Research On ACE Inhibition Mechanism Of Food-Derived Peptides Based On The Membrane Phase Separation Regulation

The research of this paper relies on the National Natural Science Foundation of China’s general project,"The mechanism of egg white peptides regulating lipid phase in synthetic cell membrane-coupled ACE system(NO.31972096)".Angiotensin Iconverting enzyme(ACE)is a key target in the human blood pressure regulation system.Using food-derived peptides to inhibit ACE activity is an important strategy for regulating human blood pressure.The ACE inhibitory mechanisms of food-derived peptides currently reported mainly focuses on their regulation of ACE and its upstream and downstream signaling pathway proteins,ignoring the influence of cell membrane on ACE.Cell membrane phospholipid bilayers will spontaneously phase-separate into sordered(liquid-ordered,Lo)and disordered(liquid-disordered,Ld)phases;ACE as a membrane protein,the interaction between food-derived peptides and cell membrane lipids leads to changes in the phase separation of the cell membrane,which is the basis for the indirect regulation of membrane ACE activity by peptides;however,the mechanism of inhibition of ACE activity by food-derived peptides based on phase separation regulation is still unclear.Therefore,if the ACE inhibitory mechanism of food-derived peptides involved in phase separation can be clarified,the production efficiency of ACE inhibitory peptides will be significantly improved,which is of great significance for the functional development and processing screening of food-derived peptides.In this paper,human umbilical vein endothelial cells(Human umbilical vein endothelial cells,HUVECs)were first used to construct a cell membrane ACE evaluation system,and the ability of the peptides to regulate membrane ACE and cell membrane was confirmed;the in vitro giant unilamellar vesicles(Giant unilamellar vesicles,GUVs)model was used to construct phase separation regulation evaluation system,and the phase separation regulation effect and mechanism of the peptides were clarified;combined with HUVECs and GUVs models,the correlation between the membrane ACE inhibitory activity of the peptides and the phase separation regulation ability was systematically studied,and the phase separation regulation effect and mechanism of membrane ACE inhibitory activity of the peptides was clarified;finally,the peptide-bilayer membrane system and the peptide-phase separation simulated membrane system were constructed using molecular dynamics simulation technology,and the interaction mode between membrane ACE inhibitory peptides and membrane lipid molecules and phase separation mechanism were explored from the shallower to the deeper.The main research content and results of this paper are as follows:(1)HUVECs were used to construct a cell membrane ACE evaluation system;methyl-β-cyclodextrin(MβCD)was used to study the relationship between ACE activity and cell membrane phase separation.The effect of peptides on cell survival was determined by the MTS method;the expression and localization of ACE on the cell membrane were analyzed by combining Western blot and immunofluorescence staining;the cell membrane was labeled with fluorescent molecular probes to determine the cell membrane properties such as membrane fluidity,electric potential,and permeability.The results showed that the peptides QVPLW and LCAY showed high inhibitory activity of cell membrane ACE;the IC50 values in the control group were 238.46 ±11.35 μM and 31.55 ± 2.64 μM,respectively,and in the MβCD group were 45.43 ±6.15 μM and 34.69 ± 1.59 μM.QVPLW and LCAY have no inhibitory and toxic effects on cell growth and can inhibit ACE activity by preventing ACE from recruiting to the Lo phase.In addition,QVPLW and LCAY can ultimately promote the translocation of ACE between the Lo and Ld phases of the cell membrane by changing the properties of the cell membrane,including fluidity,potential,and permeability.(2)GUVs were used to construct a phase separation evaluation system;the morphology and integrity of the GUVs membrane encapsulating calcein were observed;binding of the peptide to the membrane was measured;fluorescence resonance energy transfer(F?rster resonance energy transfer,FRET)efficiency and generalized polarization(Generalized polarization,GP)values were measured to analyze the effect of peptides on the arrangement and order of lipid molecules;phase separation was evaluated by fluorescent staining and statistical analysis;steady-state fluorescence quenching was used to determine the localization of the peptides in the membrane,and the relationship between the phase separation regulation of the peptides and the membrane localization was further clarified by correlation analysis.Five peptides(LCAY,QVPLW,YYK,CLA,and SLW)can increase the Lo phase,and one peptide,WLQ,was found to decrease the Lo phase.LCAY and QVPLW can bind to GUVs and localize in the amphipathic region of the membrane,where they increase the Lo phase by rearranging lipids and ordering the Ld phase.Through hydrogen bonds and hydrophobic interactions,LCAY and QVPLW can play a role similar to cholesterol in regulating the phase separation.The ability of peptides to regulate phase separation depends on their localization in the amphiphilic region of the membrane.For the three peptides YYK,CLA and SLW,the more the peptide is positioned in the amphiphilic region of the membrane,the stronger the ability to increase the Lo phase.(3)HUVECs and GUVs were used to explore the correlation between the membrane ACE inhibitory activity of the peptides and the ability to regulate phase separation,and the HUVECs model was used to evaluate the cell membrane ACE inhibitory activity of the peptides;the Lo phase stained with a specific fluorescent dye was observed by laser confocal microscopy to evaluate the phase separation regulation of the peptides;the effect of the modified peptides on the membrane phase state of GUVs was detected by an environment-sensitive fluorescent probe;the relationship between the membrane ACE inhibitory activity of the peptides and their phase separation,and membrane phase regulation ability were explored by correlation analysis.The results showed that the phase separation regulation of tripeptides was independent of their membrane ACE inhibitory activity,while the phase separation regulation of tetrapeptides and pentapeptides was highly correlated with their membrane ACE inhibitory activity.For tetrapeptides and pentapeptides(YLFKD,IWNLN,LCAY,GTYW,and QVPLW),the more they increased the Lo phase,the higher the membrane ACE activity,and correspondingly the lower their membrane ACE inhibitory activity.For sequence-modified tetrapeptides,the membrane phase regulation ability of the peptides was related to their membrane ACE inhibitory activity;the stronger their ability to increase membrane phase and lipid order,the higher the membrane ACE activity,and correspondingly the lower their membrane ACE inhibitory activity.(4)Molecular dynamics simulation technology was used to study the specific molecular mechanism of phase separation regulation.Different simulation systems from ns to μs were used to gradually solve the interaction mode between peptides and membrane bilayer lipid molecules and separate regulatory mechanisms.The research results show that the leucine(Leu,L)and alanine(Ala,A)of the alkyl side chain of LCAY were deeply inserted into the membrane;the peptide chain of QVPLW was inserted into the membrane in a "V" shape;the modified peptide FCAY N-terminal phenylalanine(Phe,F)and A were inserted into the membrane.LCAY and QVPLW can aggregate cholesterol in the upper membrane,and the aggregation effect of LCAY on cholesterol was more evident than that of QVPLW.The peptide was located in the amphiphilic region of the bilayer membrane,and the lipids surrounding the peptide were mainly phosphatidylcholine head groups and saturated palmitoyl chain tails.The terminal amino acids of the peptides,especially the N-terminal amino acids,contributed the most to the hydrogen bonds between peptide membranes.The N-terminal amino acids leucine L and glutamine(Gln,Q)of LCAY and QVPLW contributed 46% and 68%of the total hydrogen bonds respectively.The N-terminal phenylalanine F of peptide FCAY contributed the most to the binding energy,which was-43.27±2.83 KJ/mol.It played the most important role in the binding process of peptides and lipids.FCAY always tended to distribute the Ld phase at the phase interface to promote the further formation of phase separation.