A polypeptide for inhibiting intracellular P-body formation and its application

Abstract

This invention discloses a polypeptide for inhibiting the formation of P-bodies in cells and its applications. The amino acid sequence of the polypeptide is shown in SEQ ID NO.4. This invention identifies the key domains required for P-body formation through structural analysis of the key proteins of the P-body; it designs an inhibitory peptide for the key P-body proteins using AlphaFold 3 and bioinformatics methods; and it obtains the polypeptide with the amino acid sequence shown in SEQ ID NO.4 by combining a cell-penetrating peptide with the designed inhibitory peptide, thereby enhancing its intracellular bioavailability. This polypeptide can effectively inhibit and block the formation of intracellular P-bodies, and at a concentration of 30 μM, it can significantly reduce the expression of inflammatory factors in endothelial cells, playing an important role in protecting the blood-brain barrier and having significant implications for the treatment of cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and biotechnology, and in particular to a polypeptide for inhibiting the formation of P-bodies in cells and its applications. Background Technology

[0002] The P-body (processing body) is a non-membrane organelle located in the cytoplasm, primarily involved in the metabolic processes of mRNA, including degradation, storage, and translation regulation. The P-body has a granular structure and several key characteristics: (1) Composition: The P-body is composed of various RNA-binding proteins and RNA, which play important roles in its function; (2) Degradation site: The P-body is the main site of mRNA degradation, effectively regulating gene expression; (3) Translational repression: mRNA can be isolated within the P-body, thereby inhibiting its translation; (4) Environmental response: The P-body can respond to changes in the intracellular environment, such as stress or nutrient deficiency, thereby regulating RNA fate and participating in stress responses; (5) Dynamic characteristics: The P-body is not a fixed structure; it dynamically changes according to the physiological state of the cell and changes in the external environment.

[0003] The main protein components that make up P-bodies include DDX6, EDC4, 4E-T, LSM14a, and PATL1. These protein molecules bind together through key structural domains to form P-bodies. Each of these proteins is essential for P-body assembly. Current literature has demonstrated the structural domains through which protein molecules bind in P-bodies and has explored gene modification methods to inhibit P-body formation. However, there are currently no studies or products on peptides that inhibit P-body formation in cells.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a polypeptide for inhibiting the formation of P-body in cells and its application, aiming to solve the problem of the lack of polypeptide drugs for inhibiting the formation of P-body in cells in the prior art.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, a polypeptide for inhibiting the formation of P-bodies in cells is provided, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] In a second aspect, a nucleic acid is provided, said nucleic acid encoding a polypeptide as described in the first aspect.

[0009] Thirdly, a recombinant vector is provided that expresses the polypeptide as described in the first aspect.

[0010] Fourthly, a composition is provided comprising the polypeptide as described in the first aspect.

[0011] In a preferred embodiment, the composition further includes a pharmaceutically acceptable solvent.

[0012] In a further preferred embodiment, the solvent is selected from one or more of water, physiological saline, glucose aqueous solution, and dimethyl sulfoxide.

[0013] In a preferred embodiment, the composition further includes a pharmaceutically acceptable carrier.

[0014] In a further preferred embodiment, the pharmaceutically acceptable carrier is selected from one or more excipients, fillers, binders, lubricants, disintegrants, and stabilizers.

[0015] Fifthly, the use of a polypeptide as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant vector as described in the third aspect, and a composition as described in the fourth aspect in the preparation of a reagent that inhibits the formation of P-bodies in cells.

[0016] In a sixth aspect, the use of the polypeptide as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant vector as described in the third aspect, and the composition as described in the fourth aspect in the preparation of a medicament for treating cardiovascular and cerebrovascular diseases is provided.

[0017] Beneficial Effects: This invention, through structural analysis of key proteins in the P-body, identified the crucial domains required for P-body formation. Using AlphaFold 3 and bioinformatics techniques, an inhibitory peptide for the key P-body protein was designed. By combining the cell-penetrating peptide with the designed inhibitory peptide, a polypeptide with the amino acid sequence shown in SEQ ID NO.4 was obtained, enhancing its intracellular bioavailability. This polypeptide can effectively inhibit and block the formation of intracellular P-bodies, and at a concentration of 30 μM, it can significantly reduce the expression of inflammatory factors in endothelial cells, playing an important role in protecting the blood-brain barrier and having significant implications for the treatment of cardiovascular and cerebrovascular diseases. Attached Figure Description

[0018] Figure 1 This is a graph showing the binding analysis of peptide-1 and DDX6 protein.

[0019] Figure 2 This is a diagram showing the binding analysis of peptide-2 and DDX6 protein.

[0020] Figure 3 This is a diagram showing the binding analysis of peptide-3 and DDX6 protein.

[0021] Figure 4 This is a diagram showing the binding analysis of peptide-4 and 4E-T protein.

[0022] Figure 5 This is a graph showing the effects of peptides 1-4 on the cell viability of bEend.3 cells.

[0023] Figure 6 This is a graph showing the change in the intensity of polypeptide-1-Cy5 entering bEend.3 cells over incubation time.

[0024] Figure 7 This is a graph showing the effect of polypeptide-1 on the number of P-bodies in bEend.3 cells.

[0025] Figure 8 This is a graph showing the effect of polypeptide-2 on the number of P-bodies in bEend.3 cells.

[0026] Figure 9 This is a graph showing the effect of polypeptide-3 on the number of P-bodies in bEend.3 cells.

[0027] Figure 10 This is a graph showing the effect of polypeptide-4 on the number of P-bodies in bEend.3 cells.

[0028] Figure 11 This is a graph showing the effect of peptide-5 on the number of P-bodies in bEend.3 cells.

[0029] Figure 12 This is a diagram showing the results of peptide-4 alleviating BMEC damage caused by OGD / R.

[0030] Figure 13 This is a graph showing the results of peptide-4 reducing the mRNA levels of inflammatory factors in bEnd.3 cells induced by OGD / R. Detailed Implementation

[0031] This invention provides a polypeptide for inhibiting the formation of P-body in cells and its application. To make the purpose, technical solution and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0032] P-bodies play a crucial role in the fine-tuning of intracellular RNA regulation and gene expression. Dysregulation of P-body function and RNA regulation is associated with a variety of diseases, including cancer and neurodegenerative diseases. Understanding P-body RNA regulation is essential for elucidating the complexity of gene regulation and cellular responses to environmental changes. However, there are currently no reagents or drugs available to regulate the structure and function of P-bodies.

[0033] Based on this, embodiments of the present invention provide a polypeptide for inhibiting the formation of P-bodies in cells, the amino acid sequence of which is shown in SEQ ID NO.4.

[0034] This invention performs structural analysis on key proteins of the P-body, identifying the critical domains required for P-body formation. Using AlphaFold 3 and bioinformatics techniques, corresponding repressive peptides (repressive peptides-1–4) were designed for each key protein molecule, along with a non-specific peptide as a null control (null control peptide-5). To improve the cell membrane permeability of the repressive peptides, a transmembrane-penetrating peptide (TAT) with the amino acid sequence YGRKKRRQRRR was linked to the N-terminus of the aforementioned P-body repressive peptides. By combining the transmembrane-penetrating peptide with the designed repressive peptide, a complex of peptides-1–4 (SEQ ID NO. 1–4) was formed to enhance their intracellular bioavailability. Studies using peptides-1–4 showed that the peptides with the added transmembrane-penetrating peptide sequence accumulated intracellularly, reaching peak concentration at 12 hours. Meanwhile, peptide-4, with the amino acid sequence shown in SEQ ID NO.4, effectively inhibited P-body formation at a concentration of 30 μM and significantly reduced the expression of inflammatory factors in endothelial cells, playing an important role in protecting the blood-brain barrier. Other peptides, including peptide-1, peptide-2, and peptide-3, failed to effectively inhibit P-body formation. Therefore, peptide-4 has significant implications for the treatment of cardiovascular and cerebrovascular diseases.

[0035] This invention provides a nucleic acid that encodes a polypeptide as described above.

[0036] This invention provides a recombinant vector that expresses the polypeptide as described above.

[0037] This invention provides a composition comprising the polypeptides described above.

[0038] In one embodiment, the composition further includes a pharmaceutically acceptable solvent.

[0039] In a more specific embodiment, the solvent is selected from one or more of water, physiological saline, glucose aqueous solution, and dimethyl sulfoxide (DMSO).

[0040] In one embodiment, the composition further includes a pharmaceutically acceptable carrier.

[0041] In a more specific embodiment, the pharmaceutically acceptable carrier is selected from one or more excipients, fillers, binders, lubricants, disintegrants, and stabilizers.

[0042] This invention provides the application of the polypeptide, nucleic acid, recombinant vector, and composition described above in the preparation of a reagent that inhibits the formation of P-bodies in cells.

[0043] This invention provides the application of the polypeptide, nucleic acid, recombinant vector, and composition described above in the preparation of a medicament for treating cardiovascular and cerebrovascular diseases.

[0044] The present invention will be further described below through specific embodiments.

[0045] Example 1

[0046] I. Experimental Design

[0047] 1. Design peptides to inhibit P-body formation

[0048] Protein sequences of key P-body binding proteins collected by NCBI, with information from GeneBank, include:

[0049] (1)DDX6 (Mus musculus): P54823;

[0050] (2)4E-T (Mus musculus): Q9EST3;

[0051] (3)LSM14a (Mus musculus): Q8K2F8;

[0052] (4)PATL1 (Mus musculus): Q3TC46.

[0053] Based on their interaction sites, this embodiment designed five repressive peptides, as follows:

[0054] (1) The inhibitory peptide-1 occupying the DFDF and TGF structural pockets (from the 219-238 site sequence of mouse 4E-T) in the RECA2 region of DDX6: EWFSAGPTSQSETIELTGFD;

[0055] (2) Repressor peptide-2 occupying the TGF structural pocket (from the 27-40 site sequence of mouse PATL-1): EEIDQFNDDTFGSG;

[0056] (3) Repressive peptide-3 occupying the DFDF structural pocket (from the 290-304 site sequence of mouse LSM14-A): KFEKDFDFESANAQFNK;

[0057] (4) Inhibitory peptide-4 occupying the structural pocket of DDX6 (from the 317-334 site sequence of mouse DDX6): TERQKVHCLNTLFSRLQI;

[0058] (5) Non-specific null control peptide-5: DKENQDFEFEAKEFRKEHINFSAD.

[0059] Based on this, a cell-penetrating peptide sequence (YGRKKRRQRRR) was added to the N-terminus of the initial peptide to construct a peptide for inhibiting the formation of P-bodies within cells. The sequences of the five peptides are as follows:

[0060] (1) 4E-T based peptide-1: YGRKKRRQRRREWFSAGPTSQSETIELTGFD (SEQ ID NO.1);

[0061] (2) PATL-1-based peptide-2: YGRKKRRQRRREEIDQFNDDTFGSG (SEQ ID NO.2);

[0062] (3) LSM14-A-based peptide-3: YGRKKRRQRRRKFEKDFDFESANAQFNK (SEQ ID NO.3);

[0063] (4) DDX6-based peptide-4: YGRKKRRQRRRTERQKVHCLNTLFSRLQI (SEQ ID NO.4);

[0064] (5) Peptide-5 of the invalid control: YGRKKRRQRRRDKENQDFEFEAKEFRKEHINFSAD (SEQ ID NO.5).

[0065] NCBI protein sequence comparison ensured that these designed peptides had no homologous sequences in the mouse proteome. Simultaneously, AlphaFold 3 was used to simulate these peptides with their corresponding P-body domains to determine the spatial locations of the peptides within these domains.

[0066] 2. Dissolution and preservation of peptides

[0067] Custom-synthesized peptides 1-5 and a fluorescently tagged peptide 1-Cy5 were prepared. Peptides 1 and 1-Cy5 were dissolved in DMSO, prepared into storage solutions, aliquoted, and frozen at -80°C. Peptides 2-5 were dissolved in ultrapure water, prepared into storage solutions, filtered, aliquoted, and frozen at -80°C.

[0068] 3. Construction of plasmids and viral vectors

[0069] The DNA sequence of Ddx6 (NM_001357703.1) was cloned into the pLentiV-EGFP vector to construct the Ddx6-egfp recombinant plasmid (using BamHI / AfeI restriction sites). The recombinant plasmid, along with pMD2.G and psPAX2, was co-transfected into HEK293T cells using Lipo-3000 reagent. Four hours after transfection, the medium was replaced with complete medium, and the cells were cultured for another 48 hours. Subsequently, the cell supernatant was collected, filtered through a 0.22 μm filter, and centrifuged at 3000 rpm for 10 minutes to collect viral particles. The collected virus was stored at -80°C.

[0070] 4. Cell line construction

[0071] The collected virus was added to 10% (v / v) FBS-DMEM complete medium containing 1% (v / v) polybrene and co-incubated with bEnd.3 cells. After 48 hours, the presence of EGFP signal in the cells under 488nm excitation was confirmed by microscopy (Carl Zeiss, Axio Observer 7, Germany). Cells were then digested with trypsin to prepare single-cell suspensions. Monoclonal cell sorting was performed using a BD FACSAria SORP flow cytometer with a 100μm nozzle, and the sorted cells were seeded into 96-well plates. After 14 days, successfully surviving monoclonal cells were selected for further culture.

[0072] 5. Cytotoxicity studies

[0073] The bEnd.3 vascular endothelial cell line was divided into 1×10 4 Cells were seeded at a density of 1 / mL in 96-well plates and cultured in DMEM complete medium containing 10% (v / v) FBS. When the cells reached 70%–80% confluence, the medium was replaced with medium containing the peptide designed in this embodiment at concentrations of 0.003 μM, 0.03 μM, 0.3 μM, 3 μM, 15 μM, and 30 μM. The control group contained an equal volume of solvent. After 24 hours of incubation, 10 μL of CCK-8 solution was added to each well, and after 1 hour of incubation, the absorbance was measured using a microplate reader to calculate cell viability.

[0074] 6. Pharmacological studies on blocking P-body formation

[0075] The bEnd.3 vascular endothelial cell line expressing DDX-6-EGFP obtained in step 4 was cultured in DMEM complete medium containing 10% (v / v) FBS at a concentration of 1×10⁻⁶. 4Cells were seeded at a density of 1 / mL in glass-bottomed culture dishes. When the cells grew to 70%–80% confluence, they were photographed under a microscope, and then the culture medium was replaced with one containing the peptide designed in this embodiment at concentrations of 0.3 μM, 3 μM, and 30 μM; the control group received an equal amount of peptide-5. Cells were incubated in the new medium for 5 hours, and then photographed again after 24 hours to begin the oxygen-glucose deprivation (OGD) experiment. After removing the medium, the cells were washed twice with DPBS, and then treated with 95% (v / v) nitrogen and 5% (v / v) carbon dioxide-free EBSS solution. The cells were placed in a hypoxic chamber, and a mixture of 95% (v / v) nitrogen and 5% (v / v) carbon dioxide was introduced for 15 minutes to reduce the oxygen concentration in the chamber to below 0.5% (v / v). After stopping the gas supply, the chamber containing the cells was placed in an incubator at 37°C for 4 hours. After treatment, the chambers were opened and cells were fixed with 4% (w / v) paraformaldehyde, followed by microscopic imaging. Another group of cells, after undergoing oxygen-glucose deprivation, was replaced with complete culture medium containing the peptides designed in this embodiment, incubated for another 4 hours, fixed with 4% (w / v) paraformaldehyde, and photographed. Finally, P-body counting and statistical analysis were performed using ImageJ software.

[0076] 7. Study on the protective effect of polypeptide-4 on primary mouse brain microvascular endothelial cells (BMEC)

[0077] Primary vascular endothelial cell extraction: Ten adult mice (3-4 months old) were anesthetized and sacrificed. Brain tissue was removed, meninges were removed, and cortical tissue was separated and minced. Type II collagenase (1 mg / mL) was added, and the mixture was digested at 37°C with shaking for 40 minutes. The mixture was then centrifuged at 1000g for 5 minutes at 4°C, the supernatant was discarded, and 25 times the volume of 20% (w / v) BSA solution was added. After mixing, the mixture was centrifuged again at 1000g for 20 minutes at 4°C, and the supernatant was discarded. Next, type II collagenase-dispersant enzyme (1 mg / mL) was added, and the mixture was digested at 37°C with shaking for 40 minutes. The mixture was then centrifuged again at 1000g for 5 minutes at 4°C, and the supernatant was discarded. A small amount of DMEM was added, and the mixture was placed in 30% (w / v) Percoll solution and centrifuged at 1000g for 10 minutes at 4°C. Take the red precipitate near the bottom and the yellowish-white portion above it, dilute with DMEM, and then centrifuge at 1000g for 10 minutes at 4°C. Seed the obtained brain microvascular segments into fibronectin-coated culture plates. During the 48-hour culture period, puromycin (4 μg / mL) was added to the culture medium to remove non-endothelial cells, and then the medium was replaced with normal culture medium (containing high-glucose DMEM, 20% (v / v) FBS, 100 μg / mL heparin sodium, 2 ng / mL bFGF, 2 mM glutamine, and pen-strep antibiotics).

[0078] Cell Culture and Treatment: After culturing primary vascular endothelial cells for 14 days, the medium was replaced with medium containing 30 μM of peptide-4 (as described in this example), and incubated for 24 hours. The medium was then removed, and the cells were washed twice with DPBS. DMEM solution (glucose-free) treated with 95% (v / v) nitrogen and 5% (v / v) carbon dioxide was added. The cells were placed in a hypoxic chamber, and a mixture of 95% (v / v) nitrogen and 5% (v / v) carbon dioxide was introduced for 15 minutes to reduce the oxygen concentration in the chamber to below 0.5% (v / v). After stopping the gas infusion, the chamber containing the cells was placed in a 37°C incubator for 4 hours. After treatment, the medium was replaced with complete medium, and incubation continued for 24 hours. 10 μL of CCK-8 solution was added to each well, and after incubation for 1 hour, the absorbance was measured using a microplate reader to calculate cell viability.

[0079] 8. Effect of peptide-4 on the expression of inflammatory factors mRNA in OGD / R-induced bEnd.3 cells

[0080] The peptide-4 dose was 30 μM, and the control group was supplemented with an equal volume of solvent. After incubation with bEnd.3 cells for 24 h, OGD treatment was performed for 6 h. During OGD treatment, 30 μM peptide-4 or solvent was added to the sugar-free DMEM medium. After OGD treatment, the medium was restored to DMEM containing glucose and oxygen, and 30 μM peptide-4 or solvent was added again, and incubated for 2 h or 4 h. Cells were collected at three time points: OGD-6 h, OGD-6 h / R-2 h, and OGD-6 h / R-4 h. Total RNA was extracted from the cells using a PCR extraction kit, and the mRNA levels of TNF-α, G-CSF, GM-CSF, CXCL1, CXCL2, ICAM1, and VCAM1 in the cells were measured using a Real-Time PCR detection kit and instrument.

[0081] II. Experimental Results

[0082] 1. Peptide structure

[0083] Alpha Fold 3 was used to analyze the binding of peptide-1, peptide-2, and peptide-3 to DDX6 protein, and the binding of peptide-4 to 4E-T protein. The results are as follows: Figures 1-4 As shown. Figures 1-4 In the diagram, orange represents DDX6, silver represents 4E-T, bright purple represents the binding site identified in the literature, and green / cyan / blue / orange represent peptides 1 through 4, respectively. Figures 1-4 It is known that peptides 1 through 4 can all bind to their associated proteins.

[0084] 2. The peptide showed no cytotoxicity to bEend.3 cells.

[0085] Cytotoxicity study results such as Figure 5 As shown. By Figure 5 It was found that the five synthesized peptides had no significant effect on the survival rate of bEend.3 cells at concentrations ranging from 0.003 to 30 μM, indicating that these five peptides have good safety.

[0086] 3. Determination of the time window for fluorescently labeled peptides entering bEend.3 cells

[0087] In this embodiment, peptide-1 was labeled with a Cy5 fluorescent tag and co-incubated with bEend.3 cells at different time points. Fluorescence images of bEend.3 cells were acquired using a fluorescence microscope under the same exposure parameters, and the results are as follows. Figure 6 As shown. By Figure 6 It can be seen that the fluorescence intensity reaches its maximum value after 12 hours of incubation.

[0088] 4. Peptide-4 significantly inhibits P-body formation.

[0089] The experimental results of the five peptides at different doses and during the oxygen-glucose deprivation / recovery (OGD / R) process are as follows: Figures 7-11 As shown. By Figure 7 It was found that peptide-1 could not prevent the formation of P-bodies under non-OGD / R conditions, but it could significantly inhibit the formation of P-bodies in cells after OGD at concentrations of 3 μM and 30 μM. Figure 10 It was found that peptide-4 failed to prevent P-body formation under non-OGD / R conditions, but at a concentration of 30 μM, it significantly inhibited P-body formation in cells during OGD and recovery processes. Figure 8 , 9 As shown in Figures 1 and 11, peptides 2, 3, and 5 cannot prevent the formation of P-bodies in either non-OGD / R or OGD / R conditions. Therefore, peptide 4 significantly inhibits the formation of P-bodies during OGD / R, demonstrating a good blocking effect.

[0090] 5. Peptide-4 alleviates BMEC damage caused by OGD / R.

[0091] The results of the study on the protective effect of peptide-4 on BMEC are as follows: Figure 12 As shown. By Figure 12 It is known that BMEC cells are damaged during OGD / R, with cell viability decreasing by 30%. Administration of peptide-4 can significantly alleviate BMEC cell damage, doubling cell viability while reducing it by only 15%. Therefore, peptide-4 can alleviate BMEC damage caused by OGD / R.

[0092] 6. Peptide-4 reduces the levels of inflammatory factors expressed in bEend.3 cells induced by OGD / R.

[0093] The effect of peptide-4 on the expression of inflammatory factors mRNA in OGD / R-induced bEnd.3 cells is as follows: Figure 13 As shown. By Figure 13 It was found that the mRNA levels of all inflammatory factors significantly increased during OGD / R. Using 30 μM peptide-4 significantly inhibited the mRNA levels of multiple inflammatory factors expressed in bEnd.3 cells, including G-CSF, VCAM1, and ICAM1. Therefore, peptide-4 has a significant inhibitory effect on the expression of inflammatory factors in vascular endothelial cells induced by OGD / R.

[0094] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A polypeptide for inhibiting the formation of P-bodies in cells, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

4.

2. A nucleic acid, characterized in that, The nucleic acid encodes the polypeptide as described in claim 1.

3. A recombinant vector, characterized in that, The recombinant vector expresses the polypeptide as described in claim 1.

4. A composition, characterized in that, The composition comprises the polypeptide as described in claim 1.

5. The composition according to claim 4, characterized in that, The composition also includes a pharmaceutically acceptable solvent.

6. The composition according to claim 5, characterized in that, The solvent is selected from one or more of water, physiological saline, glucose aqueous solution, and dimethyl sulfoxide.

7. The composition according to claim 4, characterized in that, The composition also includes a pharmaceutically acceptable carrier.

8. The composition according to claim 7, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: excipients, fillers, binders, lubricants, disintegrants, and stabilizers.

9. The use of a polypeptide as described in claim 1, a nucleic acid as described in claim 2, a recombinant vector as described in claim 3, or a composition as described in any one of claims 4 to 8 in the preparation of a medicament for treating cardiovascular and cerebrovascular diseases.

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