was

added and digested in a water bath at 37 ◦C for 1 h. After inactivated in

100 ◦C water bath for 10 min, it was ltered with 0.22

M lter mem-

brane and analyzed by RP-HPLC. The degradation of the synthesized

peptides under different conditions was determined based on the peak

areas and peak emergence times of the liquid phase proles.

2.6. Molecular dynamics simulations

All-atom MD simulation via NAMD package [34] was carried out

based on molecular docking results. The protonation state of each his-

tidine residue was determined according to their respective chemical

environments. TIP3P water model, incorporated by 154 mM NaCl, was

used to imitate the solvent environment. Topology and force eld pa-

rameters were assigned from the CHARMM36 protein parameter set

[35]. In each MD simulation, periodic boundary conditions were utilized

to avoid boundary effect caused by nite size. The van der Waals and

short-range electrostatic interactions were gradually reduced to 0 in the

range of 10 to 12 Å. The particle mesh Ewald method was employed to

compute long-range electrostatic interactions [36]. The simulation sys-

tems were rst subjected to 50,000 steps of energy minimization with

the protein heavy atoms and ligand’s backbone atoms harmonically

constrained by a force constant of 0.5 kcal/mol Å

−2

. Following the

minimization, at the same restraint, 5 ns additional MD simulation in the

canonical (NVT) ensemble were implemented to ensure the temperature

stability by Langevin thermostat [37]. Thereafter, 200 ns MD simula-

tions in the NPT ensemble were run to adequately sample the

J. Liu et al.

文档加载中……请稍候！
如果长时间未打开，您也可以点击刷新试试。

下载文档到电脑，查找使用更方便

1 知币 4人已下载

立即下载

A combined in vitro and in silico study of the inhibitory mechanism of angiotensin-converting enzyme with peanut peptides.pdf

共13页,预览4页

还剩页未读，继续阅读

A combined in vitro and in silico study of the inhibitory mechanism of angiotensin-converting enzyme with peanut peptides

相关推荐

开通VIP享超值会员特权

作者详情

相关内容

推荐作者

热门标签

举报选择: