Polypeptides specifically binding to DDX24 helicase and their applications

Abstract

The present invention discloses a series of polypeptides that specifically recognize and bind to the DDX24 helicase and their applications. The polypeptide sequences include the following amino acid signatures: 1. N-terminal-SQETFSDLWKLLPEN-carboxyl terminus; 2. N-terminal-S(p)QET(p)FSDLWKLLPEN-carboxyl terminus; 3. N-terminal-LTFEHYWAQLTS-carboxyl terminus, where the English letters represent common natural amino acids and (p) represents phosphorylation of the hydroxyl group of S or T. Currently, no polypeptides specifically recognize DDX24. The present polypeptides, derived from the p53 protein transcriptional domain, demonstrate for the first time that these polypeptides can specifically bind and recognize the DDX24 helicase.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a series of polypeptides specifically binding to DDX24 helicase and applications thereof. Background Art

[0002] DDX24 is an ATP-dependent RNA helicase edited by the DDX24 gene, which belongs to the DDX protein family. Proteins in this family are involved in cellular processes that change the secondary structure of RNA, such as protein translation initiation, mitochondrial RNA shearing, and the assembly of ribosomes and spliceosomes (Staley and Guthrie 1998, Cell 92: 315-325; Berthelot, Muldoon et al, 2004 Molecular Microbiology 51 (4): 987-1001; Linder 2006, Gene & Development 19: 2122-2137). DDX24 is overexpressed in a variety of human cancer cells. Other articles point out the connection between DDX24 and multi-organ venous and lymphatic malformations, suggesting that DDX24 plays an important role in endothelial cells and participates in the formation of vascular malformations (Pang, Hu et al, 2019, Hepatology 69 (2): 803-816). Therefore, finding a probe that specifically binds to DDX24 helicase is of great significance for understanding the role of DDX24 in the occurrence and development of diseases, and then applying it to early disease detection, molecular imaging, and targeted drug delivery.

[0003] Current studies have shown that DDX24 may be a negative regulator of the tumor suppressor gene p53 (Shi, Dai et al, 2016, Oncogene 35(4):528-536). DDX24 inhibits p300-mediated p53 acetylation, thereby promoting the p53-mediated transcriptional target p21 and upregulating the activation of the apoptosis regulator (PUMA). DDX24 is overexpressed in human breast cancer cells. If the DDX24 protein level is reduced by RNA interference, it will cause cell cycle arrest and senescence, and this effect is p53-dependent (Shi, Dai et al, 2016, Oncogene 35(4):528-536). These results suggest that DDX24 and p53 may have a close interaction in diseased cells, and this interaction has an important impact on the occurrence and development of the disease. This patent first discovered that peptides derived from the transcriptionally active region of p53 (Kussie, Gorina et al. 1996, Science 274:948-953) can directly bind to recombinant DDX24 protein in vitro. Therefore, peptide probes based on p53 (Hu, Gilkes et al. 2007, Cancer Res 67(18):8810-8817) can be used to specifically identify DDX24 helicase. Peptides from the transcriptionally active region of p53 can interact with DDX24, and modifications of certain domains (such as phosphorylation) may increase the affinity between p53 and other interacting proteins (Feng, Jenkins et al. 2009, Structure 17(2):202-210). Based on the above studies, we designed a series of peptides with highly specific binding to DDX24 helicase. Summary of the Invention

[0004] The purpose of the present invention is to provide a polypeptide with high specificity for binding to DDX24 helicase and its application, wherein the polypeptide can specifically recognize DDX24 helicase.

[0005] A polypeptide that specifically recognizes DDX24 helicase, wherein the amino acid sequence of the polypeptide includes the characteristic sequence portion shown below:

[0006] 1. Nitrogen terminus - SQETFSDLWKLLPEN - carboxyl terminus, where the English letters represent common natural amino acids;

[0007] 2. N-terminal - S(p)QET(p)FSDLWKLLPEN - carboxyl terminus, where the English letters represent regular natural amino acids, and (p) represents that the hydroxyl group of S or T is phosphorylated;

[0008] 3. N-terminal-LTFEHYWAQLTS-carboxyl-terminal, where the English letters represent common natural amino acids;

[0009] 4. N-terminal-SGQHSHGYDDCWHQP-carboxyl-terminal, where the English letters represent common natural amino acids. The polypeptide of this sequence has the same length as 1-3 above and serves as a negative control in the experiment.

[0010] Furthermore, the tumor cells are selected from the SMMC-7721 liver cancer cell line with high expression of DDX24.

[0011] The p53 polypeptide is derived from the transcriptionally active region of the p53 protein and is obtained through conventional peptide chemical synthesis or specific chemical modification. The p53 polypeptide can bind to, block, or antagonize the DDX24 helicase. The p53 polypeptide is labeled with chemiluminescence, fluorescence, or radionuclide for use in the preparation of drugs or detection reagents related to DDX24 helicase.

[0012] The beneficial effect of the present invention is that the polypeptide of the present invention can specifically recognize DDX24 helicase, which is of great significance for understanding the role of DDX24 in the occurrence and development of diseases, and further applying it to early detection of diseases, molecular imaging, and targeted drug delivery.

[0013] The present invention also includes applying the p53 polypeptide to the design and preparation of drugs or detection reagents for diseases related to DDX24. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The binding affinity of the fluorescently labeled peptide sequence to DDX24 in vitro was determined using surface plasmon resonance (SPR) analysis. Test conditions: pH 10 mM Acetate 5.5, flow rate 10 μl / min, flow rate 30 μl / min, peptide concentrations 0.78125 / 1.5625 / 6.25 / 12.5 / 25 / 50 μg / ml, association time 120 s, dissociation time 120 s. The results showed that the K of peptide 1 was 0. d =7.15+3.6μM, K of peptide 2 d =1.83+0.2μM, K of peptide 3 d =0.069+0.03 μM, while comparative polypeptide 4 had no measurable binding activity.

[0015] Figure 2In Figure 2A, peptides 1-3 and control peptide 4 are fluoresced with green FITC. SMCC-7721 cells (labeled DDX24H) and 97L cells (labeled DDX24L) were seeded in a 96-well black fluorescent plate. Experimental peptides 1-3 and control peptide 4 were diluted to a final concentration of 10 μM in cell culture medium. After incubation and elution, the specific binding signals within the cells were read using a fluorescence detector (EM: 528, EX: 485). The results showed that peptides 1-3 were significantly more actively taken up by living cells than control peptide 4. In a comparison of DDX24-overexpressing SMMC-7721 cells and DDX24-underexpressing 97L cells, peptides 1-3 were significantly more actively taken up by DDX24-overexpressing SMMC-7721 cells than by DDX24-underexpressing 97L cells. In Figures 2B and 2C, cells in the 96-well plate were incubated on ice for 30 minutes, and the fluorescence signals were read again to observe the difference in binding compared to incubation at 37°C. After reading, the cells were returned to 37°C for 2 hours, and the fluorescence signal was read again. The results showed that the fluorescence intensity changes of SMMC-7721 cells (DDX24H) were more obvious than those of 97L cells (DDX24L), with the fluorescence intensity increasing at low temperatures and decreasing upon returning to 37°C.

[0016] Figure 3 Peptide 3 was labeled with green fluorescence (FITC) and added to the culture medium at a final concentration of 40 μM. SMCC-7721 cells were incubated at 37°C for 4 hours. The plates were then washed three times for 5 minutes each with 0.1% BSA / PBS and fixed with 4% paraformaldehyde for 30 minutes at either 37°C or 4°C. The plates were then washed and blocked with 3% BSA / PBS for 30 minutes at room temperature. The cells were then incubated with a DDX24 antibody (ThermoFisher, PA5-51721) and a red fluorescent secondary antibody (ThermoFisher, A21428) for 1 hour at room temperature in the dark. Nuclear staining with DAPI was performed. Finally, images were acquired and observed under a fluorescence microscope. The results showed that the phase shift observed after peptide 3 bound to intracellular DDX24 (intranuclear or intracellular granular aggregates, indicated by yellow arrows, were more pronounced at 4°C) was consistent with staining with a DDX24-specific antibody. DETAILED DESCRIPTION

[0017] In order to explain the technical content, structural features, achieved objectives and effects of the present invention in detail, they are described in detail below in conjunction with the embodiments.

[0018] The present invention provides a p53 polypeptide that can specifically bind to DDX24 helicase and its application. The sequence of the p53 polypeptide includes:

[0019] 1. N-terminal - SQETFSDLWKLLPEN - carboxyl terminus, where the letters represent common natural amino acids; 2. N-terminal - S(p)QET(p)FSDLWKLLPEN - carboxyl terminus, where the letters represent common natural amino acids, and (p) represents that the hydroxyl group of S or T is phosphorylated;

[0020] 3. N-terminal-LTFEHYWAQLTS-carboxyl-terminal, where the English letters represent common natural amino acids;

[0021] 4. N-terminal-SGQHSHGYDDCWHQP-carboxyl-terminal, where the English letters represent common natural amino acids. The polypeptide of this sequence has the same length as 1-3 above and serves as a negative control in the experiment.

[0022] Specific embodiment 1: Direct binding of polypeptide to DDX24 recombinant protein (SPR in vitro binding experiment).

[0023] Purification of DDX24 Protein: To obtain recombinant DDX24 protein in vitro, the DDX24 gene (28-859 aa, based on PubMed library ID 57062) was ligated into the pSumo vector via the Bamh I and Hind III restriction sites. The recombinant plasmid was generated in DH5α bacteria, verified by sequencing, and transformed into BL21(DE3) expression bacteria for protein purification. The plasmid was transformed into BL21(DE3) and shaken at 37°C at 200 rpm to an absorbance of 0.6-0.8. IPTG (Solybol, SBJ-F2160) was added to a final concentration of 0.1 mM. Expression was induced at 16°C and harvested by centrifugation at 4°C / 5000g for 15 minutes. The cells were resuspended in 30 ml of Solution A per liter of culture medium. Bacteria were disrupted using a high-pressure, low-temperature disruptor (JNBIO, JN-mini) and harvested at 14,000 g for 35 minutes at 4°C. The supernatant was purified using a nickel column (Nanjing Senbeijia Biotechnology Co., Ltd., SBJ-FL2160) with elution using Solution B. The eluted protein was digested with ULP1 for 16 hours at 4°C and dialyzed overnight in Solution C (Solarbo, ya1043 dialysis tubing). The dialyzed protein was then applied to a reverse-loaded nickel column (Nanjing Senbeijia Biotechnology Co., Ltd., Cat. No. SBJ-FL2160). The flow-through was collected and concentrated by ultrafiltration (Millipore, Cat. No. UFC903096) and then passed through a HiLoad 16 / 600 Superdex 200 PrepGrade column (GE, Cat. No. 28-9920-17AC). The target protein was eluted using Solution D, concentrated, and lyophilized (lyophilization equipment manufacturer: Guangzhou Zhixiang Biotechnology Co., Ltd., model: TF-LFD-1). Solution A: 50mM Tris-HCl pH 8.0, 500mM NaCl, 20mM Imidazole; Solution B: 50mM Tris-HCl pH 8.0, 500mM NaCl, 300mM Imidazole; Solution C: 50mM Tris-HCl pH 8.0, 150mM NaCl; Solution D: PBS. The typical protein extraction yield per 10 liters of expression bacterial culture is 0.1mg of 90% pure DDX24 protein.

[0024] The binding affinity of the fluorescently labeled peptide sequence to DDX24 in vitro was determined using surface plasmon resonance (SPR) analysis. SPR binding analysis: The dissociation constant (K) between DDX24 and the peptide was determined on a Biacore T200 instrument. d The target peptide was biotinylated using biotin (Pierce). Streptavidin was bound to the surface of the sensor chip SA, and the conditions were adjusted, and then the biotinylated peptide was fixed to the surface. DDX24 was injected into the sensor chip. The maximum response under equilibrium binding conditions was then plotted against the protein concentration to determine the K dTest conditions: pH: 10mM Acetate 5.5, flow rate: 10μl / min, flow rate: 30μl / min, peptide concentration: 0.78125 / 1.5625 / 6.25 / 12.5 / 25 / 50μg / ml, association time: 120s, dissociation time: 120s. The results are Figure 1 As shown, the K d =7.15+3.6μM, K of peptide 2 d =1.83+0.2μM, K of peptide 3 d =0.069+0.03 μM, while no measurable binding was observed for the comparative peptide 4. The results showed that peptides 1-3 could specifically bind to DDX24 in vitro.

[0025] Specific embodiment 2: The polypeptide selectively binds to living cells with high expression of DDX24 (living cell fluorescence binding experiment).

[0026] To further verify that the listed peptides can bind to intracellular DDX24 in living cells, the peptides 1-3 and control peptide 4 were labeled with green FITC fluorescence (synthesized by Shanghai Botai Company). The same number of peptides (1×10 5 ) of SMCC-7721 cells with high DDX24 expression (labeled as DDX24H), and 97L cells with low DDX24 expression (labeled as DDX24L). Experimental peptides 1-3 and control peptide 4 were diluted to the same final concentration (10 μM) with cell culture medium, and the same concentration was repeated in 2 wells. After adding the sample, the total fluorescence intensity of each well was measured by a fluorescence reader (Synergy HTX Company) (EM: 528, EX: 485) to determine whether the total fluorescence signal of each well was flat. After recording, the cells were placed in a 37°C incubator and incubated for 12 hours. The culture medium containing the peptide was then aspirated, and the cells were gently washed twice with fresh culture medium without the peptide, each time for 5 minutes. Finally, the signal of the peptide specifically bound to the cell was read by a fluorescence detector (EM: 528, EX: 485). The experimental results are as follows. Figure 2 As shown in A, the amount of 1-3 peptide taken up by living cells was significantly higher than that of control peptide 4. Comparing DDX24-overexpressing SMMC-7721 cells with DDX24 overexpression and DDX24-underexpressing 97L cells, the 1-3 peptide taken up by DDX24-overexpressing SMMC-7721 cells was significantly higher than that by DDX24-underexpressing 97L cells.

[0027] To further verify that DDX24 family proteins are prone to intracellular phase transfer and that low temperature promotes their phase transfer phenomenon (Nott, Petsalaki et al. 2015, Mol Cell 57(5):936-947), cells in 96-well plates were incubated on ice for 30 minutes and the fluorescence signal was read again to observe the difference compared with the case of incubation at 37°C. After reading the data, the cells were returned to 37°C for incubation for 2 hours and the fluorescence signal was read again. The experimental results are as follows Figure 2 As shown in Figures B and C, the fluorescence intensity changes in SMMC-7721 cells (DDX24H) are more pronounced than those in 97L cells (DDX24L), with an increase in fluorescence intensity at low temperatures and a decrease upon return to 37°C. This suggests that the fluorescence signals traced by specifically bound peptides 1-3 accurately reflect the unique biophysical property of intracellular DDX24, which is prone to phase transition at low temperatures.

[0028] Specific embodiment 3: The polypeptide selectively binds to DDX24 high-expressing cells (immunofluorescence).

[0029] In order to further verify whether the above-mentioned peptide can specifically bind to DDX24 in living cells, a peptide cell binding experiment was performed using liver cancer cells with high expression of DDX24. The peptide was labeled with green fluorescence (FITC), and DDX24 high-expressing SMCC 7721 cells (1×10 5 / well, 8-well chamber slides (ThermoFisher, 154941), added to the culture medium of the polypeptide 3 at a final concentration of 40 μM, and then eluted with 0.1% BSA / PBS solution 3 times, 5 minutes each time. Then add 4% paraformaldehyde and fix at 37°C or 4°C for 30 minutes. After elution, block with 3% BSA / PBS solution for 30 minutes. Add DDX24 antibody (ThermoFisher, PA5-51721) and incubate in a 4°C wet box overnight. After elution, add red fluorescent secondary antibody (ThermoFisher, A21428) and incubate at room temperature in the dark for 1 hour. After elution, add DAPI to the slide and incubate in the dark for 5 minutes. Observe and collect images under a fluorescence microscope. As Figure 3 As shown, it was found through observation that the phase shift phenomenon (intranuclear or intracellular granular aggregation indicated by yellow arrows is more obvious at 4°C) displayed by polypeptide 3 of the present invention (polypeptide 3 is used in this example, but the implementation of the present invention is not limited to polypeptide 3) after binding to intracellular DDX24 was completely consistent with the results of staining with a DDX24-specific antibody.

Claims

1. Use of a polypeptide that specifically recognizes and binds to DDX24 helicase in the preparation of a DDX24 helicase detection reagent, wherein the amino acid sequence of the polypeptide is: LTFEHYWAQLTS.

2. The use according to claim 1, characterized in that The polypeptide that specifically recognizes and binds to the DDX24 helicase is labeled with chemiluminescence, fluorescence or radionuclide and is used in the preparation of a DDX24 helicase detection reagent.

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