Identification and molecular mechanism of novel ACE inhibitory peptides from goat milk protein A combined in silico and in vitro study

Fig.1 General description of the proteolytic system of goat milk and the distribution of bioactive peptide species based on online simulation. (A)The enrichment ability of different enzymatic hydrolysis systems to inhibit the function of ACE peptides. (B, C) Distribution of bioactive peptide species after enzymatic hydrolysis of casein and whey proteins. Fig.2 Study of the screening of conditions and components of goat milk proteolysis. (A-D) Optimal digestion conditions for casein in proteinase K and papain. (E-H) Optimal digestion conditions for whey proteins in proteinase K and papain. (I, K) Effect of enzyme addition sequence on the degree of protein hydrolysis and ACEI rate in goat milk. (J, L) Distribution of ACEI rates for different components, casein and whey proteins. Fig.3 The 19 potential ACEI peptides were virtually screened from 698 active peptides. (A) The distribution of 698 active peptides. (B) Potential ACEI peptides were virtually screened by PeptideRanker, and CDocker. (C) Molecular weight, logarithmic value of peptide identification score and intensity by LC-MS/MS, and source distribution of enzymatic hydrolysis of 224 potential ACEI peptides. (D) Molecular weight, sequence, logarithmic value of peptide identification score by LC-MS/MS, and source distribution of potential ACEI peptides for 19 of the 698 peptides. Fig.4 Underlying molecular mechanisms of identified peptides from goat milk against ACE revealed by molecular docking. (A-I) Three-dimensional and two-dimensional diagrams of FKF (A), FRY (B), MPFPK (C), RWL (D), WFK (E), WKP (F), VPP (G), IPP (H), and lisinopril (I) binding to ACE, respectively. (J) The statistics of non-bonded interaction types of ligands (FKF, FRY, MPFPK, RWL, WFK, WKP, VPP, IPP, and lisinopril) interacting with ACE. (K) Heatmap analysis of the number of conventional hydrogen bonds interacting with ACE (S1, S2, S1’, and Zn (II) are the core residues of active sites with ACE). Numerical values indicate the quality and number of conventional hydrogen bonds interactions with active pockets in ACE. Fig.5 Changes in systolic blood pressure (SBP) in spontaneously hypertensive rats after administration of Saline Solution (A), Captopril (5 mg/kg ip, B) or synthetic peptides (100 mg/kg ip): FKF (C) and FRY (D).图1 山羊乳蛋白水解系统的总体描述及基于在线模拟的生物活性肽种类分布。(A) 不同酶解体系对血管紧张素转换酶（Angiotensin-Converting Enzyme，ACE）抑制肽功能的富集能力。(B、C) 酪蛋白与乳清蛋白经酶解后所得生物活性肽的种类分布。 图2 山羊乳蛋白水解条件与组分筛选研究。(A-D) 蛋白酶K与木瓜蛋白酶对酪蛋白的最优消化条件。(E-H) 蛋白酶K与木瓜蛋白酶对乳清蛋白的最优消化条件。(I、K) 酶添加顺序对山羊乳蛋白水解度及血管紧张素转换酶抑制率（Angiotensin-Converting Enzyme Inhibitory Rate，ACEI rate）的影响。(J、L) 不同组分（酪蛋白与乳清蛋白）的ACEI率分布。 图3 从698条活性肽中虚拟筛选得到19条潜在ACE抑制肽。(A) 698条活性肽的分布情况。(B) 通过PeptideRanker与CDocker虚拟筛选潜在ACE抑制肽的结果。(C) 224条潜在ACE抑制肽的分子量、液相色谱-串联质谱（Liquid Chromatography-Tandem Mass Spectrometry，LC-MS/MS）鉴定得分的对数值与强度分布，以及其酶解来源分布。(D) 698条肽中筛选得到的19条潜在ACE抑制肽的分子量、序列、LC-MS/MS鉴定得分的对数值及其酶解来源分布。 图4 分子对接揭示山羊乳来源鉴定肽对抗ACE的潜在分子机制。(A-I) 分别为FKF、FRY、MPFPK、RWL、WFK、WKP、VPP、IPP与赖诺普利与ACE结合的三维与二维作用示意图。(J) 配体（FKF、FRY、MPFPK、RWL、WFK、WKP、VPP、IPP与赖诺普利）与ACE相互作用的非键相互作用类型统计。(K) 与ACE活性位点核心残基（S1、S2、S1’与Zn(II)）相互作用的常规氢键数量热图分析，数值代表与ACE活性口袋结合的常规氢键相互作用的质量与数量。 图5 自发性高血压大鼠经生理盐水(A)、卡托普利（5 mg/kg 腹腔注射，ip，B）或合成肽（100 mg/kg ip）FKF(C)与FRY(D)给药后的收缩压（Systolic Blood Pressure，SBP）变化。