Milk protein source urokinase-type plasminogen activator inhibitor peptide and use thereof

Abstract

The application provides a milk protein source urokinase-type plasminogen activator inhibitory peptide and application thereof. Through a combination of virtual screening by molecular docking and in-vitro experiment verification, two u-PA inhibitory activity polypeptides PEVMGVSK and HIQKEDVPSE derived from bovine milk casein are obtained. The two polypeptides can be stably combined with the u-PA active site through multiple intermolecular forces, have a significant inhibitory effect on u-PA-mediated plasminogen activation, can effectively improve the storage period stability of ultra-high temperature instant sterilized milk, delay protein hydrolysis and aging gel formation, and can inhibit the plasmin clot lysis and MDA-MB-231 human breast cancer cell migration in a concentration-dependent manner. The polypeptides obtained by the application have application potential and practical value in the fields of dairy product quality improvement, hemostatic drugs and anti-cancer drug research, etc. as natural source active ingredients.

Description

Technical Field

[0001] This invention belongs to the field of bioactive polypeptide technology, and relates to a milk protein-derived urokinase plasminogen activator inhibitory peptide and its application. Specifically, it relates to a milk protein-derived polypeptide with urokinase plasminogen activator (u-PA) inhibitory activity, and the application of this polypeptide in food processing, hemostatic drugs and anticancer drug development. Background Technology

[0002] The fibrinolytic system is a core component in maintaining the body's coagulation-fibrinolysis balance, and its stable function is crucial for ensuring vascular patency and promoting tissue repair. This system mainly consists of plasminogen, plasminogen activator, plasmin, and various inhibitory factors. Plasmin, as a key effector, exerts its thrombolytic effect by degrading fibrin thrombi, and this activation process requires the catalytic action of plasminogen activator. Urokinase plasminogen activator (u-PA), as one of the most potent plasminogen activators, plays a central regulatory role in physiological processes such as thrombolysis and extracellular matrix remodeling.

[0003] In the medical field, abnormal activation of u-PA is closely related to the occurrence and development of various diseases. On the one hand, in scenarios such as trauma and major surgery, excessive fibrinolysis mediated by u-PA can lead to coagulation disorders, resulting in secondary bleeding. Currently, most u-PA inhibitors used clinically are chemically synthesized amino acid analogs, which have problems such as poor biocompatibility and significant adverse reactions. On the other hand, numerous studies have shown that u-PA can significantly promote the invasion and metastasis of tumor cells by inducing extracellular matrix degradation. Especially in malignant tumors such as breast cancer and lung cancer, high expression of u-PA is positively correlated with tumor malignancy and poor prognosis. Therefore, the development of highly effective and low-toxicity u-PA inhibitors is of great significance for the innovative development of hemostatic and anti-tumor drugs.

[0004] In the food processing industry, the plasmin system in cow's milk and dairy products can enter milk through the mammary gland cell wall. Plasmin primarily acts on casein, initiating protein hydrolysis, bitter peptide formation, and aging gel formation. This is a major cause of quality deterioration in dairy products such as directly ultra-high temperature (dUHT) sterilized milk during storage. Currently, the main method for controlling plasmin activity in cow's milk is to intensify heat treatment, but this method can cause off-flavors from cooking and loss of nutrients, severely affecting product quality. Currently, there are no research reports on the application of milk-derived u-PA inhibitory peptides in dairy product processing. Developing natural and safe milk-derived u-PA inhibitory peptides has significant practical application value for improving the storage stability of dairy products and expanding the deep processing pathways of milk proteins. Based on this background, this invention aims to screen for milk protein-derived peptides with highly efficient u-PA inhibitory activity through a combination of computer virtual design and in vitro experimental verification, providing a new technical solution for food industry improvement and novel drug development. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide two milk protein-derived polypeptides with highly active u-PA inhibitory effects, clarify their amino acid sequences, inhibitory activities and mechanisms of action, and expand their applications in food processing, hemostatic drugs and anticancer drugs, thereby solving problems such as poor biocompatibility of chemical inhibitors and short shelf life of dairy products in existing technologies.

[0006] In one aspect, the present invention provides a milk protein polypeptide with urokinase-type plasminogen activator inhibitory activity, the amino acid sequence of which is PEVMGVSK, as shown in SEQ ID NO.1.

[0007] On the other hand, the present invention provides a milk protein polypeptide with urokinase-type plasminogen activator inhibitory activity, the amino acid sequence of which is HIQKEDVPSE, as shown in SEQ ID NO.2.

[0008] Preferably, the polypeptide is derived from milk β-casein or milk αs1-casein. More preferably, the polypeptide is derived from cow's milk, sheep's milk, camel's milk, yak's milk, or buffalo's milk.

[0009] On the other hand, the present invention provides a method for preparing the above-mentioned polypeptide, comprising the following steps: The peptide sequence screening step includes: virtual screening based on the amino acid sequence database of bovine milk casein using Discovery Studio molecular docking technology. Using the crystal structure of a urokinase-type plasminogen activator encoded as Q05589 in the UniProt database as a target, the three-dimensional structure of potential milk-derived peptides was constructed using the NovoPro platform, and peptide sequences with high binding potential to the u-PA active site were screened. The peptides are derived from milk β-casein.

[0010] Preferably, the procedure also includes the aforementioned peptide sequence screening, sequencing, and / or inhibitory peptide activity detection steps.

[0011] On the other hand, the present invention provides an application of milk protein peptides in food processing. Preferably, in this application, the aforementioned protein-derived peptides are added to dairy products as quality improvers. Preferably, the application is to improve the product's shelf-life stability.

[0012] Preferably, the dairy product is directly ultra-high temperature instantaneous sterilized milk or dairy product with fibrinolytic enzyme activity; the quality improver is used to delay protein hydrolysis and / or aging gel formation in the dairy product.

[0013] Furthermore, this invention verified the beneficial effects of the two peptides on the storage stability of dUHT milk. By monitoring the changes in particle size and degree of hydrolysis of dUHT skim milk with added PEVMGVSK and HIQKEDVPSE during a 6-month storage period, it was found that PEVMGVSK and HIQKEDVPSE can effectively maintain the occurrence of protein hydrolysis during the storage period of dUHT skim milk.

[0014] On the other hand, the present invention provides an application of milk protein polypeptide in drug development, that is, using the above-mentioned polypeptide as an active ingredient to prepare antifibrinolytic enzyme active drugs or anticancer drugs.

[0015] In the application of the above-mentioned milk protein polypeptides in the preparation of anti-fibrinolytic active drugs, preferably, the anti-fibrinolytic active drugs are wound hemostatic agents.

[0016] Among them, the above-mentioned milk protein polypeptide is used in the preparation of anticancer drugs, which block tumor cell migration by inhibiting u-PA activity; preferably, the tumor cells include MDA-MB-231 human breast cancer cells.

[0017] The beneficial effects of this invention include: 1. Based on molecular docking technology, this invention has discovered two milk-derived polypeptides that can bind to u-PA. The binding effect is good, and they can bind tightly to u-PA through hydrogen bonds, electrostatic forces, salt bridges, etc. The amino acid sequences are PEVMGVSK and HIQKEDVPSE, respectively.

[0018] 2. This invention verified the in vitro inhibitory activity of the above two peptides against u-PA using the S-2251 substrate chromogenic method, and their IC50 values ​​were [not specified]. 50 The values ​​were 8.763±0.244 mM and 10.577±0.359 mM, respectively.

[0019] 3. The two peptides mentioned above can improve the storage stability of dUHT milk and delay the occurrence of aging gel.

[0020] 4. The two polypeptides mentioned above can significantly delay the dissolution rate of tissue protein clots by plasmin by inhibiting the activation of plasminogen by u-PA, suggesting that they have potential application value in the development of drugs against uPA (such as hemostatic agents, sclerosing agents for the treatment of venous malformations, etc.).

[0021] 5. The two peptides mentioned above can inhibit the activity of u-PA in MDA-MB-231 human breast cancer cells, thereby inhibiting the migration of these cancer cells, suggesting that they are of great significance for the development of anti-cancer drugs. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the docking results between the peptide PEVMGVSK and u-PA. Figure 2 This is a schematic diagram of the docking results between the peptide HIQKEDVPSE and u-PA. Figure 3 The effects of peptides PEVMGVSK and HIQKEDVPSE on the average particle size of dUHT milk during storage. Figure 4 The effects of peptides PEVMGVSK and HIQKEDVPSE on zeta potential during storage of dUHT milk. Figure 5 The effects of peptides PEVMGVSK and HIQKEDVPSE on the degree of hydrolysis during the storage of dUHT milk. Figure 6 The inhibitory effects of peptides PEVMGVSK and HIQKEDVPSE on uPA-induced fibrin clot dissolution. Figure 7 The inhibitory effects of peptides PEVMGVSK and HIQKEDVPSE on the migration of MDA-MB-231 BAG human breast cancer cells. Figure 8 The inhibitory effects of peptides PEVMGVSK and HIQKEDVPSE on uPA activity in MDA-MB-231 BAG human breast cancer cells. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] Example 1: Molecular docking of peptide ligand and u-PA 1. Download the u-PA protein crystal structure coded Q05589 from the UniProt database, remove ligand molecules and water molecules from the structure using Discovery Studio software, hydrogenate the protein, and determine the active site region of u-PA using the software's built-in algorithm.

[0025] 2. Based on the amino acid sequence of bovine casein, potential peptide fragments were designed, and the three-dimensional structures of each peptide were generated using the NovoPro platform. After importing them into Discovery Studio software, energy minimization processing was performed to obtain stable peptide conformations.

[0026] 3. The CDOCKER module was used to dock the peptide conformation with the u-PA active site, and the binding energy of each peptide with u-PA was calculated. Combined with the interaction mode analysis, two peptides, PEVMGVSK (SEQ ID NO.1) and HIQKEDVPSE (SEQ ID NO.2), with high binding energy and multiple interactions with key residues of the active site were screened out.

[0027] The results are as follows Figures 1-2 As shown, the peptide PEVMGVSK can be linked to amino acid sites such as Arg149, Arg73, Arg45, and Gln25 on u-PA via hydrogen bonds, to Asp157, Lys70, and Glu35 via hydrophobic interactions, and to Thr147, His183, Met75, and Gly64 via van der Waals forces. Similarly, the peptide HIQKEDVPSE can be linked to amino acid sites such as Lys304, His67, Asp157, and Gln82 on u-PA via hydrogen bonds, to Arg45 and Arg73 via hydrophobic interactions, and to Tyr77, Pro68, Thr38, and Lys246 via van der Waals forces. Both peptides exhibit binding activity to Asp in u-PA, suggesting a potential to inhibit u-PA activation of plasminogen, thereby inhibiting plasmin-induced proteolysis in milk and delaying the aging gel's effect.

[0028] Example 2: In vitro detection of the inhibitory activity of peptides against u-PA 1. Reagent preparation: Detection buffer: 0.1 M Tris-HCl, 8 mM EACA, 0.4 M NaCl, pH=8.0, autoclaved before use.

[0029] Peptide solution: Accurately weigh the target peptide, dissolve it in the detection buffer, and prepare a series of concentration solutions with final concentrations of 0, 5, 10, 15, 20, and 25 mM.

[0030] u-PA solution: Dissolve u-PA lyophilized powder in detection buffer to prepare a stock solution of 500 U / mL.

[0031] Plasminogen solution: Dissolve bovine plasminogen in detection buffer to prepare a 2000 U / mL solution.

[0032] S-2251 substrate solution: Dissolve the S-2251 substrate in deionized water to prepare a 0.8 mM solution. Store the prepared solution at 4°C for later use.

[0033] 2. Testing Steps Add 100 μL of peptide solutions of different concentrations to centrifuge tubes. Add 100 μL of detection buffer to the control group. Then add 100 μL of u-PA solution to each tube, vortex to mix, and incubate at 37°C for 15 min. Add 200 μL of plasminogen lysate solution to the above mixture, vortex to mix, and incubate at 37°C for another 15 min. Add 50 μL of the incubated mixture to a 96-well plate, and add 50 μL of S-2251 substrate solution to each well. Incubate at 37°C. Measure the absorbance at 405 nm every 5 min using a microplate reader for 35 min. The rate of change of absorbance over time reflects plasmin activity. Calculate the inhibition rate at each peptide concentration. Use GraphPad Prism software to fit the dose-response curve and calculate the IC50. 50 value.

[0034] Table 1. Binding energy of peptides to u-PA and in vitro inhibition of IC50. 50 Value determination As shown in Table 1, binding energy reflects the potential binding strength between the receptor and ligand; a higher absolute value indicates a stronger likelihood of binding. The binding energies of PEVMGVSK with HIQKEDVPSE and u-PA are -107.56 kcal / mol and -97.23 kcal / mol, respectively, indicating that PEVMGVSK has a stronger potential binding activity with u-PA. To further verify this, two peptides were synthesized to investigate their effects on u-PA's in vitro activation of plasminogen. It was found that both peptides inhibited plasmin activation, with PEVMGVSK showing the highest IC50 value. 50 The IC value of HIQKEDVPSE is 8.763 ± 0.244 mM. 50 It is 10.577±0.359 mM.

[0035] Example 3: Improvement of the storage stability of dUHT milk by peptides Commercially available dUHT skim milk (treated at 147℃ for 3 seconds) was selected, and 10 mM of PEVMGVSK and HIQKEDVPSE peptides were added respectively. After vortexing and mixing, the mixture was dispensed into sterile bottles. dUHT milk without added peptides was used as a control. The samples were stored at 25℃ in the dark for 6 months. 200 mL samples were taken each month to monitor the particle size distribution, zeta potential, and degree of hydrolysis.

[0036] 1. Particle size distribution and zeta potential were determined using a Malvern particle size analyzer. The specific operating steps were as follows: monthly samples were diluted with distilled degassed water, and the zeta potential and average particle size were determined using a Malvern particle size analyzer. The particle refractive index was set to 1.45, the dispersant refractive index to 1.33, and the laser shielding value to 5.5-8.0%.

[0037] 2. The degree of hydrolysis was determined using the 1,2-Phthalic dicarboxaldehyde (OPA) method, following these steps: ① Preparation of OPA reagent: Dissolve 7.620 g sodium tetraborate and 200 mg SDS in 150 mL deionized water, add 4 mL of 40 mg / mL OPA ethanol solution, then add 176 mg DTT, and bring the volume to 200 mL; ② Construction of standard curve: Prepare a 0.9516 mM serine standard solution, react different volumes of the standard solution with the OPA reagent, and measure the absorbance at 340 nm after 2 min to construct a standard curve; ③ Sample determination: Weigh an appropriate amount of sample, react it with the OPA reagent, and measure the absorbance. Calculate the number equivalent of serine amino groups based on the standard curve, which is the degree of hydrolysis index. Serine NH2 – the amino equivalent of serine per gram of protein X — Sample mass (g) P – Protein content in the sample (%), in this experiment P is 0.8 In the results of sample particle size distribution, such as Figure 3 As shown, the average particle size in dUHT milk gradually increased within 6 months of storage, which may be related to plasmin-induced casein micelle hydrolysis, instability, reorganization, and polymerization. Compared with the control group, the particle size increase in dUHT milk with added peptides was slower, indicating that peptides can delay the process of casein instability and reorganization. Among them, PEVMGVSK showed a significantly better inhibitory effect on particle size increase than HIQKEDVPSE.

[0038] In the results of the Zeta potential, such as Figure 4As shown, the Zeta potential reflects the stability of casein micelles in a milk system; a higher absolute value indicates greater stability. Within 6 months of storage, the absolute value of the Zeta potential in dUHT milk gradually decreased, indicating a gradual decline in micelle stability. Compared to the control group, the absolute value of the Zeta potential in dUHT milk with added peptides was higher, indicating that the peptides can improve the stability of casein micelles. Among them, PEVMGVSK showed a significantly better effect on improving casein stability than HIQKEDVPSE.

[0039] The results are as follows Figure 5 As shown, the degree of hydrolysis in dUHT milk gradually increased within 6 months of storage, indicating that milk proteins underwent hydrolysis during storage, presumably mainly related to the action of plasmin. Compared with the control group, the degree of hydrolysis in dUHT milk with the addition of three peptides increased more slowly, indicating that peptides can delay the hydrolysis of casein. Among them, PEVMGVSK showed a significantly better inhibitory effect on protein hydrolysis than HIQKEDVPSE.

[0040] Example 4: Effect of peptides on uPA-activated plasmin on fibrinolysis levels Fibrin clots (containing 12.5 μM bovine fibrinogen and 55 nM thrombin) were incubated for 3 h at 37 °C in lysis buffer (0.01% Tween 20, 30 mM Tris, 75 mM NaCl, 3 mM CaCl2). Lysis was initiated by adding 100 μL of lysis buffer containing a mixture of Plg and uPA in the presence of both peptides at 0–25 mM. The clot dissolution process was monitored using a turbidimeter, read every minute at 37 °C, to obtain the 50% clot dissolution time. The results were normalized, and the inhibitory activity of the peptides on clot dissolution was calculated.

[0041] The results are as follows Figure 6 As shown, both peptides inhibited the dissolution of u-PA-activated fibrin clots in vitro, and the inhibition was concentration-dependent. PEVMGVSK showed the strongest inhibitory effect, with a 46.54% inhibition rate at 25 mM, followed by HIQKEDVPSE, with a 35.23% inhibition rate at 25 mM.

[0042] Example 5: Effect of peptides on the migration of uPA-activated MDA-MB-231 human breast cancer cells MDA-MB-231 cells in logarithmic growth phase were resuspended in serum-free DMEM high-glucose medium and the cell density was adjusted to 102. 6 mL -1Add 100 µL of cell suspension to the upper chamber of a Transwell plate and 500 µL of DMEM complete medium to the lower chamber. Then, add 0.1–5 mM of peptides PEVMGVSK and HIQKEDVPSE to each chamber. Incubate at 37°C and 5% CO2 for 48 h. Remove the upper chamber, wipe off cells from the membrane with a cotton swab, wash three times with PBS, and add 500 µL of complete medium containing 5 g / L MTT. Incubate at 37°C for 4 h. Add 300 µL of DMSO to each well and shake for 10 min to fully dissolve the MTT. Remove the chamber and analyze using a microplate reader.

[0043] The results are as follows Figure 7 As shown, the two peptides at 0.1-5 mM exhibited inhibitory activity against the migration of MDA-MB-231 human breast cancer cells in a concentration-dependent manner. Among them, PEVMGVSK showed a significantly higher inhibitory effect on the migration of MDA-MB-231 human breast cancer cells than HIQKEDVPSE. The inhibition rates of the two peptides at 5 mM against the migration of MDA-MB-231 human breast cancer cells were (48.32±3.29)% and (37.94±4.26)%, respectively.

[0044] MDA-MB-231 cells were cultured in complete medium (1% penicillin-streptomycin, 10% fetal bovine serum, 89% DMEM) containing 2 mM peptides PEVMGVSK and HIQKEDVPSE and incubated at 37°C with 5% CO2 for 48 h. Cells were frozen at -20°C and then rapidly thawed at room temperature, repeated three times to induce cell swelling and lysis. The supernatant was collected by centrifugation, and uPA levels were detected using an ELISA kit, strictly following the kit's instructions.

[0045] The results are as follows Figure 8 As shown, the uPA activity of MDA-MB-231 human breast cancer cells treated with both peptides was significantly downregulated, which was associated with a decrease in cell migration rate, suggesting that the peptides inhibit cell matrix dissolution by inhibiting uPA activity, thereby inhibiting cell migration.

[0046] In summary, this invention successfully obtained two u-PA inhibitory peptides, PEVMGVSK and HIQKEDVPSE, derived from bovine milk casein, through a combination of molecular docking virtual screening and in vitro experimental verification. Experiments confirmed that both peptides can stably bind to the u-PA active site through multiple intermolecular forces, significantly inhibiting u-PA-mediated plasminogen activation, with IC50 values ​​of 8.763±0.244 mM and 10.577±0.359 mM, respectively. Simultaneously, they can effectively improve the storage stability of dUHT milk, delay proteolysis and aging gel formation, and inhibit fibrin clot lysis and the migration of MDA-MB-231 human breast cancer cells in a concentration-dependent manner.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A milk protein polypeptide with urokinase-type plasminogen activator inhibitory activity, characterized in that, Its amino acid sequence is PEVMGVSK, as shown in SEQ ID NO.

1.

2. A milk protein polypeptide with urokinase-type plasminogen activator inhibitory activity, characterized in that, Its amino acid sequence is HIQKEDVPSE, as shown in SEQ ID NO.

2.

3. The use of the milk protein polypeptide as described in claim 1 or 2 in the preparation of u-PA inhibitors.

4. The application of the milk protein polypeptide as described in claim 1 or 2 in food processing or drug development.

5. The application as described in claim 4, characterized in that, The application is as a quality improver in food processing.

6. The application as described in claim 5, characterized in that, The quality improver is used to add to dairy products to enhance their storage stability.

7. The application as described in claim 6, characterized in that, The dairy products mentioned are direct ultra-high temperature instantaneous sterilized milk or dairy products containing fibrinolytic enzyme activity.

8. The application as described in claim 6, characterized in that, The quality improver is used to delay protein hydrolysis, bitter peptide formation, and / or aging gel formation in dairy products.

9. The application as described in claim 4, characterized in that, The application is as an active ingredient in the preparation of antifibrinolytic active drugs in drug development, wherein the antifibrinolytic active drug is a wound hemostatic agent.

10. The application as described in claim 4, characterized in that, The application is as an active ingredient in the preparation of anticancer drugs in drug development, which are used to block the invasion and metastasis of tumor cells.

Images