Polypeptides targeting antagonizing pCAV1 and uses thereof

Abstract

The application discloses a polypeptide for targeting antagonizing pCAV1 and application thereof, and relates to the technical field of biology.The polypeptide is any one of the amino acid sequences shown in (1) and (2): (1) has the amino acid sequence shown in SEQ ID NO:1: VHPKQHRGGSKGCGGYGRKKRRQRRRGGFIYVNQSFAPK; and has the amino acid sequence shown in SEQ ID NO:1, and a polypeptide derivative obtained by modifying, substituting or adding one or more amino acid sites in the amino acid sequence.The application first excavates an antagonistic short peptide (namely CBM polypeptide) which can target p-caveolin-1 (pCAV1), can specifically block the combination of pCAV1 and ATG12-ATG5 compound to activate the autophagy process of endothelial cells, and can reduce the amount of low-density lipoprotein (LDL) crossing the endothelial cells, and it is proved that the polypeptide can effectively delay the progress of early atherosclerotic plaques.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a polypeptide that targets and antagonizes pCAV1 and its applications. Background Technology

[0002] Lipid retention beneath the endothelial cell (EC) layer of the vascular lining 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 transcellular action.

[0003] Caveolin-1 (CAV1) is a 21-24 kDa membrane protein essential for caveolae formation. As a substrate of Src kinases, CAV1 is phosphorylated at tyrosine 14 by the Src tyrosine kinase family (c-Abl, Fyn). Transmission electron microscopy (TEM) has revealed an increased number of caveolaes in the CAV1-Y14D phosphorylation mimic mutant treatment group (mimicking CAV1 phosphorylation overexpression), stimulating low-density lipoprotein (LDL) transcellularity. p-Caveolin-1 (pCAV1) has also been shown to directly increase the uptake of oxidized LDL in human umbilical vein endothelial cells (HUVECs). In endothelial cells, pCAV1 has a direct atherosclerotic effect; however, there are currently no reports of targeted therapies against pCAV1 to delay early atherosclerosis.

[0004] Autophagy is a highly conserved cellular circulation mechanism mediated by the lysosomal system to degrade damaged cytoplasmic material. Autophagosome formation requires the synergistic action of two ubiquitin-like systems: the ATG12 and LC3 / GABARAP (or human ATG8) systems. The product of the ATG12 ubiquitin-like conjugate system (i.e., the ATG12-ATG5-ATG16L1 complex) acts as an E3-like enzyme in the second conjugate system, guiding LC3 / GABARAP to the autophagosome membrane and catalyzing the covalent binding of LC3 / GABARAP family members to the lipid phosphatidylethanolamine to participate in the elongation and closure of the autophagosome membrane. Activation of autophagy is a protective factor in early atherosclerosis, and it has been shown to inhibit LDL transcellularity in endothelial cells.

[0005] Currently, the applicant has designed an antagonistic peptide that can target pCAV1 based on the CBM (caveolin-1 binding motif) structure on ATG12, and is observing the role of this peptide in the development of atherosclerosis. This is an urgent problem that needs to be solved in this invention. Summary of the Invention

[0006] This invention provides a polypeptide that targets and antagonizes pCAV1 and its application, aiming to solve the problems existing in the above-mentioned background art.

[0007] To achieve the above-mentioned 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, wherein the polypeptide is any one of the amino acid sequences shown in (1) and (2):

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

[0010] VHPKQHRGGSKGCGGYGRKKRRQRRRGGFIYVNQSFAPK;

[0011] (2) A polypeptide derivative having amino acid sites in the amino acid sequence shown in SEQ ID NO: 1, obtained by modification, substitution or addition of one or more amino acid sites.

[0012] In a preferred embodiment of the present invention, the polypeptide derivative is acetylated at the N-terminus and amidated at the C-terminus of 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] Thirdly, 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 plasmids, bacteriophages, bacteria, or host cells.

[0016] Fourthly, the present invention discloses the use of the polypeptide as described in the first aspect in the preparation of a drug that targets and antagonizes pCAV1.

[0017] Preferably, the pCAV1-targeting drug is a drug for treating atherosclerosis.

[0018] Fifthly, the present invention discloses a drug for treating atherosclerosis, comprising a polypeptide that targets and antagonizes pCAV1 as described in the first aspect.

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

[0020] This invention is the first to discover an antagonistic short peptide (i.e., CBM peptide) that can target pCAV1, specifically blocking the binding of pCAV1 to the ATG12-ATG5 complex, thereby reducing the formation of the pCAV1-ATG12-ATG5 complex to activate endothelial cell autophagy and reducing the transendothelial cell transcellular amount of LDL. It has been shown to effectively delay the progression of early atherosclerotic plaques. Attached Figure Description

[0021] Figure 1 Statistical graph for the detection of CBM peptide toxicity and half-life;

[0022] Figure 2 This is a double immunofluorescence pattern showing the specific binding of CBM peptide to pCAV1.

[0023] Figure 3 Image of immunoprecipitation of CBM peptide bound to pCAV1;

[0024] Figure 4 A statistical graph showing the expression of autophagy-related proteins after CBM peptide application;

[0025] Figure 5 A statistical graph showing the effect of CBM peptide introduction on LDL transcellularity in endothelial cells;

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

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

[0028] Figure 8 ApoE after CBM peptide introduction - / - Images and statistical graphs of plaque area at the root of the mouse aorta and throughout the aorta. Detailed Implementation

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

[0030] The applicant has invented a novel antagonistic short peptide that targets pCAV1 based on the CBM (caveolin-1 binding motif) structure on ATG12. This peptide activates autophagy by specifically binding to pCAV1 and inhibiting the formation of the pCAV1-ATG12-ATG5 complex, thereby reducing the amount of LDL penetrating into endothelial cells and thus delaying the progression of early atherosclerosis.

[0031] Specifically, in this invention, the polypeptide targeting and antagonizing pCAV1 is named CBM polypeptide (also known as VHPK-TAT-CBM peptide), which mainly consists of three parts: a VHPK targeting peptide, a TAT membrane-penetrating peptide, and a 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 (an endothelial cell targeting peptide with high affinity for VCAM-1 (vascular cellular adhesion molecule-1) on the surface of endothelial cells) and TAT (a cationic cell-penetrating peptide). 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] This invention provides a novel perspective on how CBM peptides target pCAV1 in endothelial cells to prevent early atherosclerosis. After entering cells via the VCAM-1 receptor on the endothelial cell membrane, the CBM peptides target and block the interaction between pCAV1 and the ATG12-ATG5 complex. This effectively activates autophagy in endothelial cells and reduces the transcellular penetration of LDL, thereby delaying the formation of early atherosclerotic plaques.

[0033] The following is a description through specific embodiments.

[0034] Example 1: CBM Peptide Synthesis Steps

[0035] The ATG12-ATG5 system is a ubiquitin-like system required for autophagy formation. Based on the CBM structure on ATG12, a CBM peptide (VHPK-TAT-CBM peptide) was designed as an antagonistic short peptide targeting pCAV1. It mainly consists of three parts: a VHPK targeting peptide, a TAT membrane-penetrating peptide, and a CBM target peptide. The peptide sequence is as follows: VHPKQHRGGSKGC-GG-YGRKKRRQRRR-GG-FIYVNQSFAPK. This peptide sequence was acetylated at the N-terminus and amidated at the C-terminus, and methylated at the 29th amino acid N to form a peptide derivative, improving its stability. The improved sequence is: Ac-VHPKQHRGGSKGC-GG-YGRKKRRQRRR-GG-F(N-Me)IYVNQSFAPK-NH2 (In this invention, all examples were conducted using the improved peptide sequence). VHPK is an endothelial cell targeting peptide that specifically binds to VCAM-1 on the surface of endothelial cells. In addition, the cell-penetrating peptide TAT (a short cationic peptide rich in arginine) has high cell membrane permeability, almost no killing effect on cells, and can improve the delivery effect.

[0036] In this invention, the Fmoc solid-phase synthesis method is used to synthesize CBM peptides. Fmoc has the advantages of being able to absorb ultraviolet light and being easy to detect, and it is widely used because it has mild reaction conditions, high yield and simple operation.

[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 reagent

[0041] Benzotriazole-N,N,N',N'-Tetramethylurea 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 degree of substitution of 0.38 mmol / g

[0046] 5. Deprotection reagent

[0047] Piperidine

[0048] 6. Test reagents:

[0049] Phenol reagent, pyridine reagent, ninhydrin reagent

[0050] 7. Cutting reagent

[0051] Trifluoroacetic acid (TFA), triisopropylsilane (TIS), 1,2-ethylenedithiol (EDT), anhydrous diethyl 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 (HPLC)

[0057] 3. Freeze dryer

[0058] 4. Centrifuge

[0059] (3) Synthesis steps:

[0060] I. Resin swelling

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

[0062] 2. Continuing with the first amino acid

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

[0064] III. Deprotection

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

[0066] IV. Testing

[0067] Remove the piperidine solution, take a dozen or so resin grains, wash them three times with ethanol, add one drop each of ninhydrin, potassium cyanide (KCN), and phenol solution, and heat at 105℃-110℃ for 5 minutes. A deep 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] VI. Condensation

[0071] Add 3 times molar excess of Fmoc to protect amino acids, 3 times molar excess of HBTU, then add 10 times molar excess of DIEA, and finally add DMF to dissolve. Shake for 45 minutes.

[0072] VII. Testing

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

[0074] 8. Washing

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

[0076] 9. Repeat steps three through eight, connecting from right to left, until the Fmoc protecting group of the last amino acid is removed, and then acetylate to cap the end.

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

[0078] DMF (10 mL / g) twice, DCM (10 mL / g) three times, methanol (10 mL / g) four times, and then the mixture was dried under vacuum for 10 minutes.

[0079] 11. Cutting

[0080] Preparation of cutting fluid (10 mL / g): TFA 95%; Water 2%; EDT 2%; TIS 1%

[0081] Cutting time: 180 min

[0082] 12. Dry and wash

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

[0084] 13. Purification and preparation.

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

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

[0087] 3. Preparation using a C18 reversed-phase chromatography 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. Take a small amount of the target peak solution in a 1.5 mL centrifuge tube for mass spectrometry confirmation and purity detection.

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

[0092] XV. Appraisal

[0093] Small amounts of the finished peptide were taken and subjected to molecular weight determination by MS and purity determination by HPLC.

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

[0095] Example 2: Detection of CBM peptide toxicity and half-life

[0096] like Figure 1 As shown, firstly, dosage optimization was performed using different concentrations (2.5, 5, 10, and 20 μM) and time points (6, 12, 24, and 48 h) of CBM peptide. No statistically significant differences in cytotoxicity to HUVECs were observed among the different concentrations of CBM peptide. Furthermore, the application of 20 μM CBM peptide showed no significant differences in cytotoxicity at any time point, indicating that CBM peptide is non-toxic to normal endothelial cells and has high biosafety. The CBM half-life (t...) 1 / 2 The experimental results (40.582h) showed that its effect reached its peak at 6h and remained stable for 24h before starting to degrade, indicating that the CBM peptide has a relatively long stability.

[0097] Example 3: Investigating the blocking effect of CBM peptides

[0098] To examine whether the CBM peptide possesses targeting ability for pCAV1 and can exert a pCAV1 blocking effect, Cy3-labeled CBM peptide and a plasmid encoding CAV1-Y14D-Flag were introduced into endothelial cells. The binding of the CBM peptide to overexpressed pCAV1 was observed using a double immunofluorescence assay under confocal microscopy. Figure 2 As shown, the results indicated that the CBM peptide co-localized with pCAV1 overexpressed by CAV1-Y14D in the cell membrane and part of the cytoplasm of endothelial cells, suggesting that the CBM peptide has targeting ability for pCAV1.

[0099] Secondly, such as Figure 3 As shown, the interaction between CBM peptide and CAV1-Y14D was further confirmed by immunoprecipitation experiments. Similarly, after adding CBM peptide to endothelial cells inoculated with the CAV1-Y14D plasmid, CBM peptide successfully pulled down Flag-Y14D, and Flag-Y14D successfully pulled down CBM, demonstrating the existence of the interaction between CBM and Y14D. In addition, compared with the CAV1-Y14D group, the introduction of CBM peptide reduced the interaction between Y14D-ATG12-ATG5, indicating that CBM peptide can specifically bind to pCAV1 and weaken the interaction between pCAV1-ATG12-ATG5.

[0100] Example 4 verifies the role of pCAV1 antagonistic short peptide (CBM polypeptide) in endothelial cells.

[0101] To verify the role of the pCAV1 antagonistic peptide (CBM peptide) in endothelial cells, autophagy and LDL translocation were measured after introducing the CBM peptide into the cells. Firstly, the CBM peptide activated endothelial cell autophagy and reduced the expression levels of CAV1 and pCAV1, while simultaneously restoring the inhibition of autophagy flux and the increase in CAV1 and pCAV1 expression induced by CAV1-Y14D overexpression. Figure 4 Secondly, the exogenous introduction of CBM peptides also reduced the amount of LDL penetrating into endothelial cells and could eliminate the increase in LDL penetrating amount caused by overexpression of CAV1-Y14D. Figure 5 In summary, our findings suggest that CBM peptides can block the effects of pCAV1-induced autophagy inhibition and increased LDL transcellularity by directly binding to pCAV1, indicating that the CBM structure may have a potential protective effect against the early progression of atherosclerosis.

[0102] Example 5 investigated the expression of CBM peptide in the mouse aorta.

[0103] To further confirm the in vitro study results and verify whether the pCAV1 antagonistic peptide functions in vivo, AAV9 adeno-associated virus encoding CAV1-Y14D (AAV-Y14D) was introduced into ApoE. - / - In a mouse model of atherosclerosis, CBM peptides (labeled with Cy3) were used for experimental treatment to evaluate their effect on the progression of atherosclerotic plaques in vivo. Simultaneously, in ApoE... - / - Adeno-associated virus vector (AAV-Vector) and TAT were introduced into mice as negative controls. Thirty-two mice were randomly divided into four groups (n = 8 / per group): 1. AAV-Vector + TAT; 2. AAV-Vector + CBM peptide; 3. AAV-Y14D + TAT; 4. AAV-Y14D + CBM peptide. All groups were fed a high-fat diet (21% fat + 0.15% cholesterol). At week 8, mice were randomly assigned to the AAV-Vector and AAV-Y14D groups (n = 16 / per group) at a dose of 5 × 10⁻⁶. 11 Mice were administered AAV-Vector and AAV-Y14D once daily via tail vein injection (vg / mouse). Starting from week 16, mice were randomly assigned to TAT control and CBM peptide treatment subgroups (n=8 / per group), and were administered CBM peptide and TAT control peptide at a concentration of 1.5 mg / kg via tail vein injection (v) daily until week 21.

[0104] At week 21, blood was collected from mice under anesthesia, and the hearts and aortas were fixed with 4% paraformaldehyde. In vivo fluorescence imaging was performed before sacrifice, such as... Figure 6 As shown, CBM-Cy3 fluorescence was distributed in mouse heart and aortic tissues, indicating that the CBM peptide can specifically act on the heart and aorta. In vitro animal vascular imaging also verified that the CBM-Cy3 peptide can specifically act on the aortic vessels.

[0105] Example 6 investigated the effect of CBM peptides on atherosclerotic plaque deposition in mice.

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

[0107] Oil Red O is a lipid-soluble dye that specifically stains neutral lipids such as triglycerides in cells or tissues, making them appear red or orange-red. In pathological conditions, Oil Red O staining is commonly used to detect atherosclerosis and fat embolism, revealing lipid deposition beneath endothelial cells and lipids within fat emboli. Compared to AAV-Vector mice, ApoE mice receiving AAV-Y14D showed... - / - Oil Red O lipid staining in mice showed increased atherosclerotic lesions. Figure 8 The presence of atherosclerotic plaques is manifested by lipid deposition in the subendothelial space of the aortic root and a significant increase in the area of ​​aortic lesions. However, CBM peptides can 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, both in vitro and in vivo experiments have demonstrated that intervention with the pCAV1 antagonistic short peptide (CBM peptide) can significantly reduce atherosclerotic plaque deposition induced by pCAV1 overexpression. The CBM peptide also has an effect on ApoE. - / - The CBM peptide significantly inhibited plaque progression in mice by specifically binding to pCAV1 and inhibiting the formation of the pCAV1-ATG12-ATG5 complex, thereby activating endothelial cell autophagy and reducing the transendothelial cell transcellular amount of LDL to decrease atherosclerotic lesions. These results suggest that CBM peptide delivery may be a promising therapeutic approach for delaying early AS progression.

[0109] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A polypeptide that targets and antagonizes pCAV1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1: VHPKQHRGGSKGCGGYGRKKRRQRRRGGFIYVNQSFAPK.

2. A polypeptide derivative that targets and antagonizes pCAV1, characterized in that, The polypeptide derivative is a polypeptide derivative in which the N-terminus of SEQ ID NO: 1 as described in claim 1 is acetylated and the C-terminus is amidated, and the 29th amino acid N is methylated.

3. A polynucleotide encoding a polypeptide that targets and antagonizes pCAV1 as described in 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 plasmids or bacteriophages.

6. The use of the polypeptide of claim 1 or the polypeptide derivative of claim 2 in the preparation of a drug targeting and antagonizing pCAV1, wherein the drug targeting and antagonizing pCAV1 is a drug for treating atherosclerosis.

7. A drug for treating atherosclerosis, characterized in that, Contains a polypeptide that targets and antagonizes pCAV1 as described in claim 1 or a polypeptide derivative that targets and antagonizes pCAV1 as described in claim 2.

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