Functional site of targeting ACAP4 protein for inhibiting tumor cell migration and application of functional site

Abstract

The invention belongs to the technical field of protein engineering, and particularly relates to a functional site for inhibiting tumor cell migration of a targeted ACAP4 protein and application of the functional site. And the site is any one or a combination of more of Patch2, Pair 2, Pair 3 and Pair 4. The ACAP4BAR crystal is obtained through crystal screening, the structure of the ACAP4BAR crystal is analyzed, seven positive charge enrichment regions on the surface of ACAP4BAR-PH are analyzed and found, and four key sites influencing cell migration are found. The four sites provided by the invention have a certain regulation effect on cell membrane localization of ACAP4 protein, can regulate the cell migration ability, and provide a molecular basis and a theoretical basis for treatment of diseases, cancer invasion and metastasis and the like caused by related protein dysfunction.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and particularly relates to a functional site targeting ACAP4 protein to inhibit tumor cell migration and its application. Technical Background

[0002] Tumor-related protein ACAP4 is a member of ArfGAPs. It is hardly expressed in normal tissue cells, but is abnormally highly expressed in a variety of cancer tissue cells and participates in regulating the occurrence of many cancers and the metastasis process of tumor cells. Previous studies have shown that ACAP4 can promote cell migration and growth by participating in cell dynamic regulation processes such as cell membrane remodeling and cytoskeleton rearrangement. Specifically, ACAP4 catalyzes the cycle of the GTP / GDP binding mode of Arf protein through its ArfGAP domain, affects the activity of small G proteins and then regulates the rearrangement of cytoskeletal proteins; ACAP4 senses the cell membrane curvature through its BAR-PH domain, and then changes the morphology of the cell membrane to exert the dynamic regulation function of the cell membrane; ACAP4 connects the cell membrane and the cytoskeleton network through its interaction with effector proteins such as the actin-binding protein Ezrin.

[0003] However, due to the lack of relevant biochemical research and structural information, the internal regulatory mechanism of the ACAP4 interaction signal regulation network and the occurrence mechanism of a series of downstream cell dynamic regulation events caused by this network are still unclear. Studying the structural basis and molecular regulatory mechanism of ACAP4 and its complex in the cell membrane-cytoskeleton cell dynamic regulation process is crucial for understanding the function of ACAP4 in promoting physiological processes such as cell migration and the role it plays in pathological events such as cancer occurrence and metastasis. So far, there has been no relevant report on the structure of ACAP4 and the mechanism of promoting cancer cell migration. Summary of the Invention

[0004] To solve the problems in the prior art, one of the purposes of the present invention is to provide a functional site targeting ACAP4 protein to inhibit tumor cell migration.

[0005] A functional site for targeting ACAP4 protein to inhibit the migration of tumor cells, which is any one or a combination of more than one of Patch 2, Pair 2, Pair 3, and Pair 4. Patch 2, Pair 2, Pair 3, and Pair 4 are located in the BAR-PH domain of the ACAP4 protein, and the sequence of the BAR-PH domain is shown in SEQ ID NO.1. Among them, Patch 2 consists of 3 amino acids at positions 145, 149, and 152 of the amino acid sequence of the BAR-PH domain, Pair 2 consists of 2 amino acids at positions 311 and 323 of the amino acid sequence of the BAR-PH domain, Pair 3 consists of 2 amino acids at positions 317 and 318 of the amino acid sequence of the BAR-PH domain, and Pair 4 consists of 2 amino acids at positions 336 and 341 of the amino acid sequence of the BAR-PH domain.

[0006] The second object of the present invention is to provide an application of the functional site as described above in inhibiting the migration of tumor cells.

[0007] Preferably, any one or more sites of Patch 2, Pair 2, Pair 3, and Pair 4 are edited, and the editing includes mutation, deletion, or insertion.

[0008] Preferably, the editing is point mutation.

[0009] Preferably, the point mutation mutates the amino acids in any one or more sites of Patch 2, Pair 2, Pair 3, and Pair 4 into glutamic acid, aspartic acid, glycine, or alanine respectively.

[0010] Preferably, mutating the amino acids in any one or more sites of Patch 2, Pair 2, Pair 3, and Pair 4 into glutamic acid includes the following steps:

[0011] S1. Obtain the complete DNA containing the mutation site by two-step PCR. Among them, the primer pair sequences used for the Patch 2 site are shown in SEQ ID NO.2 - SEQ ID NO.5 respectively, the primer pair sequences used for the Pair 2 site are shown in SEQ ID NO.6 - SEQ ID NO.9 respectively, the primer pair sequences used for the Pair 3 site are shown in SEQ ID NO.10 - SEQ ID NO.13 respectively, and the primer pair sequences used for the Pair 4 site are shown in SEQ ID NO.14 - SEQ ID NO.17 respectively;

[0012] S2. Use restriction endonucleases NotⅠ and XholⅠ to digest the target vector and the PCR product, ligate them by homologous recombination method, transform competent cells, and verify by sequencing.

[0013] Preferably, the PCR reaction program is as follows: 50 ng of template DNA, 15 μL of high-fidelity PCR mixture, 1.2 μL of each upstream and downstream primer, and make up to 30 μL with ddH2O; pre-denature at 95 °C for 3 min; denature at 95 °C for 15 s, anneal at 58 °C for 30 s, extend at 72 °C, cycle 35 times; final extension at 72 °C for 5 min, cool down. After the amplification is completed, recover the target fragment.

[0014] The beneficial effects of the present invention are as follows:

[0015] This application comprehensively uses various research methods such as biochemistry, structural biology, and cell biology to analyze the crystal structure of ACAP4 BAR and reveals the mechanism of ACAP4 binding to membrane lipids and promoting cell migration.

[0016] By designing and constructing the pEGFP-C3 eukaryotic expression vector of truncated proteins of different domain fragments of ACAP4. And through transient transfection overexpression and fluorescence microscopy imaging technology, observe the localization of ACAP4-related fragment proteins in HeLa cells, and find that the BAR-PH tandem domain is the key domain for ACAP4 to affect cell migration.

[0017] Crystals of ACAP4 were obtained through crystal screening BAR and its structure was analyzed. It was found that there are 7 positively charged enrichment regions on the surface of ACAP4 BAR-PH . Corresponding mutants of ACAP4 with 7 positively charged enrichment regions were constructed respectively. By overexpressing the corresponding proteins in HeLa cells to simulate the abnormal high expression of the protein in cancer cells, and using the cell scratch wound healing experiment to characterize the effect of the protein on cell migration ability, four key sites affecting cell migration were found, namely Patch2, Pair2, Pair 3, Pair 4. BAR-PH The four sites provided by this application have a certain degree of regulatory effect on the cell membrane localization of ACAP4 protein and can regulate cell migration ability. This application provides a molecular basis and theoretical basis for the treatment of pathological conditions such as the occurrence of diseases caused by the dysfunction of related proteins, cancer invasion and metastasis, provides new ideas for drug design and the development in the field of bioengineering, and provides new strategies for the research of related diseases.

[0018] Description of the Drawings

[0019] Figure 1 ​The localization of different truncated fragments of ACAP4 in HeLa cells. In the left three columns of the figure are cell localizations, and in the right figure is the longitudinal fluorescence intensity change along the white line of the cells selected from the white dotted box in the left figure (both are transient transfection overexpressions; cells: HeLa, scale bar: 10 μm).

[0020] Figure 2 shows ACAP4 BAR-PH The positively charged region on the surface, where Panel A is the charge analysis of the ACAP4 BAR structure. The rectangular enlarged region is the positively charged region on the surface of one monomeric BAR domain, and Patch1 (R54, K56, K57, R60), Patch2 (K145, K149, K152), and Patch 3 (K160, K163) are circled with black dotted lines respectively; on the left of Panel B is the resolved ACAP4 BAR structure (light blue, 9JIH) compared with the predicted ACAP4 structure (light green) (RMSD = 0.836); on the right is the charge of ACAP4 PH The charge situation in the rectangle is an enlarged view of the charge of the PH domain (the secondary structure is marked in black, and the disordered structure is marked in green).

[0021] Figure 3 For the cell localization of ACAP4 BAR-PH mutant proteins, in the left three columns of the figure are cell localizations, and in the right figure is the longitudinal fluorescence intensity of the cells in the white dotted box in the left figure along the white line. (Scale bar: 10 μm).

[0022] Figure 4 For the cell scratch assay, in the figure, Panel A is the cell scratch assay of cells overexpressing GFP-C3, Panel B is the cell scratch assay of cells overexpressing GFP-ACAP4 FL ; Panel C is the cell scratch assay of cells overexpressing GFP-ACAP4 BAR-PH ; Panels D - J are the cell scratch assays of cells overexpressing the corresponding mutants of GFP-ACAP4 BAR-PH (Scale bar: 200 μm, cell line: HeLa).

[0023] Figure 5 For Figure 4 the corresponding statistical chart of the scratch assay in (n = 4, ***P < 0.001, *P < 0.1, ns > 0.1, mean ± SEM, the red line represents the mean). Detailed implementation manners

[0024] For the convenience of understanding, the technical solutions of the present invention will be described in more detail below in combination with experiments.

[0025] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art.

[0026] 1. Domain positioning and structural analysis

[0027] (1) Domain positioning

[0028] By designing and constructing the pEGFP-C3 eukaryotic expression vectors for truncated proteins of different ACAP4 domains, we observed the localization of ACAP4-related fragment proteins in HeLa cells by transient overexpression and fluorescence microscopy. Figure 1 .

[0029] The results showed that the full-length GFP-ACAP4 was diffusely distributed in the cytoplasm ( Figure 1 Middle A). By gradually truncating some of its domains from the C-terminus, it was found that deleting the disordered structure and ANK domain had no significant effect on its cellular localization ( Figure 1 (B-C) When the ArfGAP domain is further deleted, GFP-ACAP4 BAR-PH showed obvious cell membrane localization ( Figure 1 D), and after deleting the PH domain, it was found that GFP-ACAP4 alone BAR Diffused in the cytoplasm and no longer able to maintain cell membrane localization ( Figure 1 Middle E). Overexpression of GFP-ACAP4 alone ANK-GAP-LOOP When the protein is in a diffuse state, it does not show any connection with the cell membrane ( Figure 1 Middle F).

[0030] (2) Analysis of BAR domain structure

[0031] 1) Crystal screening sample preparation

[0032] The TRX-His-ACAP4-BAR protein was expressed using a prokaryotic protein expression system and purified using Ni-NTA and Superdex 200 columns. The purified protein was concentrated to approximately 10 mL and digested overnight at 4°C with 100 μL of HRV3C protease to remove the tag protein. The target protein and the tag protein were separated using a Superdex 75 column. After purification, the protein quality was verified by SDS-PAGE gel electrophoresis. The protein was concentrated to 5–20 mg / mL and used for crystal screening.

[0033] 2) Crystal screening

[0034] Protein crystals grow based on the principle of the vapor diffusion method. That is, a high-concentration protein sample is dissolved in a solvent, and at this time, the solvent concentration in the sample is lower than the original solvent concentration. When the sample is placed in a closed environment containing the original solvent, due to vapor-phase equilibrium, the solvent concentration in the sample will slowly increase, thereby gradually reducing the solubility of the protein. When the protein approaches the state of spontaneous precipitation, crystals will form.

[0035] Crystal screening is carried out in the sitting-drop manner. The reservoir solution conditions for screening are adopted from the Hampton crystallization kits: CrystalScreen I / II, PEGs&Ion, PEG Rx, Salt Rx, Index, Jena PEP, Jena JCSG. The reservoir solution is aliquoted into the bottom sample wells of a 96-well plate, 80 μL per well; 1 μL of the reservoir solution and 1 μL of the protein sample are added to the spotting wells respectively. Generally, two concentrations, high and low, are set for the protein sample.

[0036] After the crystal plate is sealed, it is placed in a constant-temperature crystal cabinet (16 °C), and the crystal growth is observed regularly. The crystal growth conditions are observed and optimized to obtain the required crystals for experiments.

[0037] 3) Structure determination

[0038] The crystal structure is determined by X-ray diffraction, that is, the distribution of atoms in the unit cell is obtained. The diffraction spots reflect the result of the Fourier transform of the electron density, and the electron density function of the unit cell can be obtained through the Fourier function:

[0039]

[0040] In the formula, x, y, z refer to the spatial coordinates of the actual nodes of the protein, h, k, l refer to the diffraction spot coordinates, v refers to the volume of the unit cell, F refers to the structure factor, and Φ refers to the phase.

[0041] The diffraction pattern is processed by denzo and scalepack to directly obtain the unit cell parameters of the protein crystal and the structure amplitudes of the diffraction spots. Since the phase angle problem of the structure factor, that is, the phase information cannot be directly obtained, the phase is solved first. The structure of the BAR domain of ACAP4 is determined by the molecular replacement method. By comparison, the structure of ACAP4 predicted by Alphafold BAR-PH is selected as the model. After obtaining the initial structure, the structure is alternately refined by Coot and PHENIX, and finally the R work / R free , stereochemical deviation, bond length, bond angle and other parameters all reach the standard level.

[0042] 2. Screening of sites

[0043] (1) Site selection

[0044] According to the resolved structure, it is shown on ACAP4 BAR On each monomer on the surface of the dimer, there are three regions rich in positively charged amino acids, which are respectively named Patch 1 (R54, K56, K57, R60), Patch 2 (K145, K149, K152), and Patch 3 (K160, K163) ( Figure 2 in A). The structure of ACAP4 was predicted by Alpha-fold2, and the properties of the PH domain were also analyzed accordingly, showing some positively charged amino acids that are close to each other pairwise and protrude from the surface. They were respectively named Pair 1 (K283, K284), Pair 2 (K311, K323), Pair 3 (R317, R318), and Pair 4 (H336, R341) ( Figure 2 in B).

[0045] The amino acid sequence of the BAR-PH domain is shown in SEQ ID NO.1.

[0046] Using two-step PCR, the positively charged amino acids in the above 7 regions rich in positively charged amino acids were mutated to negatively charged amino acids - glutamic acid, and the corresponding mutants of ACAP4 were constructed BAR-PH , which were respectively called BAR-PH Patch 1mut , BAR-PH Patch 2mut , BAR-PH Patch 3mut , BAR-PH Pair 1mut , BAR-PH Pair 2mut , BAR-PH Pair 3mut , and BAR-PH Pair 4mut .

[0047] The method is as follows:

[0048] 1) RCR amplification

[0049] The working concentration of the primer is 10 μM. The PCR reaction system is shown in Table 1 below:

[0050] Table 1 PCR reaction system

[0051]

[0052] During the experiment, the wild-type ACAP4 gene was used as a template. First, two reaction systems were configured according to the primer pairs of upstream primer 1 and downstream primer 1, and upstream primer 2 and downstream primer 2. After the reaction systems were configured, the PCR tubes were placed in a PCR instrument, and the program was set according to Table 2 below.

[0053] Table 2 PCR reaction program

[0054]

[0055]

[0056] In this example, the primer information used is shown in Table 3 below:

[0057] Table 3 Primer information

[0058]

[0059] After the amplification was completed, the target gene was determined according to the results of agarose gel electrophoresis, and the target band was cut out for recovery and purification.

[0060] Then, using the two recovered genes as vectors, a reaction system was configured with the primer pair of upstream primer 1 and downstream primer 2, and the PCR amplification reaction was carried out again.

[0061] 2) Enzyme digestion of the vector

[0062] The vector was linearized by double enzyme digestion, and the reaction system is as follows in Table 4:

[0063] Table 4 Vector double enzyme digestion reaction system

[0064]

[0065] The restriction endonucleases NotⅠ and XholⅠ were selected. After the reaction system was configured, it was gently pipetted evenly and centrifuged to prevent residues on the tube wall. The EP tube was placed in a 37°C metal bath and reacted for 2 - 4 h. Then, according to the results of agarose gel electrophoresis, the vector band was determined, and the target band was cut out for recovery. The gel recovery process was the same as the steps for recovering the PCR gene product described above.

[0066] Finally, the mutant gene was ligated to the vector by homologous recombination:

[0067] Table 5 Homologous recombination system

[0068]

[0069] After the reaction system was configured, it was gently pipetted evenly and centrifuged to prevent residues on the tube wall. The EP tube was placed in a 37°C metal bath and incubated for 30 min. Then it was transferred into competent cells. After the reconstructed plasmid was extracted and sequenced correctly, it was transformed into competent cells for amplification culture, induced protein expression, and purification.

[0070] These mutants were introduced into cells by transient transfection, and the subcellular localization of these mutant proteins was observed. The method was as follows:

[0071] 1) The adherent cell types included HEK293, HeLa, and MDCK, and the cell culture methods were the same:

[0072] ① After the cells stored in the culture flask were sent to the laboratory, they were left standing in a 37 °C, 5% CO2 incubator for more than 4 h to stabilize the cell state; ② Observe the cell growth condition, and change the culture medium or perform subculture according to the cell contact and confluence; ③ Subculture: Aspirate the original culture medium, add 0.25% trypsin, and perform a digestion reaction in a 37 °C incubator. Terminate the digestion reaction when the cell-cell contact disappears; ④ Centrifuge at 1000 rpm for 2 min, discard the waste liquid, pipette the cells with fresh culture medium, and add the cell suspension to a new culture dish suitable for the experimental specifications at a ratio of 1:3 for culture; ⑤ The cells were stored in cell cryopreservation solution and cryopreserved in an -80 °C refrigerator for later use.

[0073] 2) Transfection

[0074] Taking a six-well plate as an example, the transfection operation was as follows: ① When the cells reached 70 - 80% confluence, change the fresh culture medium; ② Prepare the transfection complex: Add 100 μL of MEM-OPTI to two EP tubes. Add 2 μg of the target gene to tube A and 6 μg of PEI to tube B. After mixing well and standing for 5 min respectively, add the mixture in tube A to tube B, mix well and stand for 15 min; ③ Add the transfection complex to the culture dish, and change the fresh culture medium 8 - 12 h later. Prepare the transfection complex according to the proportion for culture dishes of different specifications.

[0075] 3) Slide preparation and observation

[0076] Observation was carried out by making slides 36 - 48 h after cell transfection. The specific steps are as follows: ① After discarding the culture medium in the culture dish, wash it once with PBS; ② Add 4% paraformaldehyde methanol to fix for 10 - 20 min and then wash with PBS; ③ Add PBS-T2 to permeabilize cells for 30 min and then wash with PBS-T; ④ Add PBS-T containing 1% BSA and incubate on a shaker at room temperature for 30 - 60 min for blocking; ⑤ Add the corresponding primary antibody and incubate on a shaker at room temperature for 1 h or overnight at 4 °C (the primary antibody is diluted with the blocking solution); ⑥ After washing with PBS-T, add the corresponding secondary antibody and incubate on a shaker at room temperature in the dark for 1 h (the secondary antibody is diluted with the blocking solution); ⑦ After washing with PBS-T, add DAPI and incubate in the dark for 10 min (DAPI is diluted with PBS); ⑧ After washing with PBS, take out the cover glass, place the side with cells facing down to touch the cover glass (drop anti-quenching agent), and pay attention to preventing the generation of bubbles; ⑨ Fix the cover glass with nail polish, pay attention to avoiding light and preventing the cover glass from rubbing and sliding on the glass slide; ⑩ Microscopic imaging (in this study, cell microscopic imaging was all performed using Zeiss LSM980 laser confocal microscope).

[0077] The results are shown in Figure 3 . The results showed that compared with wild-type GFP-ACAP4 BAR-PH membrane localization ( Figure 3 A in Patch 1mut ), the membrane localization of mutants was disrupted to varying degrees. Among the mutants in the three positively charged enrichment regions in the BAR domain, GFP-BAR-PH Patch 3mut was able to partially maintain membrane localization, while the membrane localization of GFP-BAR-PH Patch 2mut was completely lost, and the protein was diffused throughout the cell ( Figure 3 B - D in Pair 1mut ); among the mutants of the four pairs of positively charged amino acids in the PH domain, only GFP-BAR-PH Figure 3 E in Pair 2mut was still able to partially maintain membrane localization ( Pair 3mut ), while GFP-BAR-PH Figure 3 F, G in Pair4mut was completely diffused in the cytoplasm ( Figure 3 H in

[0078] ), and GFP-BAR-PH Pair4mut even all entered the nucleus ( Figure 3 H in

[0078] ). These phenomena indicate that the positively charged amino acid enrichment region on the surface of BAR-PH plays a decisive role in the electrostatic interaction with the cell membrane and plays an important role in maintaining its intracellular membrane localization.

[0078] (2) Influence of the locus on cell migration ability

[0079] By overexpressing the corresponding protein in HeLa cells to simulate the abnormally high expression of the protein in cancer cells, the cell scratch wound healing experiment was used to characterize the influence of the protein on cell migration ability.

[0080] 1) The migration experiment method is as follows: The migration system consists of a multi-well plate and a cell insert. During the experiment, cells and serum-free culture medium are added to the insert, and complete culture medium is added to the multi-well plate. The cell insert has a multi-pore membrane that allows cells to pass through. The migration ability of cells is compared according to the number of cells passing through the multi-pore membrane.

[0081] In this example, an insert with an 8-μm pore size was used. The specific steps are as follows: ① Add 600 μL of complete medium to the Transwell multi-well plate, place the cell insert, and incubate in an incubator for more than 1 h; ② Culture the control group and the cells with the ACAP4 gene knocked down until the confluence rate reaches 80%; ③ Add trypsin for digestion, centrifuge to collect the cells, and resuspend the cells with serum-free culture medium; ④ Count the cells, add 50,000 cells to each insert, and supplement with serum-free medium to 200 μL; ⑤ After culturing for 18 - 24 h, aspirate the culture medium in the well plate and the insert, wash with PBS, and gently wipe off the non-migrated cells on the inner wall of the insert and the pore membrane with a cotton swab; ⑥ Fix with 4% paraformaldehyde for 10 - 20 min; ⑦ Stain with crystal violet for 20 - 30 min after washing twice with PBS; ⑧ After washing three times with PBS, wipe off the remaining cells and dye on the inner wall of the insert with a cotton swab, air dry, observe microscopically, and randomly select fields for photography; ⑨ Statistically analyze the number of cells migrated outside the membrane.

[0082] 2) The cell scratch wound healing experiment, the specific steps are as follows:

[0083] ① Draw 5 parallel lines through each well on the bottom of the six-well plate, inoculate cells, and when the cell confluence rate reaches 80%, transfect the corresponding target gene and continue to culture for 48 h to fully express the target protein; ② When the cell confluence rate reaches 100%, use a 10-μL pipette tip to vertically pierce the bottom of the well plate, and quickly scratch with the same force and angle to make the scratch perpendicular to the marked line; ③ After washing with PBS to remove the suspended cells, supplement with fresh culture medium; ④ Observe under a microscope and photograph to record the wound area when the cells have not migrated (0 h); ⑤ Put the cells back into the incubator, observe the wound healing at 12 h and 24 h respectively and take pictures. Before each picture taking, wash three times with PBS to fully remove the suspended cells to prevent affecting the results; ⑥ Statistically analyze the wound areas at different time points. In this study, the cell healing rate within 24 h was used to characterize the cell migration ability, that is, (wound area at 0 h - wound area at 24 h) / 24 h.

[0084] The results are shown inFigure 4 , Figure 5 .

[0085] The cell migration ability of cells overexpressing the full-length GFP-ACAP4 protein is improved to a certain extent compared with the control group ( Figure 4 A and B in BAR-PH ). When overexpressing GFP-ACAP4 alone, Figure 4 C in

[0086] The research results on the effect of the positively charged amino acid-rich region on the cell migration ability of ACAP4 show that, first of all, among the three positively charged Patches in the BAR domain, the cell migration rate of cells overexpressing GFP-BAR-PH Patch 1mut has no obvious difference from that of cells overexpressing GFP-ACAP4 BAR-PH , but is significantly enhanced compared with that of cells overexpressing GFP-C3, indicating that the sites in Patch 1 have no obvious regulatory effect on cell migration ( Figure 4 C and D in Figure 5 ); while the cell migration rate of cells overexpressing GFP-BAR-PH Patch 2mut is significantly lower than that of cells overexpressing GFP-ACAP4 BAR-PH , and has no obvious difference from that of cells overexpressing GFP-ACAP4 FL , indicating that the sites in Patch2 are very important for cell migration ( Figure 4 C and E in Figure 5 ); the experimental results of cells overexpressing GFP-BAR-PH Patch 3mut are similar to those of the experimental group overexpressing GFP-BAR-PH Patch 1mut , indicating that the sites in Patch3 also have no obvious regulatory effect on cell migration ( Figure 4 C and F in Figure 5 ).

[0087] The positively charged Pair 1 is located in the connecting region between the BAR domain and the PH domain. The scratch experiment of overexpressing the corresponding mutant shows that the cell migration rate is not only lower than that of cells overexpressing GFP-ACAP4 BAR-PH , but also significantly lower than that of cells overexpressing GFP-ACAP4 FL ( Figure 4 C and G in Figure 5 ), indicating that GFP-BAR-PH Pair 1mutCompletely lost the ability to promote cell migration, that is, loop1 connecting BAR-PH plays a key regulatory role in cell migration. The corresponding mutants of the other three positively charged Pairs located in the PH domain cannot achieve the promotion effect on cell migration as overexpressing wild-type GFP-ACAP4 BAR-PH at the same level, but are close to the cell migration rate of overexpressing GFP-ACAP4 FL (C, H, I, J in Figure 4 ). Figure 5 )

[0088] These experimental results indicate that the ability of ACAP4 to promote cell migration is mainly achieved through its BAR-PH tandem domain. Among them, the four positively charged enrichment regions (Patch 2, Pair 2, Pair 3, and Pair 4) that play important roles in cell membrane localization also have regulatory effects on the property of ACAP4 BAR-PH to promote cell migration, and these amino acid pairs in these regions also have significant regulatory effects on the in vitro phase separation ability.

[0089] In summary, through crystallographic analysis, the present application discovered 7 positively charged enrichment regions on the surface of ACAP4 BAR-PH , all of which have a certain degree of regulatory effect on the cell membrane localization of ACAP4 protein. Among them, the four positively charged enrichment regions Patch 2, Pair2, Pair 3, and Pair 4 can regulate the cell migration ability. The present application provides a molecular basis and theoretical basis for understanding related physiological processes and the occurrence of diseases, provides new ideas for drug design and the development in the field of bioengineering, and provides new strategies for the research of related diseases.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the present invention; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A functional site for targeting the ACAP4 protein to inhibit the migration of tumor cells, said site being any one or a combination of more than one of Patch2, Pair 2, Pair 3, and Pair 4, wherein Patch 2, Pair 2, Pair 3, and Pair 4 are located in the BAR-PH domain of the ACAP4 protein, and the sequence of the BAR-PH domain is as shown in SEQ ID NO.1, wherein, Patch 2 is composed of 3 amino acids at positions 145, 149, and 152 of the amino acid sequence of the BAR-PH domain. Pair 2 is composed of 2 amino acids at positions 311 and 323 of the amino acid sequence of the BAR-PH domain. Pair 3 is composed of 2 amino acids at positions 317 and 318 of the amino acid sequence of the BAR-PH domain. Pair 4 is composed of 2 amino acids at positions 336 and 341 of the amino acid sequence of the BAR-PH domain.

2. Use of the functional site as described in claim 1 in inhibiting the migration of tumor cells.

3. The application according to claim 2, characterized in that Edit any one or more of the sites in Patch 2, Pair 2, Pair 3, and Pair 4, and the editing includes mutation, deletion, or insertion.

4. The application according to claim 3, characterized in that, The editing is point mutation.

5. The application according to claim 4, characterized in that, The point mutation mutates the amino acids in any one or more of the sites in Patch 2, Pair 2, Pair 3, and Pair 4 into glutamic acid, aspartic acid, glycine, or alanine respectively.

6. The application according to claim 5, wherein Mutating the amino acids in any one or more of the sites in Patch 2, Pair 2, Pair 3, and Pair 4 into glutamic acid includes the following steps: S1. Obtain the complete DNA containing the mutation site by two-step PCR. Among them, the primer pair sequences used for the Patch 2 site are shown in SEQ ID NO.2 - SEQ ID NO.5 respectively, the primer pair sequences used for the Pair 2 site are shown in SEQ ID NO.6 - SEQ ID NO.9 respectively, the primer pair sequences used for the Pair 3 site are shown in SEQ ID NO.10 - SEQ ID NO.13 respectively, and the primer pair sequences used for the Pair 4 site are shown in SEQ ID NO.14 - SEQ ID NO.17 respectively; S2. Digest the target vector and the PCR product with restriction enzymes NotⅠ and XholⅠ, connect them by homologous recombination method, transform competent cells, and verify by sequencing.

7. The application according to claim 6, wherein The PCR reaction program is as follows: 50 ng of template DNA, 15 μL of high-fidelity PCR mixture, 1.2 μL of each upstream and downstream primer, and make up to 30 μL with ddH2O; pre-denature at 95 °C for 3 min; denature at 95 °C for 15 s, anneal at 58 °C for 30 s, extend at 72 °C, cycle 35 times; final extension at 72 °C for 5 min, cool down. After the amplification is completed, recover the target fragment.

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