Targeted antagonistic pCAV1 polypeptide and application thereof

Abstract

The invention discloses a targeted antagonistic pCAV1 polypeptide and application thereof, and relates to the technical field of biology. The polypeptide is any one of amino acid sequences as shown in (1) and (2): (1) the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1: VHPKQHRGGSKGCGGYGRKKRRQRRRGGFIYVNQSFAPK; the polypeptide derivative is a polypeptide derivative which is obtained by modifying, substituting or adding one or more amino acid sites in the amino acid sequence shown in SEQ ID NO: 1. An antagonistic short peptide (namely CBM polypeptide) capable of targeting p-caveolin-1 (pCAV1) is excavated for the first time, combination of the pCAV1 and an ATG12-ATG5 compound can be specifically blocked so as to activate the autophagy process of endothelial cells, the cross-endothelial cell penetrating quantity of low-density lipoprotein (LDL) is reduced, and it is proved that the antagonistic short peptide can effectively delay the progress of early atherosclerotic plaques.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide for targeting and antagonizing pCAV1 and an application thereof. Background Art

[0002] Atherosclerosis (AS) or coronary artery disease (CAD) is still the leading cause of death worldwide. Lipid retention under the endothelial cell (EC) monolayer on the inner wall of blood vessels is also an early step in the formation of atherosclerosis. Lipoproteins containing apolipoprotein B (ApoB) are absorbed and transported in endothelial cells through receptor-mediated endocytosis or through transcytosis.

[0003] Caveolin-1 (CAV1) is a 21-24 kDa membrane protein that is essential for caveolae formation. As a substrate of Src kinase, CAV1 can be phosphorylated by the Src tyrosine kinase family (c-Abl, Fyn) at tyrosine 14. Under transmission electron microscope (TEM), an increase in the number of caveolae in the CAV1-Y14D phosphorylation mimic mutant treatment group (mimicking phosphorylated CAV1 overexpression) was observed to stimulate the transcytosis of low-density lipoprotein (LDL). p-Caveolin-1 (pCAV1) has also been shown to directly increase the uptake of oxidized low-density lipoprotein in human umbilical vein endothelial cells (HUVECs). In endothelial cells, pCAV1 has a direct atherogenic effect. However, there is no literature reporting targeted therapeutic measures against pCAV1 to delay early atherosclerosis.

[0004] Autophagy is a highly conserved cell cycle mechanism for the degradation of damaged cytoplasmic materials mediated by the lysosomal system in cells. The formation of autophagosomes requires the synergistic action of two ubiquitin-like systems, namely the ATG12 and LC3 / GABARAP (or human ATG8) systems. The product of the ATG12 ubiquitin-like conjugation system (i.e., the ATG12-ATG5-ATG16L1 complex) acts as an E3-like enzyme of the second conjugation system, directing LC3 / GABARAP to the autophagosome membrane and catalyzing the covalent binding of LC3 / GABARAP family members to lipid phosphatidylethanolamine to participate in the extension and closure of the autophagosome membrane. The activation of autophagy is a protective factor for early atherosclerosis, which has been shown to inhibit the permeation of LDL in endothelial cells.

[0005] At present, the applicant has designed an antagonistic peptide that can target pCAV1 based on the CBM (caveolin-1 binding motif) structure on ATG12, and observed the role of the peptide in the development of atherosclerosis, which is an urgent problem to be solved by the present invention. Summary of the invention

[0006] The present invention provides a polypeptide for targeting and antagonizing pCAV1 and its application, aiming to solve the problems existing in the above-mentioned background technology.

[0007] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0008] In a first aspect, the present invention discloses a polypeptide that targets and antagonizes pCAV1. The polypeptide is any one of the amino acid sequences shown in (1) and (2):

[0009] (1) having the amino acid sequence shown in SEQ ID NO: 1:

[0010] VHPKQHRGGSKGCGGYGRKKRRQRRRGGFIYVNQSFAPK;

[0011] (2) A polypeptide derivative obtained by modifying, replacing or adding one or more amino acid sites in the amino acid sequence shown in SEQ ID NO: 1.

[0012] In a preferred embodiment of the present invention, the polypeptide derivative is acetylated at the N-terminus and amidated at the C-terminus in the amino acid sequence shown in SEQ ID NO: 1, and methylated at the 29th amino acid N.

[0013] In a second aspect, the present invention discloses a nucleotide sequence encoding a polypeptide targeting and antagonizing pCAV1 as described in the first aspect.

[0014] In a third aspect, the present invention discloses an expression vector containing the nucleotide sequence as described in the second aspect.

[0015] Preferably, the expression vector is selected from any one of a plasmid, a phage, a bacterium or a host cell.

[0016] In a fourth aspect, the present invention discloses a use of the polypeptide as described in the first aspect in preparing a drug for targeted antagonism of pCAV1.

[0017] Preferably, the targeted antagonist of pCAV1 is a drug for treating atherosclerosis.

[0018] In a fifth aspect, the present invention discloses a drug for treating atherosclerosis, comprising the polypeptide targeting and antagonizing pCAV1 as described in the first aspect.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention has discovered for the first time an antagonistic short peptide (i.e., CBM polypeptide) that can target pCAV1, which can specifically block the binding of pCAV1 to the ATG12-ATG5 complex, reduce the formation of the pCAV1-ATG12-ATG5 complex to activate endothelial cell autophagy, and reduce the amount of LDL transendothelial cell permeation, which has been shown to effectively delay the progression of early atherosclerotic plaques. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a statistical chart of CBM peptide toxicity and half-life detection;

[0022] Figure 2 The immunofluorescence double labeling image shows that the CBM polypeptide specifically binds to pCAV1;

[0023] Figure 3 The co-immunoprecipitation images of CBM peptides binding to pCAV1;

[0024] Figure 4 This is a statistical chart of the expression of autophagy-related proteins after application of CBM peptides;

[0025] Figure 5 This is a statistical diagram of the LDL transcytosis in endothelial cells after the introduction of CBM polypeptide;

[0026] Figure 6 ApoE after CBM peptide introduction - / - In vivo fluorescence imaging of mice;

[0027] Figure 7 Transmission electron microscopy images of aorta vessels of mice in each group after CBM peptide treatment;

[0028] Figure 8 ApoE after CBM peptide introduction - / - Images and statistical graphs of plaque area in the aortic root and entire aorta of mice. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] Based on the CBM (caveolin-1 binding motif) structure on ATG12, the applicant invented a novel antagonistic short peptide that can target pCAV1. It specifically binds to pCAV1 to hinder the formation of the pCAV1-ATG12-ATG5 complex to activate cell autophagy, reduce the amount of LDL transcytosis in endothelial cells, and thus delay the progression of early atherosclerosis.

[0031] Specifically, in the present invention, the polypeptide targeting antagonism of pCAV1 is named CBM polypeptide (ie, VHPK-TAT-CBM peptide), which is mainly composed of three parts: VHPK targeting peptide, TAT membrane-penetrating peptide and CBM target peptide. The polypeptide sequence is as follows: Ac-VHPKQHRGGSKGC-GG-YGRKKRRQRRR-GG-F(N-Me)IYVNQSFAPK-NH2. Specific targeting and penetration of endothelial cells are achieved by adding VHPK (endothelial cell targeting peptide with high affinity to VCAM-1 (vascular cellular adhesion molecule-1) on the surface of endothelial cells) and TAT (cationic cell penetrating peptide), and then chemical modifications such as N-terminal methylation and terminal protection (N-terminal acetylation and C-terminal amidation) are applied to improve the stability of the polypeptide in vivo.

[0032] The present invention provides a new perspective on how CBM polypeptides target pCAV1 in endothelial cells to prevent early atherosclerosis. After CBM polypeptides enter cells through the VCAM-1 receptor on the endothelial cell membrane, they target and block the interaction between pCAV1 and the ATG12-ATG5 complex, thereby effectively activating autophagy in endothelial cells and reducing the amount of LDL transendothelial cell permeation, thereby delaying the formation of early atherosclerotic plaques.

[0033] The following is an explanation through specific embodiments.

[0034] Example 1 CBM polypeptide synthesis steps

[0035] The ATG12-ATG5 system is a ubiquitin-like system required for the formation of autophagosomes. According to the CBM structure on ATG12, a CBM peptide (VHPK-TAT-CBM peptide) was designed as an antagonistic short peptide targeting pCAV1. It is mainly composed of three parts: VHPK targeting peptide, TAT transmembrane peptide and CBM target peptide. The polypeptide sequence is as follows: VHPKQHRGGSKGC-GG-YGRKKRRQRRR-GG-FIYVNQSFAPK, acetylation is performed at the N-terminus of the polypeptide sequence and amidation protection is performed at the C-terminus, and methylation protection is performed on the 29th amino acid N to form a polypeptide derivative to improve its stability. The improved sequence is: Ac-VHPKQHRGGSKGC-GG-YGRKKRRQRRR-GG-F(N-Me)IYVNQSFAPK-NH2 (in the present invention, the embodiments have been tested with the improved polypeptide sequence). VHPK is an endothelial cell targeting peptide that can specifically bind to VCAM-1 on the surface of endothelial cells. In addition, the cell-penetrating peptide TAT (arginine-rich cationic short peptide) has high cell membrane permeability, has almost no killing effect on cells and can improve the delivery effect.

[0036] In the present invention, the Fmoc solid phase synthesis method is used to synthesize the CBM polypeptide. Since Fmoc itself has ultraviolet absorption, it is easy to detect and has the advantages of mild reaction conditions, high yield and simple operation, it is widely used.

[0037] (1) Synthetic raw materials and related reagents:

[0038] 1. Protect amino acid raw materials

[0039] Fmoc-Val-OH,Fmoc-His(Trt)-OH,Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH,Fmoc-Gln(Trt)-OH,Fm oc-Arg(pbf)-OH,Fmoc-Gly-OH,Fmoc-Cys(Trt)-OH,Fmoc-Tyr(tbu)-OH,Fmoc-N-Me-Phe-OH,Fmo c-Ile-OH,Fmoc-Asn(Trt)-OH,Fmoc-Ser(tbu)-OH,Fmoc-Phe-OH,Fmoc-Ala-OH,Fmoc-Lys(Bioti n)-OH

[0040] 2. Condensation Reagents

[0041] Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIEA)

[0042] 3. Solvent

[0043] N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol, acetonitrile (ACN)

[0044] 4. Resin

[0045] Rink Amide-MBHA Resin with a substitution degree of 0.38mmol / g

[0046] 5. Deprotection Reagents

[0047] Piperidine

[0048] 6. Detection reagents:

[0049] Phenol reagent, pyridine reagent, ninhydrin reagent

[0050] 7. Cutting Reagents

[0051] Trifluoroacetic acid (TFA), triisopropylsilane (TIS), 1,2-ethanedithiol (EDT), anhydrous ether

[0052] 8. Nitrogen

[0053] 9. Precision electronic balance

[0054] (2) Instruments and equipment:

[0055] 1. Twelve-channel semi-automatic peptide synthesizer

[0056] 2. High Performance Liquid Chromatography

[0057] 3. Freeze dryer,

[0058] 4. Centrifuge

[0059] (3) Synthesis steps:

[0060] 1. Resin swelling

[0061] Place Rink Amide-MBHA Resin in a reaction tube, add DCM (15 mL / g), and shake for 30 min.

[0062] 2. Connect the first amino acid

[0063] Add 20% piperidine DMF solution (15 ml / g) for 15 min. Filter out the solvent through a sand core and wash it clean. Add 3 times molar excess of Fmoc-Lys(Biotin)-OH, add DMF to dissolve, 3 times excess of HBTU, and then add 10 times molar excess of DIEA, shake for 60 min. Block with pyridine and acetic anhydride.

[0064] 3. Deprotection

[0065] Remove DMF, add 20% piperidine DMF solution (15 mL / g) for 5 min, remove and add 20% piperidine DMF solution (15 mL / g) for 15 min.

[0066] 4. Detection

[0067] Drain off the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, potassium cyanide (KCN), and phenol solution, heat at 105℃-110℃ for 5 minutes, and a dark blue color indicates a positive reaction.

[0068] 5. Wash

[0069] DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice

[0070] 6. Condensation

[0071] A 3-fold molar excess of Fmoc-protected amino acid, a 3-fold molar excess of HBTU, and a 10-fold molar excess of DIEA were added, and finally DMF was added to dissolve and shaken for 45 min.

[0072] 7. Detection

[0073] Take a dozen resin particles, wash them three times with ethanol, add one drop each of ninhydrin, pyridine and phenol solution, heat at 105℃-110℃ for 5 minutes, and colorless is a negative reaction.

[0074] 8. Wash

[0075] DMF (10 mL / g) once, methanol (10 mL / g) twice, DMF (10 mL / g) twice

[0076] 9. Repeat steps 3 to 8, connecting from right to left, until the Fmoc protecting group of the last amino acid is removed, and then acetylation is performed to cap the end.

[0077] 10. Wash the resin and drain it according to the following method

[0078] DMF (10 mL / g) was added twice, DCM (10 mL / g) was added three times, and methanol (10 mL / g) was added four times, and the mixture was pumped dry for 10 min.

[0079] 11. Cutting

[0080] Prepare cutting solution (10 mL / g): TFA 95%; water 2%; EDT 2%; TIS 1%

[0081] Cutting time: 180min

[0082] 12. Blow dry and wash

[0083] The lysate was blown dry with nitrogen as much as possible, ether was precipitated, the supernatant was removed by centrifugation, the precipitate was washed with ether six times, and then evaporated to dryness at room temperature.

[0084] 13. Purification and preparation.

[0085] 1. Take a small amount of crude product and dissolve it in H2O / ACN.

[0086] 2. Take a small amount of sample and analyze it on the HPLC analyzer to determine the peak time corresponding to the target peak.

[0087] 3. Using C18 reverse phase chromatography preparation system:

[0088] Wavelength:220nm; Flow Rate:15mL / min; Inj.Vol:20mL Column Temp:25℃

[0089] Buffer A: 0.1% TFA in water Buffer B: 0.1% TFA in Acetonitrile; collect the target peak solution.

[0090] 4. Use a 1.5mL centrifuge tube to take a small amount of the target peak solution for mass spectrometry confirmation and purity testing.

[0091] 14. Freeze-dry the qualified target peak solution to obtain the finished product.

[0092] 15. Identification

[0093] Take a small amount of the finished peptide for molecular weight identification by MS and purity identification by HPLC analysis.

[0094] 16. Seal the powdered polypeptide and store it at -80°C.

[0095] Example 2 CBM polypeptide toxicity and half-life detection

[0096] like Figure 1 As shown in the figure, first, different concentrations (2.5, 5, 10 and 20 μM) and time (6, 12, 24 and 48 h) of CBM peptides were used for dose optimization. There was no significant statistical difference in the cytotoxicity of CBM peptides to HUVECs at all different concentrations. In addition, when CBM peptides were used at a concentration of 20 μM, there was no significant statistical difference in cytotoxicity at each time point, indicating that CBM peptides were not toxic to normal endothelial cells and had high biological safety. CBM half-life (t 1 / 2 =40.582h) The experimental results showed that its effect reached a peak at 6h, remained stable within 24h, and then began to degrade, indicating that CBM polypeptides have relatively long stability.

[0097] Example 3 Investigating the blocking effect of CBM polypeptides

[0098] In order to test whether the CBM peptide has the targeting property of pCAV1 and can play the blocking effect of pCAV1, the Cy3-labeled CBM peptide and the plasmid encoding CAV1-Y14D-Flag were introduced into endothelial cells, and the binding of the CBM peptide to the overexpressed pCAV1 was observed by immunofluorescence double labeling experiment under confocal microscope. Figure 2 As shown, the results showed that the CBM polypeptide was co-localized with pCAV1 overexpressed by CAV1-Y14D in the cell membrane and part of the cytoplasm of endothelial cells, indicating that the CBM polypeptide has targeting to pCAV1.

[0099] Secondly, if Figure 3 As shown, the existence of interaction between CBM polypeptide and CAV1-Y14D was further confirmed by immunoprecipitation experiment. Similarly, after we added CBM polypeptide to endothelial cells introduced with CAV1-Y14D plasmid, CBM polypeptide could successfully pull down Flag-Y14D, and Flag-Y14D could also successfully pull down CBM, proving the existence of interaction between CBM-Y14D. In addition, compared with the CAV1-Y14D group, the introduction of CBM polypeptide can reduce the interaction between Y14D-ATG12-ATG5, indicating that CBM polypeptide can specifically bind to pCAV1 and weaken the interaction between pCAV1-ATG12-ATG5.

[0100] Example 4 Verification of the effect of pCAV1 antagonistic short peptide (CBM polypeptide) in endothelial cells

[0101] In order to verify the role of pCAV1 antagonist short peptide (CBM peptide) in endothelial cells, the degree of autophagy and LDL transcytosis were detected after the CBM peptide was introduced into the cells. First, CBM peptide can activate endothelial cell autophagy and reduce the expression of CAV1 and pCAV1. At the same time, it can restore the inhibition of autophagic flux and the increase of CAV1 and pCAV1 expression caused by overexpression of CAV1-Y14D ( Figure 4 ). Secondly, the exogenous introduction of CBM peptides also reduced the amount of LDL transcytosis in endothelial cells and eliminated the increase in LDL transcytosis caused by overexpression of CAV1-Y14D ( Figure 5 In conclusion, our findings suggest that CBM peptides can directly bind to pCAV1 and block the effects of pCAV1-induced autophagy inhibition and LDL transcytosis enhancement, indicating that CBM structures may have a potential protective effect against the early progression of atherosclerosis.

[0102] Example 5 Investigating the expression of CBM polypeptides in mouse aorta

[0103] To further confirm the in vitro results and verify whether the pCAV1 antagonist peptide works in vivo, we introduced the AAV9 adeno-associated virus (AAV-Y14D) encoding CAV1-Y14D into the ApoE - / - In the mouse atherosclerosis model, CBM peptide (labeled with Cy3) was used for experimental treatment to evaluate its effect on the progression of atherosclerotic plaques in vivo. - / - Adeno-associated virus vector (AAV-Vector) and TAT were introduced into mice as negative controls. 32 mice were randomly divided into 4 groups (n=8 / per group), divided into the following groups: 1. AAV-Vector+TAT; 2. AAV-Vector+CBM polypeptide; 3. AAV-Y14D+TAT; 4. AAV-Y14D+CBM polypeptide. All groups were fed a high-fat diet (21% fat + 0.15% cholesterol). At week 8, mice were randomly divided into AAV-Vector and AAV-Y14D groups (n=16 / per group). 5×10 11 vg / mouse was injected (iv) with AAV-Vector and AAV-Y14D once in the tail vein. Starting from the 16th week, the mice were randomly divided into TAT control and CBM polypeptide treatment subgroups (n=8 / per group), and the mice were injected (iv) with 1.5 mg / kg concentration of CBM polypeptide and TAT control polypeptide through the tail vein every day until 21 weeks.

[0104] At week 21, after blood sampling under anesthesia, the hearts and aortas of the mice were collected and fixed with 4% paraformaldehyde. In vivo fluorescence imaging was performed before the mice were sacrificed. Figure 6 As shown, CBM-Cy3 fluorescence is distributed in the mouse heart and aorta tissue, indicating that CBM peptides can specifically act on the heart and aorta. In vitro animal vascular imaging also verified that CBM-Cy3 peptides can specifically act on the aorta.

[0105] Example 6 Investigating the effect of CBM polypeptide on atherosclerotic plaque deposition in mice

[0106] Transmission electron microscopy imaging showed that CBM peptide treatment significantly increased the number of autophagic vacuoles and reduced the number of membrane pits in mouse aortic endothelial cells, while reversing the downregulation of the number of autophagic vacuoles and the increase in the number of pits in the AAV-Y14D group ( Figure 7 ). This indicates that CBM polypeptides can still effectively activate autophagy in animals, increase the number of autophagosomes in vascular endothelial cells and reduce the pit structures on the membrane.

[0107] Oil Red O is a fat-soluble dye that can specifically stain neutral lipids such as triglycerides in cells or tissues, making them appear red or orange-red. In pathological conditions, Oil Red O staining is often used to detect atherosclerosis and fat embolism, showing lipid deposits under endothelial cells and lipids in fat emboli. Compared with the AAV-Vector group, mice receiving AAV-Y14D had a higher expression of ApoE - / - Oil Red O lipid staining of mice showed increased atherosclerotic lesions ( Figure 8 ), which was manifested by a significant increase in lipid deposition in the subendothelial space of the aortic root and the area of ​​aortic lesions, while CBM peptides could significantly reduce the area of ​​plaque lesions in the aortic root and the entire aorta, indicating that CBM peptides have the effect of delaying the formation of atherosclerotic plaques in vivo.

[0108] In summary, our in vitro and in vivo experiments confirmed that the intervention of pCAV1 antagonist short peptide (CBM peptide) can significantly reduce the deposition of atherosclerotic plaques caused by pCAV1 overexpression, and CBM peptide has an important effect on ApoE - / - The plaque progression of mice was significantly inhibited by CBM, which can activate endothelial cell autophagy by specifically binding to pCAV1 and inhibiting the formation of pCAV1-ATG12-ATG5 complex, while reducing the amount of LDL transendothelial cell permeation to reduce atherosclerotic lesions. The results indicate that the delivery of CBM peptides may be a promising treatment for delaying the progression of early AS.

[0109] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A polypeptide targeting antagonism of pCAV1, characterized in that: The polypeptide is any one of the amino acid sequences shown in (1) and (2): (1) having the amino acid sequence shown in SEQ ID NO: 1: VHPKQHRGGSKGCGGYGRKKRRQRRRGGFIYVNQSFAPK; (2) A polypeptide derivative obtained by modifying, replacing or adding one or more amino acid sites in the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide targeting antagonism of pCAV1 according to claim 1, characterized in that: The polypeptide derivative is acetylated at the N-terminus and amidated at the C-terminus in the amino acid sequence shown in SEQ ID NO: 1, and methylated at the 29th amino acid N.

3. A nucleotide sequence encoding the polypeptide targeting and antagonizing pCAV1 according to claim 1.

4. An expression vector containing the nucleotide sequence of claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector is selected from any one of plasmid, bacteriophage, bacteria or host cell.

6. Use of the polypeptide according to claim 1 or 2 in the preparation of a drug for targeted antagonism of pCAV1.

7. The use according to claim 6, characterized in that: The targeted antagonistic pCAV1 drug is a drug for treating atherosclerosis.

8. A drug for treating atherosclerosis, characterized in that: Contains the polypeptide targeting and antagonizing pCAV1 as claimed in claim 1 or 2.

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