Study On The Inhibitory Mechanism Of Food-derived Bioactive Peptides On ACE N And C Domains

The research in this article is based on the National Natural Science Foundation of China Youth Science Foundation project "Study on the correlation between protein membrane affinity and ACE inhibitory activity in synthetic cell environment(NO.31601486)".Cardiovascular and cerebrovascular diseases are now the culprit threatening human health.As of 2021,the prevalence of hypertension in my country is 30.2%.It is estimated that there will be 1.05 billion hypertensive patients in the world by 2025.Angiotensin-I converting enzyme(ACE)as a key target of human blood pressure regulation,ACE inhibitors have received extensive attention.With the deepening of ACE research,it is found that the two domains of ACE have different physiological functions.The ACE C domain is the key site that governs the regulation of human blood pressure,while the ACE N domain is closely related to the pathogenesis of diseases such as pulmonary fibrosis.Therefore,effectively inhibiting the activity of each domain of ACE is an important strategy to improve and treat related diseases.Food-derived bioactive peptides have attracted more and more attention due to their mild effects and low side effects.Therefore,this article starts with the expression of ACE N and C domain proteins,and constructs the evaluation system of free ACE N and C domains and the liposome mimicking membrane-bound ACE C domain system,respectively.Using the amino acid substitution method to transform the food source obtained in the previous study ACE inhibitory peptide sequence to investigate the influence of the structure change of the active peptide on the activity of free ACE N and C domains and membrane-bound ACE C domain;with the help of particle swarm optimization support vector machine,the structure-activity relationship model of the active peptides in each system was constructed.Employ molecular docking and molecular dynamics simulations to clarify the inhibitory mechanism from the perspective of the interaction between active peptides and ACE.The main research contents and results are as follows:(1)Construct ACE N domain and ACE C domain recombinant plasmids by pc DNA3.1(+)vector,use CHO-K1 cells for protein expression in ACE N and C domain;AKTA prime plus protein purification system combined was used to purify the protein,and the specific activity of the protein was determined by HPLC.The obtained ACE N and C domain concentrations were 12.09±0.08?g/?L and 8.75±0.14?g/?L.The specific activities were 12.41±0.06 U/mg and 105.07±0.06 U/mg,respectively.The FITC labeled ACE C domain was connected to the simulated membrane,and the fluorescence intensity before and after purification was compared to confirm that the ACE C domain and membrane connections.The enzymatic kinetic parameters of the three ACE systems were determined by HPLC.The Km of the free ACE N domain system was 325.40±142.50?M,the V_max was 3.67±1.06?M/min,and the K_m of the free ACE C domain system was 114.49±29.15?M,V_max was 6.42±0.72?M/min,membrane-bound ACE C domain system Km was 158.44±43.75?M,and Vmax was 10.45±1.47?M/min.(2)From the 21 food-derived bioactive peptides identified in the laboratory,10ACE inhibitory peptides were screened out,namely MPCR,NCQG,GTYW,TYWL,FYCP,TKP,YLPR,YCPI,CVSP and RVPSL.At the same time,amino acid(Arg,Gly,Ile)replacement was performed on RVPSL,and the IC50 of the modified active peptide was mainly within 100?M.Integrating the inhibitory activities of 10 active peptides and 14 modified peptides on different domains of ACE,it is found that the active peptides containing Gly show a significant inhibitory tendency to the ACE N domain.When Gly replaces the C-terminus of the active peptide,its inhibitory ability was high.Compared with Arg and Gly replacement,the active peptide containing Ile has higher inhibitory activity on the ACE C domain.The results of molecular docking showed that the modified peptide of RVPSL and the ACE N and C domains are mainly hydrogen bonds to maintain the stability of the system.The key amino acid residues on the ACE N domain are Asp43,Ala334 and Asn494;and the key amino acids on the ACE C domain Residues are Asn66,Ala356,Asp358 and Glu403.(3)In order to explore the correlation between the amino acid sequence change of the active peptide and the inhibitory activity of the ACE N domain,the ACE inhibitory peptides LAPYK and QIGLF were further modified using Arg,Gly,and Ile,and an active peptide library containing 54 peptides was obtained.And use particle swarm optimization support vector machine(PSO-SVM)to construct a structure-activity relationship model.Through the open source software package Pa DEL-Descriptor,the structural characteristics,spatial characteristics,thermodynamic characteristics and electronic characteristics of the modified peptide were parameterized,and the measured ACE N domain inhibitory activity IC50 was used as the output item,and the PSO-SVM prediction model was established.After PSO optimization,the optimal penalty parameter c of the objective function is 8.8763,the optimal kernel function parameter g is 3.6213,the test set R² is 0.86,and the MSE value is 1.21×10^-3.There is a good relationship between the experimental value and the predicted value.The molecular docking results showed that both LAPYK and QIGLF modified peptides combined with the S1'active pocket on the ACE N domain,while the RVPSL modified peptide combined with the S1 active pocket.The basic sequence of the peptide was related to the active peptide on the ACE N domain.The binding site has a greater impact.The key amino acid residues of ACE N domain are Gln259,His331,Ala332,Thr358 and Tyr501.(4)Based on the obtained active peptide library,a particle swarm optimization support vector machine(PSO-SVM)structure-activity relationship model was constructed for the free and membrane-bound ACE C domain system.After PSO optimization,the optimal penalty parameter c of the objective function is 5.3296 and0.6621,the optimal kernel function parameter g is 2.0874 and 1.7649,the test set R² is0.72 and 0.67,and the MSE value is 1.06×10^-3 and 3.02×10^-4,there is a good correlation between the experimental value and the predicted value,and the prediction effect is reliable.The dynamic calculation system of RVPSL and free and membrane-bound ACE C domain was constructed using Gromacs.After 100 ns of calculation and balance,RVPSL in both systems had close contact with His387 and Glu411 in the HEXXH motif,and tightly bound to the active center of the ACE C domain.At the same time,the RVPSL-ACE C domain in the membrane bound system was more stable,but the aggregation of protein was weakened due to the existence of membrane structure.