Method for screening antioxidant peptide according to Keap1 protein and antioxidant peptide product

Abstract

The invention provides a method for screening antioxidant peptide according to Keap1 protein and an antioxidant peptide product, and belongs to the technical field of biological medicine. According to the method provided by the invention, the binding capacity of the peptide and the Keap1 active site is verified by utilizing a molecular docking method, and the peptide which can be mutually bound with the Keap1 active site is selected, namely the antioxidant peptide. The antioxidant peptide screened by the method is combined with a Keap1 active site through interaction of a hydrogen bond, Van der Waals force and an alkyl group, and the resistance of cells to oxidative damage induced by H2O2 is enhanced by activating a Keap1-Nrf2 pathway, so that the antioxidant effect is exerted. The antioxidant peptide has better antioxidant activity, has the characteristics of safety and no toxicity, and has very important significance for developing products with antioxidant functions and anti-aging functions.

Description

[0001] The present invention claims priority to Chinese patent application No. 2024113323349 filed on September 24, 2024, entitled “A method for screening antioxidant peptides based on Keap1 protein and antioxidant peptide products”, the entire contents of which are incorporated by reference in this application. Technical Field

[0002] The present invention belongs to the technical field of biomedicine, and specifically relates to a method for screening antioxidant peptides based on Keap1 protein and an antioxidant peptide product. Background Art

[0003] Reactive oxygen species (ROS) are byproducts of normal cell metabolism. Although a certain level of ROS is essential for physiological processes, their excessive accumulation can lead to oxidative stress and damage human health. Oxidative stress can lead to harmful effects such as protein cross-linking, lipid peroxidation, RNA and DNA damage, and thus develop into chronic diseases. Antioxidants are substances that can capture and neutralize ROS, thereby reducing their harmful damage to the human body. Therefore, supplementing antioxidants in food and medicine is the preferred strategy to combat excessive ROS damage. Considering the adverse health effects of artificial antioxidants, such as carcinogenicity and high cost, people are increasingly inclined to explore natural, efficient and non-toxic alternatives. It is particularly important to find safe antioxidant peptides in natural food sources. These peptides can be obtained through a variety of routes such as microbial fermentation, digestive enzymatic hydrolysis, and chemical synthesis, and most of the peptides with antioxidant activity are composed of 2-20 amino acids, with a molecular weight between 500-1500Da, containing a certain amount of hydrophobic amino acids and aromatic amino acids. At the same time, the presence of hydrophobic amino acids in the N-terminal or C-terminal region can also enhance the antioxidant activity of the peptide.

[0004] As a research hotspot in food science and nutrition, antioxidant peptides can play a role by scavenging free radicals, inhibiting peroxides and chelating metal ions. Therefore, how to effectively discover and screen antioxidant peptides has become a key issue in research and production. Traditional discovery methods are time-consuming and expensive, and it is also challenging to solve the quantitative structure-activity relationship of antioxidant peptides. Some new technologies, including molecular docking, artificial intelligence (AI), bioinformatics, etc., are providing new ideas as a solution to the problems brought by traditional methods. These new technologies have the effective ability to discover antioxidant peptides, that is, they have the potential for high-throughput screening. In addition, combining these new technologies can overcome the shortcomings of a single technology, thereby improving efficiency and expanding the scope of discovery. Among them, molecular docking is an effective method for evaluating antioxidant activity, elucidating antioxidant mechanisms, and predicting the quantitative structure-activity relationship of antioxidant peptides. More importantly, molecular docking predicts the ability of antioxidant peptides to bind to key targets of signaling pathways. The Keap1-Nrf2 signaling pathway is its main representative. Antioxidant peptides bind to the Kelch domain of Keap1, block the interaction of Keap1-Nrf2, and release Nrf2 to translocate to the nucleus. Nrf2 binds to the antioxidant response element (ARE), promotes the transcription of antioxidant-related genes, and exerts an antioxidant effect.

[0005] Chinese patent CN202211407894.7 discloses a screening of bovine whey protein-derived antioxidant peptides based on molecular simulation technology, Chinese patent CN202410486383.1 discloses a synthetic antioxidant peptide Pep9 and its application, and Chinese patent CN202211404356.2 discloses a screening of whey protein-derived antioxidant peptides based on a static digestion model for the elderly. Molecular docking, as a commonly used method for predicting the antioxidant capacity and mechanism of antioxidant peptides, is a powerful technology. It can reduce a large number of candidate peptides to a small number, promote more targeted experimental research in the future, not only save a lot of experimental time, but also make more effective use of limited research resources. However, there are few reports on invention patents that combine molecular docking technology with cell-level verification experiments to screen antioxidant peptides. Therefore, the development of a method for screening antioxidant peptides based on molecular docking and cell-level research methods has certain guiding significance for further understanding the antioxidant mechanism of polypeptides, and can provide a certain theoretical basis for the subsequent screening of antioxidant peptides. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for screening antioxidant peptides based on Keap1 protein and an antioxidant peptide product. The method provided by the present invention uses a molecular docking method to verify the binding ability of the peptide to the Keap1 active site, and selects a peptide that can bind to the Keap1 active site, that is, an antioxidant peptide. The antioxidant peptide screened by this method binds to the Keap1 active site through hydrogen bonds, van der Waals forces and alkyl interactions, and activates the Keap1-Nrf2 pathway to enhance the resistance of cells to H2O2-induced oxidative damage, thereby exerting an antioxidant effect. The antioxidant peptide has good antioxidant activity and is safe and non-toxic, which is of great significance for the development of products with antioxidant and anti-aging functions.

[0007] The technical solution of the present invention includes:

[0008] In a first aspect, the present invention provides a method for screening antioxidant peptides based on Keap1 protein, the method comprising: using a molecular docking method to verify the binding ability of the peptide to the Keap1 active site, and selecting a peptide that can bind to the Keap1 active site, namely, an antioxidant peptide.

[0009] Specifically, the antioxidant peptide is the antioxidant peptide VALALARE and / or the antioxidant peptide LEASPEV.

[0010] Preferably, the antioxidant peptide VALALARE has an amino acid sequence as shown in SEQ ID NO. 1. SEQ ID NO. 1: VALALARE.

[0011] Preferably, the antioxidant peptide LEASPEV has an amino acid sequence as shown in SEQ ID NO. 2. SEQ ID NO. 2: LEASPEVI.

[0012] Specifically, the Keap1 active sites include: ALA366, ALA607, ARG326, ALY466, ASN469, CYS368, GLN563, GLY509, GLY564, HIS562, ILE559, LEU365, LEU468, THR560, VAL369, VAL418, VAL463, VAL467, VAL512, VAL514, and VAL561.

[0013] Preferably, the antioxidant peptide VALALARE binds to VAL512, VAL467, GLY564, GLN563, ARG326, VAL418, LEU468, ALA607, ALA466, CYS368, ALA366, ASN469, THR560 and HIS562.

[0014] Preferably, the antioxidant peptide LEASPEV is combined with VAL514, ARG326, VAL512, VAL467, VAL561, ALA607, CYS368, ILE559, VAL369, VAL418, VAL608, VAL463, GLY509, LEU365 and ALA466.

[0015] Preferably, the binding modes of the peptide to the active site of Keap1 include one or more of hydrogen bonding, van der Waals forces and alkyl interactions.

[0016] In a second aspect, the present invention provides an antioxidant peptide screened by the above method, wherein the antioxidant peptide is the antioxidant peptide VALALARE, having an amino acid sequence as shown in SEQ ID NO.1.

[0017] In a third aspect, the present invention provides an antioxidant peptide screened by the above method, wherein the antioxidant peptide is the antioxidant peptide LEASPEVI, having an amino acid sequence as shown in SEQ ID NO.2.

[0018] In a fourth aspect, the present invention provides nucleotides encoding the above-mentioned antioxidant peptides, wherein the antioxidant peptides are the antioxidant peptide VALALARE and / or the antioxidant peptide LEASPEVI.

[0019] In a fifth aspect, the present invention provides a method for preparing the above-mentioned antioxidant peptides, wherein the antioxidant peptides are antioxidant peptides VALALARE and / or antioxidant peptides LEASPEVI.

[0020] Preferably, the preparation method is: preparation by solid phase synthesis.

[0021] In a sixth aspect, the present invention provides the use of the above-mentioned antioxidant peptides in the preparation of antioxidant products or anti-aging products, wherein the antioxidant peptides are the antioxidant peptide VALALARE and / or the antioxidant peptide LEASPEVI.

[0022] Preferably, the antioxidant product includes food additives, health products, medicines or cosmetics.

[0023] Preferably, the anti-aging product comprises a medicine or a cosmetic.

[0024] In a seventh aspect, the present invention provides an antioxidant product, comprising the above-mentioned antioxidant peptide, the antioxidant product includes a food additive, a health product, a medicine or a cosmetic, and the antioxidant peptide is the antioxidant peptide VALALARE and / or the antioxidant peptide LEASPEVI.

[0025] In an eighth aspect, the present invention provides an anti-aging product, wherein the anti-aging product comprises the above-mentioned antioxidant peptide, the anti-aging product includes a medicine or a cosmetic, and the antioxidant peptide is the antioxidant peptide VALALARE and / or the antioxidant peptide LEASPEVI.

[0026] The beneficial effects of the present invention are:

[0027] (1) The present invention uses a molecular docking method to verify the binding mode of two antioxidant peptides VALALARE and LEASPEVI with the active site of Keap1. The results show that the antioxidant peptides have a good match with the Keap1 protein and have a strong binding ability, indicating that the two peptides are potential antioxidant peptides. Among them, the binding of the peptides to the ALA607, VAL514 and ASN469 amino acid residues of Keap1 has not been reported.

[0028] (2) Most of the antioxidant peptides isolated in previous patents are mixtures, and their antioxidant activity at the cellular level is not reflected. However, the antioxidant peptides isolated and purified by the present invention have better activity evaluation at the cellular level, which proves that the functionality of the antioxidant peptides provided by the present invention is more complete, making the invention more convincing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the MS spectrum of the antioxidant peptide VALALARE.

[0030] Figure 2 This is the MS spectrum of the antioxidant peptide LEASPEVI.

[0031] Figure 3 The structure of antioxidant peptides; A in the figure is the structure of antioxidant peptide VALALARE; B is the structure of antioxidant peptide LEASPEVI.

[0032] Figure 4 Figure 1 is the binding mode of the antioxidant peptide VALALARE and the active site of Keap1; A in the figure is the 3D mode of the docking of the antioxidant peptide VALALARE and Keap1; B is the 2D mode of the docking of the antioxidant peptide VALALARE and Keap1.

[0033] Figure 5 Figure 1 is the binding mode of the antioxidant peptide LEASPEVI and the active site of Keap1; A in the figure is the 3D mode of the antioxidant peptide LEASPEVI docking with Keap1; B is the 2D mode of the antioxidant peptide LEASPEVI docking with Keap1.

[0034] Figure 6 This is the result of MTT experiment.

[0035] Figure 7For the cytoprotective effect of antioxidant peptides on cells.

[0036] Figure 8 The effect of antioxidant peptides on ROS generation. DETAILED DESCRIPTION

[0037] The present invention is described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0038] Example 1 Synthesis of Antioxidant Peptides

[0039] The amino acid sequence of the antioxidant peptide VALALARE is shown in SEQ ID NO.1, the amino acid sequence of the antioxidant peptide LEASPEVI is shown in SEQ ID NO.2, the amino acid sequence of polypeptide A is shown in SEQ ID NO.3, and the amino acid sequence of polypeptide B is shown in SEQ ID NO.4.

[0040] SEQ ID NO.3: VALA.

[0041] SEQ ID NO.4: LEAS.

[0042] We commissioned Sangon Biotech (Shanghai) Co., Ltd. to synthesize antioxidant peptides and polypeptides by solid phase synthesis. The purity of the obtained antioxidant peptide VALALARE was 98.184%, the purity of the antioxidant peptide LEASPEVI was 95.969%, and the purity of polypeptide A and polypeptide B was more than 95%. The MS spectrum of the antioxidant peptide VALALARE is shown in the figure below. Figure 1 As shown, the MS spectrum of the antioxidant peptide LEASPEVI is as follows Figure 2 shown.

[0043] Example 2 Molecular docking

[0044] The AutoDock Vina program was used to study the binding configuration of antioxidant peptides and proteins (PDB: 4L7B). The structural formula of the substrate peptide was drawn using Chemdraw software, and its energy was optimized using Chem3D software and saved as a pdb format file. The pdb file of the small peptide was charged and processed using AutoDock Tools 1.5.6, and finally saved as a pdbqt format file. The AutoDock Tools 1.5.6 graphical interface was used to add polar hydrogen atoms and charges to the protein, and the receptor PDB format file was converted to pdbqt format. Then, using the protein molecule as the search space, a suitable grid was established in the x, y and z axis directions with an exhaustiveness of 8. Finally, the molecular docking was completed according to the standard procedure.

[0045] The structures of the antioxidant peptides VALALARE and LEASPEVI are as follows Figure 3 The binding mode with the active site of Keap1 is shown in Figure 4 and Figure 5 As shown. The results showed that the antioxidant peptides can be successfully inserted into the pocket of Keap1. Among them, VALALARE can establish hydrogen bonds with VAL512, VAL467, GLY564, GLN563, ARG326 and VAL418, and form alkyl interactions with LEU468, ALA607, ALA466, CYS368 and ALA366. In addition, it forms van der Waals forces with residues such as ASN469, THR560 and HIS562. LEASPEVI interacts with VAL514, ARG326 and VAL512 of Keap1 through hydrogen bonds, and the alkyl interactions involve VAL467, VAL561, ALA607 and CYS368. In addition, van der Waals forces are formed with ILE559, VAL369, VAL418, VAL608, VAL463, GLY509, LEU365 and ALA466.

[0046] The antioxidant peptides VALALARE and LEASPEVI have a good match with Keap1 protein and strong binding ability, while peptide A and peptide B cannot dock with Keap1, indicating that in terms of molecular docking, peptides A and B have no antioxidant activity. The molecular docking results show that the antioxidant peptides VALALARE and LEASPEVI have a good match with Keap1 protein and strong binding ability, indicating that these two antioxidant peptides are potential antioxidants.

[0047] Example 3 MTT experiment

[0048] The MTT method was used to detect the cytotoxicity of antioxidant peptides on HepG2 cells. Cells in the logarithmic growth phase were collected and cultured at 7×10 3The cells were plated at a density of 100 μL / well on a 96-well plate. After 24 hours of incubation, 100 μL of antioxidant peptide VALALARE solution (0.5, 5, 50, 100 and 500 μg / mL) or antioxidant peptide LEASPEVI solution (0.5, 5, 50, 100 and 500 μg / mL) were added and incubated for 48 hours as the VALALARE group and LEASPEVI group. The control group (Control) was replaced with complete culture medium. After incubation, 20 μL of MTT solution (5 mg / mL) was added for 3 hours. The supernatant was discarded by centrifugation and dissolved in 150 μL of DMSO. The absorbance at 490 nm was then measured using an enzyme reader (Spark, TECAN, Austria) to detect cell viability.

[0049] HepG2 cells were purchased from the Cell Resource Center of Peking Union Medical College. They are derived from human liver cancer and have a high degree of differentiation ability. They are very similar to human primary hepatocytes in terms of metabolism and biotransformation activity. Therefore, HepG2 cells are widely used in in vitro testing. The results of the MTT experiment are shown in Figure 2. Figure 6 As shown, compared with the control group (Control), the cell survival rates of the VALALARE group and the LEASPEVI group were between 88.33% and 104.62% at a concentration of 0.5-500 μg / mL, proving that the two antioxidant peptides did not show obvious toxicity. According to the above method, the MTT experiment was performed on polypeptide A and polypeptide B. When polypeptide A and polypeptide B were at 0.5-500 μg / mL, the cell survival rates were between 84.82% and 108.47%, and the results showed that polypeptide A and polypeptide B did not show obvious toxicity.

[0050] Example 4 Protective effect of antioxidant peptides on H2O2-induced damage in HepG2 cells

[0051] In order to establish the oxidative damage model of HepG2 cells, 1 mM hydrogen peroxide was used. HepG2 cells were cultured at 7 × 10 3 / well were seeded in a 96-well plate and incubated for 24 hours. The damage group (Damage) and the control group (Control) were treated with culture medium (100 μL) for 24 hours, and the VALALARE group and LEASPEVI group were treated with 100 μL of antioxidant peptide VALALARE solution (25 and 100 μg / mL) or antioxidant peptide LEASPEVI solution (25 and 100 μg / mL) for 24 hours. After incubation, 50 μL of H2O2 solution was applied to the damage group, VALALARE group, and LEASPEVI group for 3 hours. The cell viability was then detected using the MTT method.

[0052] Antioxidant peptides have cytoprotective effects on cells such as Figure 7As shown. Compared with the control group (Control), the cell viability of the damage group (Damage) was significantly decreased to 49.02±3.05% (P<0.05), confirming that the cell damage model was successfully established. Compared with the damage group (Damage), the two antioxidant peptides increased cell viability to varying degrees. When the concentration of antioxidant peptides was 25μg / mL, the viability of HepG2 cells in the VALALARE group increased from 49.02±3.05% to 65.18±3.03%, and the viability of HepG2 cells in the LEASPEVI group increased from 49.02±3.05% to 66.23±4.70%. When the concentration of antioxidant peptides increased to 100 μg / mL, the viability of HepG2 cells was significantly enhanced. The viability of HepG2 cells in the VALALARE group increased from 49.02±3.05% to 71.62±6.69%, and the viability of HepG2 cells in the LEASPEVI group increased from 49.02±3.05% to 82.38±19.16%. The results showed that both antioxidant peptides had significant cell protective effects on H2O2-induced oxidative damage in HepG2 cells. The protective effects of polypeptide A and polypeptide B on H2O2-induced HepG2 cells were tested according to the above method. The results showed that the protective effects of antioxidant peptides LEASPEVI and LEASPEVI on H2O2-induced HepG2 cells were higher than those of polypeptides A and B.

[0053] Example 5 Effects of Antioxidant Peptides on ROS Production in H2O2-Induced Damage in HepG2 Cells

[0054] HepG2 cells were cultured at 7×10 3 / well were seeded in a 96-well plate and incubated for 24 hours. The damage group (Damage) and the control group (Control) were treated with culture medium (100μL) for 24 hours, and the VALALARE group and LEASPEVI group were treated with 100μL antioxidant peptide VALALARE solution (25 and 100μg / mL) or antioxidant peptide LEASPEVI solution (25 and 100μg / mL) for 24 hours. After incubation, 50μL of H2O2 solution was applied to the damage group, VALALARE group and LEASPEVI group for 3 hours. The cells were exposed to 10μM DCFH-DA solution and incubated at 37℃ for 30 minutes. After the end, the extracellular DCFH-DA was removed and the cells were rinsed three times with PBS. The fluorescence intensity was recorded using a microplate reader. The emission wavelength was 528nm and the excitation wavelength was 485nm.

[0055] Effects of antioxidant peptides on ROS generation Figure 8As shown. Compared with the control group (Control), the intracellular ROS content of the damage group (Damage) increased significantly to 332.02±31.85%, which means that H2O2 greatly increased the intracellular ROS level compared with the control group (Control) (P<0.05). Compared with the damage group (Damage), the two antioxidant peptides can reduce the intracellular ROS level to varying degrees, and the effect is more obvious when the concentration of antioxidant peptides is higher. At 25μg / mL, the antioxidant peptides VALALARE and LEASPEVI reduced the ROS level from 332.02±31.85% to 248.19±59.28% and 230.39±43.48%, respectively. At 100μg / mL, the antioxidant peptides VALALARE and LEASPEVI reduced the ROS level from 332.02±31.85% to 182.21±39.94% and 166.23±17.46%, respectively. The results showed that both antioxidant peptides could effectively inhibit the generation and accumulation of intracellular ROS, thereby protecting cells from oxidative damage. The effects of polypeptide A and polypeptide B on the generation of ROS in HepG2 cells induced by H2O2 were investigated according to the above method. The results showed that the antioxidant peptides LEASPEVI and LEASPEVI had better inhibitory effects on ROS in HepG2 cells induced by H2O2 than polypeptide A and polypeptide B.

[0056] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A method for screening antioxidant peptides based on Keap1 protein, characterized in that: The method comprises: using a molecular docking method to verify the binding ability of the peptide to the active site of Keap1, and selecting a peptide that binds to the active site of Keap1 as an antioxidant peptide; The antioxidant peptide is the antioxidant peptide VALALARE and / or the antioxidant peptide LEASPEVI; the antioxidant peptide VALALARE has an amino acid sequence as shown in SEQ ID NO.1; the antioxidant peptide LEASPEVI has an amino acid sequence as shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that The Keap1 active sites include: one or more of ALA366, ALA607, ARG326, ALY466, ASN469, CYS368, GLN563, GLY509, GLY564, HIS562, ILE559, LEU365, LEU468, THR560, VAL369, VAL418, VAL463, VAL467, VAL512, VAL514, and VAL561.

3. The method according to claim 1, characterized in that The binding modes of the peptide to the active site of Keap1 include one or more of hydrogen bond, van der Waals force and alkyl interaction.

4. The antioxidant peptide obtained by screening by the method according to any one of claims 1 to 3, characterized in that: The antioxidant peptide is the antioxidant peptide VALALARE, having an amino acid sequence as shown in SEQ ID NO.1; and / or, the antioxidant peptide is the antioxidant peptide LEASPEVI, having an amino acid sequence as shown in SEQ ID NO.

2.

5. The method for preparing the antioxidant peptide according to claim 4, characterized in that: The preparation method is: preparation by solid phase synthesis.

6. Use of the antioxidant peptide according to claim 4 in the preparation of antioxidant products or anti-aging products.

7. The use according to claim 6, characterized in that: The antioxidant products include food additives, health products, medicines or cosmetics.

8. The use according to claim 6, characterized in that: The anti-aging products include medicines or cosmetics.

9. An antioxidant product, characterized in that: The antioxidant product comprises the antioxidant peptide according to claim 4, and the antioxidant product comprises a food additive, a health product, a medicine or a cosmetic.

10. An anti-aging product, characterized in that: The anti-aging product comprises the antioxidant peptide according to claim 4, and the anti-aging product includes medicines or cosmetics.

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