P53 variants with stronger liquid-liquid phase separation ability and activity and uses thereof

Abstract

This invention discloses a p53 variant with enhanced liquid-liquid phase separation capability and activity, and its uses. Compared with wild-type p53, this novel p53 variant has the following advantages: 1) enhanced liquid-liquid phase separation capability; 2) improved targeting of p53 genes in tumor cells. CDKN1A It possesses stronger transcriptional activation capabilities; 3) It exhibits more significant killing effects on various tumor cells. Furthermore, this novel p53 variant can achieve tumor-killing function by regulating FGFR3. This invention demonstrates that improving liquid-liquid phase separation capability can enhance p53 transcriptional activation activity and induce tumor cell apoptosis, which is of great significance for improving p53-based gene / protein therapy. Moreover, the novel p53 variant gene and protein disclosed in this invention are tumor-killing drugs with promising clinical applications. At the same time, the combined use of LLPSE-p53 recombinant protein with FGFR inhibitor TAS-120 and Wnt signaling pathway inhibitor IWR-1 can enhance its tumor-killing effect, which is of great value for cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a p53 variant with enhanced liquid-liquid phase separation capability and its application in killing tumor cells. Background Technology

[0002] Transcription factor p53 is one of the most important tumor suppressor factors in cells. It is mutated and inactive in more than 50% of cancer cells, and in the remaining cancer cells, p53 activity or downstream signaling pathways are suppressed. As a "genome guard," p53 responds to intracellular and extracellular stress conditions, thereby transcribing and activating its downstream target genes. CDKN1A , PUMA, BAX Inducing DNA damage repair, cell cycle arrest, or apoptosis can inhibit tumor cell proliferation. Therefore, restoring / enhancing p53 activity in cancer cells is currently a strategy to suppress cancer cell proliferation.

[0003] Methods to restore / enhance p53 activity in cancer cells include: 1) inhibiting wild-type p53 degradation; 2) inhibiting mutant p53 protein activity; and 3) gene therapy involving the introduction of p53 cDNA. Gene therapy is the only one of these that has received clinical approval. A gene therapy (Gendicine) using a recombinant defective adenovirus to deliver wild-type p53 for the treatment of head and neck cancer has been clinically approved in China. Other gene therapies delivering wild-type p53, such as ONYX-015, are still in clinical trials. A p53 gene therapy combined with immune checkpoint inhibitors (PD-1 / PD-L1 antibodies) for the treatment of solid tumors has entered phase II clinical trials. Although direct delivery of wild-type p53 has been shown to kill cancer cells, the instability of wild-type p53 and the presence of gain-of-function mutant p53 in cancer cells prevents wild-type p53 from functioning effectively, thus limiting the therapeutic effect. Therefore, there is an urgent need to develop p53 variants with stronger tumor-suppressive effects to improve the effectiveness of p53 gene therapy. Summary of the Invention

[0004] To address the aforementioned problems, the purpose of this invention is to provide a p53 variant with enhanced liquid-liquid phase separation capability and activity, and its uses.

[0005] A p53 variant with enhanced liquid-liquid phase separation capability has an amino acid sequence rich in positive charge and histidine added to one end of the p53 sequence. This variant exhibits stronger liquid-liquid phase separation capability and stronger transcriptional activation activity compared to wild-type p53 at both the cellular and in vitro levels. The general formula for the amino acid sequence rich in positive charge and histidine is Hx(G / S / P / A / T)y(R / K)z, where H represents histidine; (G / S / P / A / T) represents a sequence composed of one or more of glycine, threonine, proline, serine, and threonine; and (R / K) represents arginine or lysine. x, y, and z represent the number of the corresponding amino acids, x, z = 0-20, y = 0-100, and x+z > 5.

[0006] The amino acid sequence rich in positive charge and histidine is further separated from one end of the wild-type p53 sequence by a linker sequence.

[0007] The p53 has transcriptional activation activity, and its DNA sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] The p53 mentioned has transcriptional activation activity, including in human and non-human species.

[0009] The p53 variant DNA sequences are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0010] The p53 variant described above can activate CDKN1A Target gene transcription, improve CDKN1A , MDM2 , PUMA , NOXA as well as RRM2B The transcriptional level of one or more downstream target genes.

[0011] The p53 variant is used in the preparation of tumor therapeutic drugs, and the applicable tumor types include non-small cell lung cancer, breast cancer, neuroblastoma, osteosarcoma, and human brain tumors; the drugs include those that exert their active effects at both the nucleic acid and protein levels.

[0012] The recombinant protein expression systems for the p53 variant include E. coli expression hosts, animal cells, and yeast protein expression systems; prokaryotic expression vectors include pET24A and pET28A(+); eukaryotic expression vectors include: 1) animal cell expression vectors such as pEGFP, pEYFP, pmcherry, pRFP, pECFP, pLenti, pLX, pCMV6, pCMV3, pcDNA3, and pcDNA6B; 2) insect cell expression vectors such as pAc5.1-EGFP; and 3) yeast expression vectors such as pPIC3 and pPIC9.

[0013] The p53 variants are available in both full-length and functionally preserved mutant forms.

[0014] The target molecules of the p53 variant that mediate tumor cell apoptosis include FGFR3 Used for treatment FGFR3 Tumors with high expression levels.

[0015] The tumor treatment drug is a drug that combines a p53 variant with the FGFR inhibitor TAS-120 and the Wnt signaling pathway inhibitor IWR-1, or a compound drug prepared to enhance its ability to kill tumor cells at lower protein concentrations.

[0016] The forms of the medicine include injections, tablets, capsules, oral liquid dosage forms, granules, and ointments.

[0017] The technical effects of this invention are as follows: (1) The innovative design of LLPSE-p53 (p53 variant) in this application has a stronger liquid-liquid phase separation effect in both intracellular and extracellular environments.

[0018] (2) This application clarifies that p53 variants with stronger liquid-liquid phase separation capabilities promote the p53 target gene. CDKN1A Transcriptional activation, etc.

[0019] (3) This application demonstrates that LLPSE-p53 has a significant killing effect on a variety of tumor cells, indicating that it has great potential for clinical application in tumor treatment. It innovatively reveals the mechanism by which p53 inhibits tumor cell proliferation from the perspective of phase separation.

[0020] (4) This application demonstrates that the combined use of low concentration LLPSE-p53 protein with drugs can enhance its tumor killing effect, providing a new idea and basis for its application in clinical cancer treatment. Attached Figure Description

[0021] Figure 1These are microscopic images comparing the ability of WT-p53 and recombinant proteins with EGFP tags added to the C-terminus (EGFP-p53) to separate in vitro.

[0022] Figure 2 The plasmids containing WT-p53 and p53 with an EYFP tag added to the C-terminus (YFP-p53) were transfected into the human non-small cell lung cancer cell line H1299. CDKN1A Transcription detection results.

[0023] Figure 3 These are confocal images of the morphology of WT-p53 and LLPSE-p53 in the human kidney epithelial cell line HEK293T.

[0024] Figure 4 These are confocal images of the morphology of WT-p53 and LLPSE-p53 in the human non-small cell lung cancer cell line H1299.

[0025] Figure 5 These are morphological confocal images of WT-p53 (NMR) and LLPSE-p53 (NMR) in the human non-small cell lung cancer cell line H1299.

[0026] Figure 6 This is a graph showing the qRT-PCR results of the regulation of downstream target gene mRNA levels of p53 by WT-p53 and LLPSE-p53 in the human kidney epithelial cell line HEK293T.

[0027] Figure 7 This is a graph showing the qRT-PCR results of the regulation of downstream target gene mRNA levels of p53 by WT-p53 and LLPSE-p53 in the human non-small cell lung cancer cell line H1299.

[0028] Figure 8 After transfecting the human non-small cell lung cancer cell line H1299 with WT-p53 and LLPSE-p53 plasmids, the effect on... CDKN1A Results of transcriptional activation detection.

[0029] Figure 9 This is a graph showing the cell viability results after transfecting the human non-small cell lung cancer cell line H1299 with WT-p53 and LLPSE-p53 plasmids, as determined by the ATP assay.

[0030] Figure 10 This is a graph showing the cell viability results after transfecting the human neuroblastoma cell line SH-SY5Y with WT-p53 and LLPSE-p53 plasmids, as determined by the ATP assay.

[0031] Figure 11This is a graph showing the cell viability results after transfection of the human osteosarcoma cell line U-2OS with WT-p53 and LLPSE-p53 plasmids, as detected by the ATP assay.

[0032] Figure 12 These are in vitro phase separation microscope images of recombinant WT-p53 and LLPSE-p53 proteins.

[0033] Figure 13 This is a graph showing the cell viability results of the human non-small cell lung cancer cell line H1299 after 72 h of transduction with recombinant WT-p53 and LLPSE-p53 proteins using the ATP assay.

[0034] Figure 14 This is a graph showing the cell viability results after transducing the human neuroblastoma cell line SH-SY5Y with recombinant WT-p53 and LLPSE-p53 proteins for 72 hours, as determined by the ATP assay.

[0035] Figure 15 This is a graph showing the cell viability results of the human breast cancer cell line SKBR3 after 72 h of transduction with recombinant WT-p53 and LLPSE-p53 proteins, as determined by the ATP assay.

[0036] Figure 16 This is a graph showing the cell viability results as determined by the CCK8 assay after transducing human osteosarcoma cell line U-2OS with recombinant WT-p53 and LLPSE-p53 proteins for 72 h.

[0037] Figure 17 This is a graph showing the cell viability results of the human brain tumor cell line SF126 after 72 h of transduction with recombinant WT-p53 and LLPSE-p53 proteins, as detected by the CCK8 assay.

[0038] Figure 18 The control group was knocked down separately. Vector )and FGFR3 Subsequently, WT-p53 and LLPSE-p53 were overexpressed respectively, and the cell viability was detected by ATP assay after 72 hours.

[0039] Figure 19 The image shows the cell viability results of the human osteosarcoma cell line U-2OS after WT-p53 and LLPSE-p53 proteins were delivered, DMSO (control), TAS-120 and IWR-1 were added and the cells were cultured for 24 hours. The results were then analyzed by the CCK8 assay. Detailed Implementation

[0040] The design, method, and technical effects of the present invention will be illustrated below with reference to specific embodiments. The present invention includes, but is not limited to, the representative embodiments disclosed below, and can be described in various other ways. This specification is intended to help those skilled in the art to comprehensively understand the implementation details of the present invention.

[0041] Regarding the DNA sequence, SEQ ID NO.1 is a human-derived, functionally normal mutant p53, SEQ ID NO.2 is a human-derived wild-type p53; the p53 in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 are human-derived p53, the p53 in SEQ ID NO.6 is p53 from silver gibbon, the p53 in SEQ ID NO.7 is p53 from rat, and the p53 in SEQ ID NO.8 is p53 from naked mole-rat.

[0042] In this invention, the amino acid sequence (including the linker) added to SEQ ID NO.3 is: GTGGGGSGGGGSGGGGSHHHHHHGGRRRRRRRRR The amino acid sequence (including the linker) added to SEQ ID NO.4 is: GGGGSHHHHHGGTGGRRRRRRR The amino acid sequence (including the linker) added to SEQ ID NO.5 is: GTGGGGSGGGGSGGGGSHHHHHHHGSGGRRRRRRRRRR The amino acid sequence (including the linker) added to SEQ ID NO.6 is: GGGGGHHHHHHHGGSPGGTGGKKKKKK The amino acid sequence (including the linker) added to SEQ ID NO7 is: GGASGHHHHHHHHGGAGGGGRRRRRRKK The amino acid sequence (including the linker) added to SEQ ID NO.8 is: GGGSGGGSGGGSHHHHGGGDPRRRRRRRRRRRR.

[0043] Example 1: Comparison of in vitro phase separation capabilities of WT-p53 and EGFP-p53 recombinant proteins Since tagged proteins can affect protein structure, to test whether simply adding a tag can promote liquid-liquid phase separation, we added EGFP as a tag to the C-terminus of p53 and then compared the in vitro liquid-liquid phase separation ability of WT-p53 without a fluorescent tag and recombinant EGFP-p53. The experimental results showed that, under the same conditions, compared with WT-p53 without a fluorescent tag, EGFP-p53 had not yet formed a distinct droplet structure when WT-p53 had already undergone liquid-liquid phase separation, indicating that the EGFP tag has a significant inhibitory effect on the liquid-liquid phase separation of p53 protein. Figure 1).

[0044] Implementation steps: 1. The two target genes were cloned into the pET-24a(+) prokaryotic expression vector, transformed into the C41 expression strain, and cultured overnight at 37°C.

[0045] 2. Select single clones and incubate at 37 ℃ and 220 rpm until OD600 = 0.6; 3. Add 200-500 μM IPTG and 100 µM ZnCl2, and incubate overnight at 25 ℃ and 220 rpm; 4. Centrifuge at 4500 rpm and 4 ℃ for 30 min; 5. Add cell lysis buffer, autoclave, and centrifuge at 17,000 rpm, 4 °C for 30 min; 6. Take the supernatant and purify it using HisTrap HP media; 7. Purify the eluent using HiTrap Heparin HP media; 8. Finally, perform gel filtration for further purification; 9. Prepare a phase separation system, take pictures under a fluorescence inverted microscope, and compare the phase separation capabilities of the two systems.

[0046] The results showed that, under the same conditions, WT-p53 had formed a distinct droplet structure, while EGFP-p53 had not formed a distinct droplet structure, indicating that the tag protein EGFP not only did not promote but actually inhibited the liquid-liquid phase separation of p53 protein.

[0047] Example 2: Comparison of transcriptional activation activities of YFP-p53 and WT-p53 To investigate whether adding any tag to the C-terminus of p53 alters its transcriptional activation activity, we added a YFP tag to the C-terminus of wild-type p53 to construct the pCMV3-YFP-p53 eukaryotic expression vector. We then transformed both pCMV3-YFP-p53 and pCMV3-p53 into cells and examined their effects on transcriptional activation. CDKN1A Effects on transcriptional activation capacity. Experimental results showed that, compared with WT-p53, YFP-p53 had a greater effect on... CDKN1A The transcriptional activation capacity tends to decrease. Figure 2 ). Implementation steps: 1. Seed an appropriate amount of cells into a 96-well cell culture plate. When the cell density reaches 60-70%, add the above-mentioned plasmid with... CDKN1A The luciferin reporter gene plasmid was co-transformed and incubated for another 24 hours. 2. Perform chemiluminescence detection using the Promega Dual-Lucent Reporter System kit and M5 microplate reader; 3. Use Graphpad5 software for data processing and analysis.

[0048] The results showed that, compared with WT-p53, YFP-p53 was more effective in treating certain conditions. CDKN1A The weak transcriptional activation activity indicates that adding the YFP tag will not only not promote, but will actually inhibit the transcriptional activation activity of wild-type p53.

[0049] Example 3: Comparison of liquid-liquid phase separation ability of WT-p53 and LLPSE-p53 (SEQ ID NO.3) in human kidney epithelial cell line HEK293T at cellular levels. To verify that the phase separation ability of the p53 variant LLPSE-p53 designed in this invention is superior to that of WT-p53, we constructed pEGFP-p53 and pEGFP-LLPSE-p53 eukaryotic expression vectors. We then detected the number and size of droplets formed in the cells by transfection into eukaryotic human embryonic kidney cells HEK293T and immunofluorescence technology.

[0050] Implementation steps: 1. One day before transfection, seed 6-8×10^5 HEK293T cells in a 35 mm confocal dish and add 2 ml of complete culture medium; 2. Once the cell density reaches 60-70%, transfect each well with 2-2.5 µg of plasmid and incubate at 37 ℃ in a 5% CO2 incubator for 24 h. 3. Discard 1 ml of complete culture medium, add 1 ml of 4% paraformaldehyde, and fix at room temperature for 10 min; 4. Discard all supernatant, add 1 ml PBS and wash 3 times, 5 min each time; 5. Add 0.2% Triton X-100 and allow to pass through at room temperature for 15 minutes; 6. Discard all supernatant, add 1 ml PBS and wash 3 times, 5 min each time; 7. Add 1 ml of 5% BSA and block at room temperature for 1 h; 8. Add diluted p53(DO-1) primary antibody (dilution ratio 1:1000), place on a horizontal shaker, and incubate overnight at 4 ℃; 9. Recover the primary antibody, add 1 ml of PBS and wash 3 times, 5 min each time; 10. Add the diluted immunofluorescence secondary antibody (dilution ratio 1:1000) and incubate at room temperature in the dark for 1 h; 11. Discard the secondary antibody, add 1 ml of PBS and wash 3 times, 5 min each time; 12. Use a confocal microscope for photographic inspection, and ensure consistent imaging parameters for different samples.

[0051] Experimental results showed that, compared with WT-p53, LLPSE-p53 formed larger and more numerous droplet structures in HEK293T cells. Figure 3 ).

[0052] Example 4: Comparison of liquid-liquid phase separation of WT-p53 and LLPSE-p53 (SEQ ID NO.4) in the non-small cell lung cancer cell line H1299 To further verify that the phase separation ability of the p53 variant LLPSE-p53 designed in this invention is superior to that of WT-p53, we constructed pCMV6-p53 and pCMV6-LLPSE-p53 eukaryotic expression vectors. After transfecting the above plasmids into the human non-small cell lung cancer cell line H1299, we used immunofluorescence technology to detect the number and size of droplets formed by the two in the cells.

[0053] Implementation steps: 1. One day before transfection, seed 6-8 × 10^5 H1299 cells in a 35 mm confocal dish and add 2 ml of complete culture medium; 2. Once the cell density reaches 60-70%, transfect each dish with 2-2.5 μg of plasmid and incubate at 37 ℃ in a 5% CO2 incubator for 24 h. 3. Discard 1 ml of complete culture medium, add 1 ml of 4% paraformaldehyde, and fix at room temperature for 10 min; 4. Discard all supernatant, add 1 ml PBS and wash 3 times, 5 min each time; 5. Add 0.2% Triton X-100 and allow to pass through at room temperature for 15 minutes; 6. Discard all supernatant, add 1 ml PBS and wash 3 times, 5 min each time; 7. Add 1 ml of 5% BSA and block at room temperature for 1 h; 8. Add the diluted p53 primary antibody (dilution ratio 1:1000), place on a horizontal shaker, and incubate overnight at 4 ℃; 9. Recover the primary antibody, add 1 ml of PBS and wash 3 times, 5 min each time; 10. Add the diluted immunofluorescence secondary antibody (dilution ratio 1:1000) and incubate at room temperature in the dark for 1 h; 11. Discard the secondary antibody, add 1 ml of PBS and wash 3 times, 5 min each time; 12. Use a confocal microscope for photographic inspection, and ensure consistent imaging parameters for different samples.

[0054] Experimental results showed that, compared with WT-p53, LLPSE-p53 formed larger and more numerous droplets in H1299 cells. Figure 4 ).

[0055] Example 5: Comparison of liquid-liquid phase separation capabilities of WT-p53 (naked mole rat) and LLPSE-p53 (naked mole rat, SEQ ID NO.8) in the non-small cell lung cancer cell line H1299. To verify whether the phase separation-enhancing sequence we constructed for p53 could also be applied to p53 in other species, we added the phase separation-enhancing sequence fragment to the naked mole-rat p53 gene (NMR-p53) and constructed pCMV6-p53 (NMR) and pCMV6-LLPSE-p53 (NMR) eukaryotic expression vectors, which were then transfected into the human non-small cell lung cancer cell line H1299. The number and size of the droplet structures formed in the cells were compared using confocal microscopy.

[0056] Implementation steps: 1. One day before transfection, seed 6-8 × 10^5 H1299 cells in a 35 mm confocal dish and add 2 ml of complete culture medium; 2. Once the cell density reaches 60-70%, transfect each dish with 2-2.5 μg of plasmid and incubate at 37 ℃ in a 5% CO2 incubator for 24 h. 3. Discard 1 ml of complete culture medium, add 1 ml of 4% paraformaldehyde, and fix at room temperature for 10 min; 4. Discard all supernatant, add 1 ml PBS and wash 3 times, 5 min each time; 5. Add 0.2% Triton X-100 and allow to pass through at room temperature for 15 minutes; 6. Discard all supernatant, add 1 ml PBS and wash 3 times, 5 min each time; 7. Add 1 ml of 5% BSA and block at room temperature for 1 h; 8. Add the diluted p53 primary antibody (dilution ratio 1:1000), place on a horizontal shaker, and incubate overnight at 4 ℃; 9. Recover the primary antibody, add 1 ml of PBS and wash 3 times, 5 min each time; 10. Add the diluted immunofluorescence secondary antibody (dilution ratio 1:1000) and incubate at room temperature in the dark for 1 h; 11. Discard the secondary antibody, add 1 ml of PBS and wash 3 times, 5 min each time; 12. Use a confocal microscope for photographic inspection, and ensure consistent imaging parameters for different samples.

[0057] Experimental results showed that, compared with WT-p53 (NMR), LLPSE-p53 (NMR) formed larger and more numerous droplets in H1299 cells. Figure 5 ).

[0058] Example 6: The p53 variant of the present invention (SEQ ID NO. 5) has a stronger ability to transcribe and activate its downstream target genes. After demonstrating that LLPSE-p53 has superior liquid-liquid phase separation capabilities compared to WT-p53, we constructed pEGFP-p53 and pEGFP-LLPSE-p53 eukaryotic expression vectors. In HEK293T cells, we used qRT-PCR to detect the transcriptional regulatory abilities of WT-p53 and LLPSE-p53 on their downstream target genes. The results showed that, compared to WT-p53, LLPSE-p53... CDKN1A , MDM2 as well as PUMA The transcriptional level of [something] was significantly increased. Figure 6 This indicates that phase-liquid separation enhances the transcriptional activity of p53.

[0059] Implementation steps: 1. Seed cells in 12-well cell culture plates. After the cell density reaches 70-80%, transfect 1 μg of plasmid and continue culturing for 24 h. 2. Total RNA was extracted from cells using the Trizol method; 3. The extracted RNA was reverse transcribed to synthesize cDNA, and a qRT-PCR system was prepared according to the standardized procedure; 4. Detection was performed using a real-time quantitative PCR instrument; 5. Use Graphpad5 software for data processing and analysis.

[0060] Experimental results show that LLPSE-p53 has stronger transcriptional activation activity compared with WT-p53.

[0061] Example 7: The p53 variant of the present invention (SEQ ID NO. 6) has a stronger ability to transcribe and activate its downstream target genes. In HEK293T cells, phase separation was demonstrated to enhance p53 transcriptional activity using qRT-PCR. We constructed pCMV6-p53 and pCMV6-LLPSE-p53 eukaryotic expression vectors and further validated them in H1299 cells. Experimental results showed that, compared to WT-p53, LLPSE-p53 enhanced p53 transcriptional activity. CDKN1A , MDM2 as well as PUMA The transcriptional level of [something] was significantly increased. Figure 7 ).

[0062] Implementation steps: 1. Seed cells in 12-well cell culture plates. After the cell density reaches 70-80%, transfect 1 μg of plasmid and continue culturing for 24 h. 2. Total RNA was extracted from cells using the Trizol method; 3. The extracted RNA was reverse transcribed to synthesize cDNA, and a qRT-PCR system was prepared according to a standardized procedure; 4. Detection was performed using a real-time quantitative PCR instrument; 5. Use Graphpad5 software for data processing and analysis.

[0063] Experimental results show that LLPSE-p53 has stronger transcriptional activation activity compared with WT-p53.

[0064] Example 8: The p53 variant of the present invention (SEQ ID NO. 7) exhibits stronger transcriptional activation activity. To further investigate whether the transcriptional activation activity of LLPSE-p53 is superior to that of WT-p53, we constructed eukaryotic expression vectors pcDNA3.1-p53 and pcDNA3.1-LLPSE-p53. In H1299 cells, a dual-luciferase reporter assay demonstrated that LLPSE-p53 could promote transcriptional activation. CDKN1A Transcriptional activation ( Figure 8 ).

[0065] Implementation steps: 1. Seed an appropriate amount of cells into a 96-well cell culture plate. When the cell density reaches 60-70%, add the above-mentioned plasmid with... CDKN1A The luciferin reporter gene plasmid was co-transformed and incubated for another 24 hours. 2. Perform chemiluminescence detection using the Promega Dual-Lucent Reporter System kit and M5 microplate reader; 3. Use Graphpad5 software for data processing and analysis.

[0066] The results showed that, compared with WT-p53, LLPSE-p53 was more effective against... CDKN1A Transcriptional activation activity was significantly enhanced.

[0067] Example 9: The p53 variant of the present invention (SEQ ID NO.3) exhibits a stronger ability to kill the non-small cell lung cancer cell line H1299. To further verify whether phase separation enhances p53 transcriptional activation, we constructed pEGFP-p53 and pEGFP-LLPSE-p53 eukaryotic expression vectors. Cell viability was assessed via plasmid transfection and ATP assay. Results showed that the phase-separation-enhanced p53 variant significantly inhibited H1299 cell proliferation. Compared to WT-p53, LLPSE-p53 more significantly inhibited H1299 tumor cell proliferation. Figure 9 ).

[0068] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 μl of complete culture medium to each well, and incubate for 12-24 h. 2. Transfect cells with 100 ng-200 ng plasmid, in triplicate per group, and incubate for 72 h. 3. Remove the cells and allow them to equilibrate to room temperature for 30 minutes. Also, bring the reaction report solution to room temperature. 4. Discard 50 μl of complete culture medium, add 50 μl of reaction reporter solution, and lyse in a horizontal shaker at room temperature for 5 min; 5. Perform chemiluminescence detection on each well for 1-3 seconds using an M5 microplate reader; 6. Use Graphpad5 software for data processing and analysis.

[0069] The results showed that LLPSE-p53 significantly inhibited the proliferation of H1299 tumor cells compared with WT-p53.

[0070] Example 10: The p53 variant of the present invention (SEQ ID NO.3) has a stronger ability to kill the neuroblastoma cell line SH-SY5Y. To investigate whether LLPSE-p53 also has a stronger killing effect on other tumors, we constructed pCMV3-p53 and pCMV3-LLPSE-p53 eukaryotic expression vectors, transfected them into SH-SY5Y cells, and examined cell viability. The results showed that the phase-separation-enhanced p53 variant significantly inhibited the proliferation of SH-SY5Y cells, with a stronger effect than wild-type p53. Figure 10 ).

[0071] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 μl of complete culture medium to each well, and incubate for 12-24 h. 2. Transfect 100-200 ng of plasmid into cells, with 3 replicates per group, and incubate for 72 h. 3. Remove the cells and allow them to equilibrate to room temperature for 30 minutes. Also, bring the reaction report solution to room temperature. 4. Discard 50 μl of complete culture medium, add 50 μl of reaction reporter solution, and lyse in a horizontal shaker at room temperature for 15 min; 5. Perform chemiluminescence detection on each well for 1-3 seconds using an M5 microplate reader; 6. Use Graphpad5 software for data processing and analysis.

[0072] The results showed that LLPSE-p53 had a stronger killing effect on SH-SY5Y cells compared with WT-p53.

[0073] Example 11 The p53 variant of the present invention (SEQ ID NO.3) has a stronger ability to kill the osteosarcoma cell line U-2OS. To further investigate the tumor-killing effect of LLPSE-p53, we constructed eukaryotic expression vectors pcDNA6B-p53 and pcDNA6B-LLPSE-p53, and also tested cell viability in U-2OS cells. The experimental results showed that the phase-separation enhanced p53 variant significantly inhibited the proliferation of U-2OS tumor cells, with a stronger effect than wild-type p53. Figure 11 ).

[0074] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 μl of complete culture medium to each well, and incubate for 12-24 h. 2. Transfect 100-200 ng of plasmid into cells, with 3 replicates per group, and incubate for 72 h. 3. Remove the cells and allow them to equilibrate to room temperature for 30 minutes. Also, bring the reaction report solution to room temperature. 4. Discard 50 μl of complete culture medium, add 50 μl of reaction reporter solution, and lyse in a horizontal shaker at room temperature for 15 min; 5. Perform chemiluminescence detection on each well for 1-3 seconds using an M5 microplate reader; 6. Use Graphpad5 software for data processing and analysis.

[0075] The results showed that LLPSE-p53 had a significant killing effect on U-2OS tumor cells compared with WT-p53.

[0076] Example 12 Comparison of in vitro phase separation ability between the p53 variant (SEQ ID NO.3) recombinant protein of the present invention and wild-type WT-p53 recombinant protein After clarifying the phase separation ability and function of LLPSE-p53 at its eukaryotic cell expression level, through prokaryotic expression and purification of WT-p53 and LLPSE-p53 recombinant proteins, as well as in vitro phase separation experiments, it was found that LLPSE-p53 had a better phase separation ability than WT-p53. Figure 12 ).

[0077] Implementation steps: 1. The two target genes were cloned into the pET-28a(+) prokaryotic expression vector, transformed into the BL21 expression strain, and cultured overnight at 37°C.

[0078] 2. Select single clones and incubate at 37 ℃ and 220 rpm until OD600 = 0.6; 3. Add 200-500 μM IPTG and 100 µM ZnCl2, and incubate overnight at 25 ℃ and 220 rpm; Centrifuge at 4,4500 rpm and 4 °C for 30 min; 5. Add cell lysis buffer, autoclave, and centrifuge at 17,000 rpm and 4 °C for 30 min; 6. Take the supernatant and purify it using HisTrap HP media; 7. Purify the eluent using HiTrap Heparin HP media; 8. Finally, perform gel filtration for further purification; 9. Prepare a phase separation system, take pictures under a fluorescence inverted microscope, and compare the phase separation capabilities of the two systems.

[0079] The results showed that LLPSE-p53 formed more and larger droplets than WT-p53, indicating that LLPSE-p53 has a stronger liquid-liquid phase separation capability than WT-p53.

[0080] Example 13: The p53 variant (SEQ ID NO.3) protein of the present invention has a stronger ability to kill human non-small cell lung cancer cell line H1299. After demonstrating in vitro that the phase separation ability of LLPSE-p53 recombinant protein is superior to that of WT-p53, we further found through protein cell delivery experiments that LLPSE-p53 protein has a stronger killing effect on H1299 tumor cells compared with WT-p53. Figure 13 ).

[0081] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 µl of complete culture medium (P / S-free), and incubate for 12-24 h. 2. Prepare a 50 μl protein delivery system with a protein concentration of 6.5-10 μM as follows: 6.5-10 μM protein + 5× transduction buffer + complete culture medium (P / S-free) 3. Sterilize the above system by passing it through a 0.22 μm filter membrane; 4. Discard the complete culture medium in the well plate, add the protein delivery system described above, and incubate for another 24 hours. 5. Discard the protein delivery system, add complete culture medium, and continue culturing for 24 h; 6. Cell viability was detected using the ATP method and an M5 microplate reader; 7. Use Graphpad5 software for data processing and analysis.

[0082] The results showed that, in H1299 tumor cells, the LLPSE-p53 recombinant protein promoted apoptosis compared to WT-p53.

[0083] Example 14: The p53 variant (SEQ ID NO.3) protein of the present invention has a stronger ability to kill the human neuroblastoma cell line SH-SY5Y. We further validated the protein level on SH-SY5Y cells and found that the LLPSE-p53 recombinant protein also had a significant killing effect on SH-SY5Y tumor cells, and the killing effect was stronger than that of wild-type p53. Figure 14 ).

[0084] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 μl of complete culture medium (P / S-free), and incubate for 12-24 h. 2. Prepare a 50 μl protein delivery system with a protein concentration of 6.5-10 μM as follows: 6.5-10 μM protein + 5× transduction buffer + complete culture medium (P / S-free); 3. Sterilize the above system by passing it through a 0.22 μM filter membrane; 4. Discard the complete culture medium in the well plate, add the protein delivery system described above, and incubate for another 24 hours. 5. Discard the protein delivery system, add complete culture medium, and continue culturing for 24 h; 6. Cell viability was detected using the ATP method and an M5 microplate reader; 7. Use Graphpad5 software for data processing and analysis.

[0085] The results showed that, in SH-SY5Y tumor cells, the LLPSE-p53 recombinant protein promoted apoptosis compared with WT-p53.

[0086] Example 15: LLPSE-p53 (SEQ ID NO.3) recombinant protein enhanced the killing effect of p53 on the human breast cancer cell line SKBR3. We further validated the protein level on SKBR3 cells and found that LLPSE-p53 recombinant protein also had a stronger killing effect on SKBR3 tumor cells compared to wild-type p53 recombinant protein. Figure 15 ).

[0087] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 μl of complete culture medium (P / S-free), and incubate for 12-24 h. 2. Prepare a 50 μl protein delivery system with a protein concentration of 6.5-10 μM as follows: 6.5-10 μM protein + 5× transduction buffer + complete culture medium (P / S-free); 3. Sterilize the above system by passing it through a 0.22 μm filter membrane; 4. Discard the complete culture medium in the well plate, add the protein delivery system described above, and incubate for another 24 hours. 5. Discard the protein delivery system, add complete culture medium, and continue culturing for 24 h; 6. Cell viability was detected using the ATP method and an M5 microplate reader; 7. Use Graphpad5 software for data processing and analysis.

[0088] The results showed that, in SKBR3 tumor cells, the LLPSE-p53 recombinant protein promoted apoptosis compared to WT-p53.

[0089] Example 16: LLPSE-p53 (SEQ ID NO.3) recombinant protein enhanced the killing effect of p53 on human osteosarcoma cell line U-2OS. We further validated the protein level on U-2OS cells and found that the LLPSE-p53 recombinant protein also had a stronger killing effect on U-2OS tumor cells compared with wild-type p53. Figure 16 ).

[0090] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 μl of complete culture medium (P / S-free), and incubate for 12-24 h. 2. Prepare a 50 μl protein delivery system with a protein concentration of 6.5-10 μM as follows: 6.5-10 μM protein + 5× transduction buffer + complete culture medium (P / S-free); 3. Sterilize the above system by passing it through a 0.22 μm filter membrane; 4. Discard the complete culture medium in the well plate, add the protein delivery system described above, and incubate for another 24 hours. 5. Discard the protein delivery system, add complete culture medium, and continue culturing for 24 hours. 6. Cell viability was detected using the CCK8 assay and an M5 microplate reader; 8. Use Graphpad5 software for data processing and analysis.

[0091] The results showed that, in U-2OS tumor cells, the LLPSE-p53 recombinant protein promoted apoptosis compared to WT-p53.

[0092] Example 17: LLPSE-p53 (SEQ ID NO.3) recombinant protein enhanced the killing effect of p53 on the human brain tumor cell line SF126. We further validated the protein level on SF126 cells and found that the LLPSE-p53 recombinant protein had a stronger killing effect on SF126 tumor cells compared with wild-type p53. Figure 17 ).

[0093] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 μl of complete culture medium (P / S-free), and incubate for 12-24 h. 2. Prepare a 50 μl protein delivery system with a protein concentration of 6.5-10 μM as follows: 6.5-10 μM protein + 5× transduction buffer + complete culture medium (P / S-free); 3. Sterilize the above system by passing it through a 0.22 μm filter membrane; 4. Discard the complete culture medium in the well plate, add the protein delivery system described above, and incubate for another 24 hours. 5. Discard the protein delivery system, add complete culture medium, and continue culturing for 24 h; 6. Cell viability was detected using the CCK8 assay and an M5 microplate reader; 7. Use Graphpad5 software for data processing and analysis.

[0094] The results showed that, compared with WT-p53, the LLPSE-p53 recombinant protein had a stronger ability to inhibit the proliferation of SF126 tumor cells.

[0095] Example 18 LLPSE-p53 (SEQ ID NO.3) through regulation FGFR3 exert its tumor-killing function To further investigate the mechanism by which LLPSE-p53 promotes tumor cell apoptosis, we screened cells using RNA-seq and found that, compared with WT-p53, overexpression of LLPSE-p53 significantly increased tumor cell apoptosis. LLPSE-p53 Able to cause FGFR3 The transcriptional level of [something] is reduced, and knockdown [something] is also possible. FGFR3 Subsequently, it was found that LLPSE-p53 lost its tumor-suppressing ability, indicating that LLPSE-p53 inhibits tumor growth through... FGFR3 Inhibit tumor cell proliferation ( Figure 18 The message indicates that LLPSE-p53 is in... FGFR3 Treatment is more effective in tumors with high gene expression levels.

[0096] Implementation steps: 1. Seed 5-6 × 10^5 cells into a 12-well cell culture plate, add 1 ml of complete culture medium and continue culturing for 12-24 h; 2. Transfect sh into cells separately. Vector and sh FGFR3 1 μg of each plasmid was placed in an incubator and cultured for another 48 h. 3. Digest and count the cells from the two groups mentioned above, and seed them into 96-well plates at a rate of 5000 cells per well, and continue culturing for 24 h; 4. Transfect 100-200 ng of plasmid into each well and incubate for 72 h. 5. Cell viability was detected using the ATP assay and an M5 microplate reader; 6. Use Graphpad5 software for data processing and analysis.

[0097] The results showed that without knocking down FGFR3 Under genetic conditions, LLPSE-p53 exhibits a stronger ability to inhibit cell activity compared to wild-type p53, especially when knocked down. FGFR3 Subsequently, LLPSE-p53 lost its ability to inhibit cell viability, indicating that LLPSE-p53 inhibits cell activity through... FGFR3 It can exert its tumor-killing function.

[0098] Example 19: The combined use of LLPSE-p53 (SEQ ID NO.3) protein with TAS-120 and IWR-1 enhances its ability to kill tumor cells. To further enhance the tumor-killing effect of LLPSE-p53 and explore its clinical application feasibility, we combined recombinant LLPSE-p53 protein with the FGFR inhibitor TAS-120 and the Wnt signaling pathway inhibitor IWR-1, and examined the proliferation of U-2OS cells. The results showed that, compared with the non-combined approach, the combination of LLPSE-p53 and TAS-120 significantly increased the tumor-proliferation inhibition effect by approximately 14.5% compared to WT-p53; the combination of LLPSE-p53 and IWR-1 significantly increased the tumor-killing effect by approximately 6.4% compared to WT-p53, indicating that TAS-120 and IWR-1 can enhance the tumor-killing ability of LLPSE-p53. Figure 19 ).

[0099] Implementation steps: 1. Seed 5000 cells / well in a 96-well cell culture plate, add 100 μl of complete culture medium (P / S-free), and incubate for 12-24 h. 2. Prepare a 50 μl protein delivery system according to a protein concentration of 0.5-2 μM, as follows: 0.5-2 μM protein + 5×transduction buffer + complete culture medium (P / S-free); 3. Sterilize the above system by passing it through a 0.22 μm filter membrane; 4. Discard the complete culture medium in the well plate, add the protein delivery system described above, and incubate for another 24 hours. 5. Discard the protein delivery system, add complete culture medium containing 5-20 μM TAS-120 and IWR-1 respectively, and add the same volume of DMSO as a control, and continue to culture for 24 h; 6. Cell viability was detected using the CCK8 assay and an M5 microplate reader; 7. Use Graphpad5 software for data processing and analysis.

[0100] Experimental results show that combined use with the FGFR inhibitor TAS-120 and the Wnt signaling pathway inhibitor IWR-1 can enhance the ability of LLPSE-p53 to kill U-2OS cells at lower protein concentrations.

[0101] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A p53 variant with enhanced liquid-liquid phase separation capability, characterized in that, Adding an amino acid sequence rich in positive charge and histidine to one end of the p53 sequence results in stronger liquid-liquid phase separation ability and stronger transcriptional activation activity compared to wild-type p53 at the cellular level and in vitro. The general formula for the amino acid sequence rich in positive charge and histidine is Hx(G / S / P / A / T)y(R / K)z, where H: is histidine; (G / S / P / A / T): is a sequence composed of one or more of glycine, serine, proline, alanine, and threonine; (R / K) is arginine or lysine, where x, y, and z are the number of the corresponding amino acids, x, z = 0-20, y = 0-100, x+z>5; the p53 variant DNA sequence is shown in SEQ ID NO.

6.

2. Use of the p53 variant according to claim 1 in the preparation of an osteosarcoma treatment medicament.

3. The use according to claim 2, characterized in that, The recombinant protein expression systems for the p53 variant include E. coli expression hosts, animal cells, and yeast protein expression systems; prokaryotic expression vectors include pET24A and pET28A(+); eukaryotic expression vectors include: 1) animal cell expression vectors such as pEGFP, pEYFP, pmcherry, pRFP, pECFP, pLenti, pLX, pCMV6, pCMV3, pcDNA3, and pcDNA6B; 2) insect cell expression vectors including pAc5.1-EGFP; and 3) yeast expression vectors including pPIC3 and pPIC9.

4. The use according to claim 3, characterized in that, The forms of the medicine include injections, tablets, capsules, oral liquid dosage forms, granules, and ointments.

Images