Method and platform for screening and identifying receptor P2Y11 of human intestinal alpha defensin 5 and application

Abstract

The invention discloses an innovative human intestinal alpha defensin 5 (HD5) receptor P2Y11 screening method, platform and application, and belongs to the field of biomedicine.The method fuses multiple omics and experimental strategies, effectively solves the problems that receptor protein is complex and interaction is difficult to independently identify, remarkably improves the accuracy and reliability of defensin receptor identification, and has good application prospects. A set of method system with high universality and wide applicability is provided for receptor research, the first cell receptor P2Y11 of HD5 is successfully identified by the method, and discovery of the P2Y11 receptor not only opens up a new perspective for deep understanding of biological functions and action mechanisms of HD5, but also provides a new potential target for treatment of related diseases; especially for infectious diseases such as bacillary dysentery caused by shigella participated in HD5, a P2Y11 receptor is used as a drug target, possibility is provided for developing a novel treatment scheme, more effective treatment choices are expected to be brought to patients, and the treatment effect and life quality are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a screening and identification method, platform and application of P2Y11, a receptor of human intestinal alpha defensin 5. Background Art

[0002] Defensins are a class of cationic small molecule polypeptides rich in β-sheet structure. They belong to the class of antimicrobial peptide molecules that exist in all biological categories and are a conservative component of the biological innate immune system. In the human body, human enteric defensin 5 (HD5) belongs to the intestinal defensin. In recent years, studies have shown that, contrary to its antiviral and antibacterial immune effects, some human α-defensins can also promote the infection of host cells by viruses and pathogens under certain physiological conditions, playing a "double-edged sword" role in the host's immune process. Studies on the involvement of human α-defensin 5 in the process of Shigella infection of intestinal epithelial cells have found that HD5 induces rapid rearrangement of the host cell cytoskeleton network, produces a large number of pseudopodia-like structures to participate in the capture of Shigella, and further promotes the invasion and infection process of Shigella. This is a new regulatory mode of host cell involvement in Shigella infection. However, the cell receptor bound by HD5 behind this mode is still unclear.

[0003] Most of the receptors of the defensin family known so far belong to G protein-coupled receptors (GPCRs), such as CCR6 and CCR2 for human β-defensin receptors, and Mrgpra2 for mouse β-defensin receptors. There are no reports on α-defensin receptors. The reason is that defensin receptors are difficult to identify using traditional biological methods. The types of proteins bound by human α-defensin are complex, and it is impossible to determine which of them is responsible for the binding. Second, the identification methods for β-defensin receptors and other receptors reported so far are all based on molecular biology experiments. The screening of inhibitors is highly random, and it is difficult to form a universal identification system for use in other receptor screening and identification studies. Third, many ligand receptors belong to GPCRs. GPCR receptors are a complex and diverse family with complex structures, signal transduction, and interactions. They often interact with other proteins such as chaperone proteins and other receptors, making it more difficult to identify specific types and functions of GPCRs alone. Summary of the invention

[0004] In view of the difficulty in identification caused by the complexity and strong randomness of defensin receptor identification methods and the diversity of GPCR receptor families, especially the current technical status of unknown α-defensin receptors, the present invention aims to provide a screening and identification method, platform and application of P2Y11, the receptor of human intestinal α-defensin 5. The present invention innovatively establishes a defensin receptor screening method and screening platform, reveals α-defensin 5 receptor P2Y11 for the first time, provides new insights into the body's recognition of pathogens and immune response mechanisms, and is expected to develop new strategies for the treatment of bacillary dysentery and highly contagious bacterial infections, which has great scientific significance and application prospects.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] The present invention provides a method for screening human intestinal α-defensin 5 receptor P2Y11, the method comprising:

[0007] S1, treating cells with human intestinal α-defensin 5 and a broad-spectrum inhibitor of G protein-coupled receptors to obtain treated cells, automatically characterizing and quantifying the filopodia produced by the treated cells, and preliminarily screening the receptor type of human intestinal α-defensin 5;

[0008] S2, based on the preliminary screening of the receptor types of human intestinal α-defensin 5, the phosphorylation levels of intracellular proteins after treatment were detected by liquid chromatography-mass spectrometry, the phosphorylated proteins and phosphorylation sites were identified, and GO functional enrichment analysis was used to screen intracellular proteins with significantly increased phosphorylation levels;

[0009] S3, based on the screening of intracellular proteins with significantly increased phosphorylation levels, the phosphorylation levels of intracellular kinases after treatment were detected using human phosphorylation kinase solid phase antibody chip, and the kinases involved in human intestinal α-defensin 5 signal transduction and its related signal pathways were screened through grayscale analysis;

[0010] S4, based on screening kinases involved in human intestinal α-defensin 5 signal transduction and their related signaling pathways, cells were treated with inhibitors of kinase-related signaling pathways, the effects of inhibitors on cell filopodia were detected, and the signaling pathways involved in human intestinal α-defensin 5-induced cytoskeletal rearrangement were verified;

[0011] S5, based on the validation of the signaling pathway involved in the cytoskeletal rearrangement induced by human intestinal α-defensin 5, cells were treated with specific inhibitors of upstream proteins in the signaling pathway that promotes cytoskeletal rearrangement by human intestinal α-defensin 5, the effects of the inhibitors on cell filopodia were detected, and the types of receptor proteins associated with human intestinal α-defensin 5 signaling were determined;

[0012] S6, based on the identification of receptor protein types associated with human intestinal α-defensin 5 signaling, human intestinal α-defensin 5 receptor P2Y11 was screened out through cell transcriptome sequencing analysis.

[0013] In S1, the automatic characterization and quantification of filopodia produced by treated cells was performed using an open source algorithm of ImageJ.

[0014] Furthermore, the length, number and cell perimeter of the cell filopodia are automatically quantified; the cells are adherent cells or suspended cells.

[0015] Furthermore, the adherent cells are any one of HeLa, Hep-G2, MS751, and SiHa.

[0016] Furthermore, the suspension cells are any one of Jurkat, THP-1, K562, and U937.

[0017] Furthermore, the G protein coupled receptor broad-spectrum inhibitor is Suramin.

[0018] Furthermore, the adherent cell culture medium is: DMEM low-glucose culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibiotics.

[0019] Furthermore, the suspension cell culture medium is: RPMI culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics.

[0020] Furthermore, the cell culture conditions are: 37° C., 5% carbon dioxide incubator for 30 minutes.

[0021] S2 includes:

[0022] S21, cells were treated with human intestinal α-defensin 5 and a broad-spectrum inhibitor of G protein-coupled receptors to obtain treated cells;

[0023] S22, cells after lysis treatment, centrifugation to obtain supernatant, protein alkylation treatment, protein precipitation, protein enzyme digestion, desalting and enrichment to obtain lysed cells;

[0024] S23, liquid chromatography-mass spectrometry analysis of protein phosphorylation levels in cells after lysis, identification of phosphorylated proteins and phosphorylation sites, and cell component enrichment analysis using the GO database to screen intracellular proteins with significantly increased phosphorylation levels.

[0025] Further, in S22, the protein is alkylated by incubating with dithiothreitol (DTT), iodoacetamide (IAA) and dithiothreitol (DTT) in sequence at room temperature for 45 to 60 minutes.

[0026] Furthermore, the final concentration of the dithiothreitol (DTT) is 10 μM, the final concentration of the iodoacetamide (IAA) is 40 μM; and the final concentration of the dithiothreitol (DTT) is 40 μM.

[0027] In S3, the human phosphorylation kinase solid phase antibody chip is an R&D SystemsTM human phosphorylation kinase solid phase antibody chip.

[0028] In S3, the grayscale analysis uses ImageJ software to screen the kinases involved in human intestinal α-defensin 5 signal transduction as kinases with significantly increased phosphorylation levels; the related signaling pathway is the cAMP / PKA signaling pathway.

[0029] In S4, the signaling pathway involved in the HD5-induced cytoskeleton rearrangement is verified to be the corresponding signaling pathway of the inhibitor that can significantly inhibit the formation of cell filopodia promoted by human intestinal α-defensin 5.

[0030] In S5, the specific inhibitor of the upstream protein of the pathway in which human intestinal α-defensin 5 promotes cytoskeleton rearrangement is NF449 or PTX.

[0031] In S6, the human intestinal α-defensin 5 receptor P2Y11 was screened out by cell transcriptome sequencing, TPM was used to measure the gene expression level, genes with TPM>1 were screened, and the intersection was integrated with the genes with cell nTPM>1 in the Human Protein Atlas database to screen out the human intestinal α-defensin 5 receptor P2Y11.

[0032] The method also includes the step of identifying the human intestinal alpha defensin 5 receptor P2Y11.

[0033] The identification of the human intestinal α-defensin 5 receptor P2Y11 is carried out by integrating bioinformatics with biochemical methods, including: constructing biotin-labeled human intestinal α-defensin 5, pulling down human intestinal α-defensin 5 with streptavidin magnetic beads to bind human intestinal α-defensin 5 to its potential receptor, verifying it with a specific antibody for P2Y11, and simultaneously searching the library with mass spectrometry to find the peptide fragment of the human intestinal α-defensin 5 receptor P2Y11, confirming that P2Y11 is the human intestinal α-defensin 5 receptor.

[0034] The present invention provides a platform for implementing the above-mentioned screening method for human intestinal α-defensin 5 receptor P2Y11, the platform comprising:

[0035] The cell culture and processing analysis module is used to provide and maintain optimal cell growth conditions, process cells and observe cell responses, and preliminarily screen the receptor types of human intestinal α-defensin 5;

[0036] Protein phosphorylation analysis module, used to detect the phosphorylation level of intracellular proteins and identify phosphorylated proteins and phosphorylation sites;

[0037] Kinase phosphorylation level detection module, used to screen kinases involved in defensin receptor signal transduction and their related signal pathways;

[0038] Signaling pathway and receptor protein verification module, which is used to verify the involvement of upstream proteins and signaling pathways in the cytoskeleton rearrangement induced by human intestinal α-defensin 5, and to determine the type of receptor protein associated with it;

[0039] The transcriptome sequencing and analysis module is used for transcriptome sequencing, analyzing changes in gene expression levels, and screening out human intestinal alpha defensin 5 receptor.

[0040] The above platform is used in screening defensin receptors and drug targets, wherein the defensin receptor includes any one of an intestinal defensin receptor, a respiratory defensin receptor and a pathogen-associated defensin receptor, and the drug target is a defensin receptor as a drug target.

[0041] Compared with the prior art, the present invention achieves the following technical effects:

[0042] The present invention provides a screening method for human intestinal α-defensin 5 receptor P2Y11, which combines multi-omics analysis with molecular experimental strategies to form a set of receptor screening and identification methods from "function-protein-gene" reverse protein translation. This method combines multi-omics and experimental strategies, and ensures the accuracy and reliability of screening through various technical means such as cell treatment, phosphorylated protein detection, kinase phosphorylation level analysis, signal pathway verification and transcriptome sequencing, avoiding the problem that receptor proteins are complex and interactions are difficult to identify separately. Compared with the existing defensin receptor identification methods, the screening method of the present invention has higher accuracy and reliability, and provides a set of widely applicable method systems for receptor identification. Through the method of the present invention, the first HD5 cell receptor P2Y11 was successfully found, which is a major breakthrough in the field of defensin receptor research.

[0043] Furthermore, the open source algorithm of ImageJ was used to automatically characterize and quantify the filopodia of cells, achieving high-throughput and high-precision preliminary screening, which not only improved the screening efficiency, but also ensured the accuracy and consistency of the data; liquid chromatography-mass spectrometry technology was used to accurately detect the phosphorylation level of intracellular proteins after treatment, and to identify phosphorylated proteins and phosphorylation sites, providing reliable data for subsequent GO functional enrichment analysis; R&D SystemsTM human phosphorylation kinase solid phase antibody chip was used to detect the phosphorylation level of kinases, ensuring the consistency and repeatability of the experiment; using human phosphorylation kinase solid phase antibody chip and grayscale analysis, the kinases involved in human intestinal α-defensin 5 signaling and its related signaling pathways were screened out, which not only revealed the molecular mechanism of HD5 signaling, but also provided potential targets for subsequent drug development; through cell transcriptome sequencing technology, the HD5 receptor P2Y11 was screened out, and TPM was used to measure the gene expression level, and compared with Human Comparison with genes in the ProteinAtlas database improved the accuracy and reliability of the screening; the identification step of the receptor P2Y11 ensured the accuracy and reliability of the screened receptor protein, providing a solid foundation for subsequent functional research and drug development.

[0044] The platform provided by the present invention integrates multiple functional modules such as cell culture and treatment, protein phosphorylation analysis, kinase phosphorylation level detection, signal pathway and receptor protein verification, and transcriptome sequencing and analysis, thereby achieving full coverage from cell treatment to gene expression analysis, and greatly improving screening efficiency; the protein phosphorylation analysis module and the kinase phosphorylation level detection module adopt advanced liquid chromatography-mass spectrometry technology and solid phase antibody chip technology, and can detect the phosphorylation level of intracellular protein and the activity of kinase with high precision; the protein phosphorylation analysis module and the kinase phosphorylation level detection module adopt advanced liquid chromatography-mass spectrometry technology and solid phase antibody chip technology, and can detect the phosphorylation level of intracellular protein and the activity of kinase with high precision; the design of the platform allows flexible adjustment according to experimental requirements, such as replacing treatment reagents, adjusting detection conditions, etc., to meet the screening requirements of different defensin receptors; the functional modules of the platform can be expanded, such as adding new detection technologies or analysis methods to cope with new defensin receptors and signal pathways that may appear in the future; the platform is equipped with a powerful data management system and analysis software, which can integrate data from different functional modules, conduct comprehensive analysis, and improve the accuracy and reliability of screening results.

[0045] The application provided by the present invention is that the platform can be used not only for screening human intestinal α-defensin 5 receptor P2Y11, but also for screening other intestinal defensin receptors, respiratory defensin receptors and pathogen-related defensin receptors, etc., which greatly broadens the screening scope. The platform can be used not only for screening human intestinal α-defensin 5 receptor P2Y11, but also for screening other intestinal defensin receptors, respiratory defensin receptors and pathogen-related defensin receptors, etc., which greatly broadens the screening scope. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The quantification results of HeLa cell pseudopodia under the action of HD5 of the present invention, wherein A is the quantification results of the filamentous pseudopodia density, average length and pseudopodia generation capacity of HeLa cells treated with HD5 and the control group by the Filoquant algorithm, B is the immunofluorescence image of the HeLa cell skeleton in the HD5 and Suramin treatment groups; C is the quantification results of the filamentous pseudopodia generation capacity of cells treated with HD5, Suramin and the control group by the Filoquant algorithm;

[0047] Figure 2 The phosphoproteomic results of HeLa cells of the present invention; wherein A is the phosphorylated protein results of HeLa cells treated with HD5, Suramin and the control group; B is the cell component of the bubble diagram of GO functional enrichment analysis; C is the molecular function of the bubble diagram of GO functional enrichment analysis;

[0048] Figure 3 The results of the phosphorylation kinase chip of HeLa cells treated with HD5 of the present invention, wherein A is the phosphorylation kinase chip of epithelial cells treated with HD5, and B is the kinase whose phosphorylation level increases after HD5 treatment;

[0049] Figure 4 The Filopodia algorithm quantification results of the filopodia growth ability of the groups treated with different signal pathway inhibitors of the present invention;

[0050] Figure 5 The laser confocal microscopic fluorescence photographs of HeLa cells acted by the Gi and Gs protein inhibitors of the present invention and the quantification of the cell filopodia generation capacity by the Filoquant algorithm, wherein A is the laser confocal microscopic fluorescence photographs, and B is the quantification of the cell filopodia generation capacity by the Filoquant algorithm;

[0051] Figure 6 is a transcriptome analysis of GPCR expression in the cell line of the present invention, wherein A is the nTPM value of the Gs-GPCR gene in the HPA database, B is the TPM value of the Gs-GPCR gene in the test cell line, and C is a schematic diagram of the process of screening P2Y11 using transcriptome data and Reactome (Human Protein Atlas Database);

[0052] Figure 7 For the identification of the P2Y11 receptor of the present invention, wherein A is the immunoblotting analysis of the protein sample pulled down by Biotin-HD5 by the P2Y11 antibody, and B is the mass spectrometry-based P2Y11 targeted peptide search;

[0053] Figure 8 This is the quantitative result of the Filopodia algorithm for the ability of NF157 of the present invention to inhibit the growth of filopodia. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0056] The present invention provides a multi-omics screening and experimental identification method for human intestinal α-defensin 5 receptor P2Y11. In the invention, considering that the reported defensin receptors all belong to GPCRs, Suramin (a broad-spectrum inhibitor of GPCRs) is used to preliminarily explore the receptor of HD5, and the Filoquant algorithm is used to provide statistical differences in pseudopodia generation ability to prove that the HD5 receptor belongs to GPCR. The process of HD5 promoting cytoskeleton rearrangement is very rapid, and cell protein phosphorylation modification is a short-time scale signal transduction method. Therefore, a part of proteins with large differences in protein phosphorylation levels in the presence of HD5 signals are screened by phosphorylation proteome, and GO enrichment analysis of the molecular functions and components of these proteins. The signal pathway cAMP-PKA activated by HD5 is screened out in combination with the phosphorylation level of protein kinase. Inhibitors are used to screen Gs-GPCR or Gi-GPCR coupled upstream of the cAMP-PKA pathway, and the range is narrowed to Gs-GPCR. Finally, a GPCR coupled to Gs protein, P2Y11, is screened out by combining the transcriptome levels of different cells after HD5 treatment with the HumanProteinAtlas database. Immunoprecipitation-mass spectrometry was used to verify the presence of a P2Y11 peptide in the pulled-down protein after HD5 interacted with cells. Identification of this receptor can help find a specific drug, NF157, that targets HD5 to promote bacterial invasion of the host. NF157 can eliminate the effect of HD5 on promoting the formation of cell filopodia.

[0057] 1. Characterization of cell filopodia morphology

[0058] (1) Adherent cell and suspension cell culture

[0059] Culture conditions for adherent cells (HeLa, Hep-G2, MS751, SiHa): Use DMEM low-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibiotics, and culture in a 37°C, 5% carbon dioxide incubator. When plating cells, 24-well plates, 4×10 5 / well, culture conditions are the same as above. When starving cells, use DMEM low-glucose medium without serum and double antibodies, and culture in a 37°C, 5% carbon dioxide incubator. Culture conditions for suspension cells (THP-1, Jurkat, K562, U937): Use RPMI medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics, and culture in a 37°C, 5% carbon dioxide incubator. When plating cells, there are no special requirements, and the culture conditions are the same as above. When starving cells, use RPMI medium without serum and double antibodies, and culture in a 37°C, 5% carbon dioxide incubator.

[0060] (2) HD5 and Suramin treatment of cells

[0061] After starvation, the cells were treated with Suramin and HD5 and cultured in a 37°C, 5% carbon dioxide incubator for 30 minutes.

[0062] (3) Cytoskeleton immunofluorescence staining

[0063] After a certain period of treatment, the culture medium was removed, and 4% paraformaldehyde was added to fix the cells at room temperature for 20 minutes, and then washed 3 times with PBS. 1% TritonX-100 diluted in PBS solution was used to punch holes at room temperature for 5 minutes, and then washed 3 times with PBS. 10 μg / mL rhodamine fluorescently labeled phalloidin was used to stain the microfilament structure of the cells in the dark for 20 minutes. After rinsing in deionized water, the slides were sealed with an anti-fluorescence quenching sealing agent containing DAPI. The samples were stored at 4°C to dry, and images of the complete edges of single cells were taken under a laser confocal microscope.

[0064] (4) Filoquant quantification

[0065] From the initial cell micrograph library, single cells with clear pseudopodia structures were selected as candidate analysis objects. The original 16-bit color depth photos were imported into ImageJ for initial processing, and the color depth was adjusted to 8 bits to meet the accuracy requirements of FiloQuant. Single cells that were not interfered by other cell images were manually selected, and "Single ImageFiloQuant" was used for automatic analysis and processing. The cell edge threshold was set to 6 pixels (pixels), the pseudopodia length range threshold was set to 0.5-25 pixels, the number of processing iterations was set to 5, and the automatic filling of cell edge curve was selected. After processing, the number of pseudopodia of the selected cells, the length of a single pseudopodia, and the total length of the cell edge can be obtained. The pseudopodia generation capacity is the length of pseudopodia that can be generated within the unit circumference of the cell. The calculation formula is: the sum of the pseudopodia lengths of a single cell / the circumference of a single cell = the pseudopodia generation capacity of a single cell. 30 cells were randomly selected to compare the average pseudopodia generation capacity of the HD5 group and the Suramin+HD5 group. For details, see the attached Figure 1 shown.

[0066] By the attached Figure 1 The data show that the density of filopodia in the HD5-treated group increased significantly compared with that in the control group, indicating that HD5 can stimulate HeLa cells to produce more filopodia; the average length of filopodia in the HD5-treated group also increased, indicating that HD5 can not only increase the number of pseudopodia, but also promote the growth of pseudopodia; quantified by the Filoquant algorithm, the pseudopodia generation ability of the HD5-treated group was significantly stronger than that of the control group, further confirming the promoting effect of HD5 on HeLa cell pseudopodia; the immunofluorescence image showed that the number of cell pseudopodia in the HD5-treated group was significantly more than that in the control group, while the Suramin-treated group showed an inhibitory effect on the promoting effect of HD5, with a relatively small number of pseudopodia; the Filoquant algorithm was used to quantify the filopodia generation ability of cells treated with HD5, Suramin and the control group, and Suramin could significantly inhibit the promoting effect of HD5 on cell pseudopodia, further verifying the inhibitory effect of Suramin.

[0067] In summary, HD5 can stimulate HeLa cells to produce more filopodia and promote the growth of pseudopodia; while Suramin can inhibit this promoting effect of HD5, which shows that the receptor of HD5 is GPCR.

[0068] 2. Screening of signaling pathways regulating host cell cytoskeleton rearrangement by HD5

[0069] (1) Phosphorylation protein spectrum analysis

[0070] Preparation of cell lysate: The second / third generation adherent or suspension cells were seeded in a six-well plate one day before the experiment. The next day, the cells were grown to 70% of the bottom of the plate, and the cells were washed 2-3 times with preheated serum-free medium to remove the serum. After treating the cells with HD5, the medium was removed and washed with ice-cold PBS. The lysis buffer was added and the cells were repeatedly blown and lysed, and the supernatant was obtained by centrifugation at 4°C.

[0071] Protein alkylation treatment: Add dithiothreitol (DTT) storage solution to the supernatant sample to a final concentration of 10 μM, vortex to mix, and incubate on a rotating incubator at room temperature for 45 to 60 minutes. Add iodoacetamide (IAA) storage solution to a final concentration of 40 μM, vortex to mix, and incubate on a rotating incubator at room temperature for 45 to 60 minutes. Add DTT storage solution with a final concentration of 40 μM again to neutralize the remaining unreacted iodoacetamide to terminate the alkylation reaction, vortex to mix, and incubate on a rotating incubator at room temperature for 45 to 60 minutes. Centrifuge at 4°C and take the supernatant in a clean centrifuge tube for later use.

[0072] To obtain protein precipitate: add acetone (precipitation solution) 5 times the original volume to the supernatant and incubate it in a -20°C refrigerator overnight. After a large amount of flocculent precipitate appears, centrifuge at 4°C and discard the supernatant. Resuspend and wash the protein precipitate with ice-cold acetone. Centrifuge at 4°C and discard the supernatant. Open the lid of the protein precipitate and dry it at room temperature to allow the remaining solution to evaporate. Use 8μM urea buffer and incubate on a 4°C rotating incubator for 10 to 30 minutes to completely dissolve the precipitate. Use 100mM NH4HCO3 solution to dilute the urea concentration in the protein extract to less than 2M. After the protein is completely dissolved, centrifuge at 4°C to obtain the supernatant and use the BCA (bicinchoninic acid) method to determine the protein concentration.

[0073] Protein digestion, desalting, and enrichment: Add protease at a ratio of 50:1 between protein and enzyme, and digest at 37°C for 16 to 18 hours. Desalt with 0.1% TFA (pH = 3). Compared with general proteomics, modified proteomics requires further enrichment at the peptide level to simplify the complexity of proteomics analysis and achieve a suitable sample size. This experiment used the TiO2 enrichment method to enrich serine and threonine phosphorylation-modified peptides, and after enrichment, a second desalting was performed to collect the peptides. Finally, liquid chromatography-mass spectrometry was used for analysis and identification.

[0074] GO functional enrichment: The protein phosphorylation data of different treatment groups in the mass spectrometry results were further processed and analyzed. The phosphorylation level under HD5 treatment was more than 2 times higher than that of the blank control group and the case of Suramin and HD5 co-treatment. GO (Gene Ontology) functional enrichment analysis was performed on the above-screened proteins. For details, see the attached Figure 2 shown.

[0075] By the attached Figure 2 The data showed that the protein phosphorylation level in the HD5-treated group was significantly higher than that in the control group and the Suramin+HD5-treated group, confirming the significant effect of HD5 on protein phosphorylation in HeLa cells; enrichment results of cellular components: multiple cellular components such as "focal adhesion", "cell-substrate junction", "Actin cytoskeleton", and lameliipodium were significantly enriched. These cellular components are closely related to functions such as cytoskeleton, cell movement, and pseudopodia extension, suggesting that HD5 treatment affects the functions of these cellular components; molecular functions such as "ATP-dependent chromatin remodeler activity", "protein kinaseA regulatory subunit binding", and "protein kinase Abinding" were significantly enriched, especially the related functions of the "protein kinaseA" (PKA) pathway were mentioned many times, indicating that the PKA pathway played a leading role in HD5 regulating cytoskeleton rearrangement.

[0076] In summary, through GO functional enrichment analysis of the screened proteins, the PKA pathway dominated the phosphorylated proteins in HeLa cells under HD5 treatment; in addition, multiple cellular components and molecular functions related to the cytoskeleton, cell movement and chromosome structure were also significantly enriched, which provided important clues and basis for further understanding the effect of HD5 on phosphorylated proteins in HeLa cells.

[0077] 3. High-throughput protein chip screening

[0078] (1) Preparation of cell lysate: Same as above.

[0079] (2) Determination of cell phosphorylation kinase content: Place the human phosphorylation kinase solid phase antibody chip in the well plate, use the kit blocking buffer to block the chip at room temperature for 1 hour, and remove the blocking buffer. Add the diluted cell lysate and incubate the chip at 4°C overnight. After the incubation, use 1× washing buffer to gently shake and wash the chip 3 times, and use the diluted detection antibody mixture to incubate the chip for 2 hours. Wash the chip again, and use diluted streptavidin-HRP to incubate the chip at room temperature in the dark for 30 minutes. Wash the chip, remove the remaining buffer on the edge of the chip with absorbent paper, and evenly drop 1mL of horseradish peroxidase chemiluminescent solution on the surface of the chip. After 1 minute of reaction, use a chemiluminescence meter to detect the chemiluminescence level.

[0080] (3) Grayscale analysis: Use ImageJ software to analyze the grayscale of chemiluminescent photos and quantify them. For details, see Appendix Figure 3 shown.

[0081] By the attached Figure 3 The data show that by identifying the kinases whose phosphorylation levels increased significantly after HD5 treatment, it was found that Akt 1 / 2 / 3, Erk1 / 2, CREB and c-Jun are all kinase effector molecules downstream of the cAMP / PKA signaling pathway.

[0082] In summary, this experiment successfully used high-throughput protein chip technology to determine the content of phosphorylated kinases in cell lysates, and identified the kinases whose phosphorylation levels significantly increased after HD5 treatment through grayscale analysis. It was found that these kinases were all kinase effector molecules downstream of the cAMP / PKA signaling pathway, indicating that HD5 can regulate the phosphorylation level of cells by affecting the cAMP / PKA signaling pathway.

[0083] (4) Validation of cell pathway inhibitors

[0084] Cell treatment: After cells were plated and allowed to adhere, they were treated with various cell pathway inhibitors and HD5, and cytoskeleton immunofluorescence staining was performed in the same manner as described above.

[0085] Laser confocal microscopy was used to capture fluorescence images of the cytoskeleton and quantify the results using Filoquant. The methods were the same as above. For specific results, see Appendix. Figure 4 shown.

[0086] By the attached Figure 4 The data showed that only the cAMP-PKA pathway inhibitors PKI 14-22 and KH7 had significant antagonistic effects, confirming that HD5 promoted cell cytoskeleton rearrangement by activating the host cell cAMP-PKA pathway.

[0087] 4. Screening of receptors that regulate host cell cytoskeleton rearrangement by HD5

[0088] (1) GPCR inhibitor screening

[0089] It is known that the cellular pathway that HD5 regulates cytoskeletal rearrangement is cAMP-PKA, and its upstream is Gs or Gi coupled GPCR, so two GPCR inhibitors were screened. Cell treatment After plating, the cells adhered to the wall and were treated with known Gs and Gi protein inhibitors NF449, PTX treatment and HD5, and cytoskeletal immunofluorescence staining was performed in the same way as above.

[0090] Laser confocal microscopy was used to capture fluorescence images of the cytoskeleton and quantify the results using Filoquant. The methods were the same as above. For specific results, see Appendix. Figure 5 shown.

[0091] By the attached Figure 5 The data show that the results of cell pseudopodia quantification found that only NF449 had a significant antagonistic effect, indicating that NF449, as an inhibitor of Gs protein, can effectively block the effect of HD5 on the cytoskeleton through the Gs-GPCR-mediated signaling pathway. In contrast, PTX, as an inhibitor of Gi protein, did not show a significant antagonistic effect, indicating that HD5 does not act through the Gi-GPCR-mediated signaling pathway. Based on the above results, it can be concluded that the receptor of HD5 belongs to the Gs-coupled GPCR, which means that HD5 activates the downstream cAMP-PKA signaling pathway by binding to Gs-GPCR, thereby promoting the rearrangement of the cytoskeleton.

[0092] (2) Cell transcriptome sequencing screening

[0093] Cell culture, collection, and transcriptome sequencing: 8 cells that can produce obvious pseudopodia in response to HD5 were selected for culture (HeLa, Hep-G2, Jurkat, K-562, MS751, SiHa, THP-1, and U-937). The culture method was as described above. Under normal physiological conditions, adherent cells were collected by trypsin digestion, centrifugation, and discarding the supernatant. Suspended cells were directly collected by centrifugation and discarding the supernatant and stored with TRizol reagent. RNA extraction, library construction, and sequencing were performed in sequence.

[0094] Download and screening of public database data: RNA-Seq data of 10 cell types (SiHa, PC-3, HEK293, THP-1, M8, D5, D6, F7, A-549, CACO-2, HT-29) were obtained from NCBI, and then the transcriptome data of 8 cells cultured, collected and sequenced by ourselves and 10 cells in the database were integrated and analyzed. TPM (Transcripts Per Kilobase Million) was used to measure gene expression levels. The calculation method is to first divide the read count of each gene by the length of the gene (in kilobases) to obtain the number of reads per kilobase of the gene; then divide this value by the sum of the number of reads per kilobase of all genes, and multiply it by one million to obtain the TPM value of the gene. Because of this, TPM can make the expression levels of genes of different lengths comparable, and it is also easier to compare between different samples. For specific results, see the attached. Figure 6 shown.

[0095] By the attached Figure 6 The data show that by screening Gs-GPCR genes with TPM>1 and relatively high gene expression levels at the transcriptome level, and combining the Gs-GPCR genes with nTPM>1 in 14 cell types (Jurkat, HEK293, SiHa, HeLa, PC-3, A-549, MS751, CACO-2, MCF-7, HT-29, K-562, Hep-G2, THP-1, U-937) in the Human Protein Atlas database, we finally screened out the only Gs-coupled GPCR—P2Y11.

[0096] Example 2

[0097] Based on Example 1, this example uses biotin-HD5 pull-down and mass spectrometry to identify the screened HD5 receptor P2Y11, which specifically includes the following steps:

[0098] Lysed cells: Prepare 1 × 10 7 Hela cells were isolated and lysed using GPCR extraction and stabilization reagents containing protease inhibitors. The supernatant contained stable protein receptors. The protein concentration was determined using the EnhancedBCAProteinAssay Kit.

[0099] Binding to biotinylated HD5: 1 mg of receptor protein was incubated with 0.4 mg of biotin-HD5 at 4°C on a rotating mixer for 12-16 hours.

[0100] Immobilized antibody / protein: Add 1 mL of washing buffer (PBS, 0.05% Tween-20, pH 7.4) to wash the magnetic beads thoroughly. Add 1 mL of biotinylated antibody / protein diluted with Buffer II, vortex and suspend thoroughly, place on a rotating mixer and incubate at room temperature for 60 minutes. Immobilize the above protein receptor mixture with 50 μL of streptavidin magnetic beads with antibodies at room temperature for 1 hour.

[0101] Pull down the interacting proteins after elution: wash 5 times with washing buffer, then elute twice with 40 μL 1X SDS-PAGE loading buffer at 95°C, and load onto 4%-20% SDS-PAGE gel.

[0102] Post-elution mass spectrometry analysis: bead reduction, alkylation and trypsin digestion were performed, and LC-MS / MS detection was performed after desalting with a C18 membrane column. The presence of the P2Y11 peptide was identified by protein target search. For specific test results, see the attached Figure 7 shown.

[0103] By the attached Figure 7 The data shows that after the in vitro pull-down experiment with biotin-bound HD5 (Biotin-HD5), a series of significant results were presented in the subsequent protein immunoblotting and protein spectrum analysis. In the protein immunoblotting experiment, when the specific antibody of P2Y11 was used for testing, the positive control group HeLaextract (input) clearly showed the presence of P2Y11. What is particularly critical is that the presence of P2Y11 was also detected in the protein pulled down by HD5 (IP group: HeLaextract+Botin-HD5+SAVBeads). At the same time, in a more in-depth protein spectrum targeted search library analysis, the peptide of P2Y11 was also successfully found. Combining the above experimental results from many aspects, it has been strongly verified that P2Y11 is a binding receptor for HD5.

[0104] NF157, a specific inhibitor of P2Y11, abolishes the effect of HD5 on promoting pseudopodia formation

[0105] As a member of the purinergic receptor family, P2Y11 has important potential application value in biomedical research. However, due to the lack of P2Y11 genes in the mouse genome, research on its function is relatively scarce. The present invention successfully identified the HD5 receptor as P2Y11, and this discovery has opened up a new way to further study the function of P2Y11.

[0106] The Filopodia algorithm was used to quantify the ability of cells to inhibit filopodia growth by pre-treating them with NF157, a known specific inhibitor of P2Y11. See Appendix. Figure 8 As shown. Figure 8 The data show that the cells in the HD5-treated group showed significant advantages in pseudopodia growth ability, which was more prominent than the control group and the NF157-treated group. After NF157 pretreatment, HD5 was added to the cell system. At this time, the pseudopodia generation ability of the cells was basically at a similar level to that of the control group. Statistical analysis showed that there was no significant difference between the two. This result shows that NF157 can eliminate the effect of HD5 in promoting pseudopodia generation, and this phenomenon provides a favorable tool for studying the regulatory mechanism of cell pseudopodia generation. Filoquant was further used to quantify the pseudopodia generation ability of the cells in the NF157+HD5 group and the HD5-treated group, and the statistical differences were compared, providing a reliable method for accurately evaluating the role of P2Y11 in cell pseudopodia generation.

[0107] The above results of the present invention show that a receptor screening and identification method combining multi-omics analysis and molecular experiments from "function-protein-gene" reverse protein translation is provided, which can be widely used. Specifically, a receptor P2Y11 for human intestinal α-defensin 5 was successfully screened. On the other hand, the method of the present invention can also be applied to the field of drug research and development. P2Y11, as a switch for HD5 to regulate cytoskeleton rearrangement, plays an important role in the involvement of HD5 in the invasion of Shigella infection. By screening new inhibitors or agonists against P2Y11, drugs with specific therapeutic effects can be developed. Specifically, it can provide possibilities for intervention strategies against pathogens that utilize HD5-induced filamentous extension, opening up new perspectives and new fields for the application of P2Y11.

[0108] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for screening human intestinal α-defensin 5 receptor P2Y11, characterized in that: The method includes: S1, treating cells with human intestinal α-defensin 5 and a broad-spectrum inhibitor of G protein-coupled receptors to obtain treated cells, automatically characterizing and quantifying the filopodia produced by the treated cells, and preliminarily screening the receptor type of human intestinal α-defensin 5; S2, based on the preliminary screening of the receptor types of human intestinal α-defensin 5, the phosphorylation levels of intracellular proteins after treatment were detected by liquid chromatography-mass spectrometry, the phosphorylated proteins and phosphorylation sites were identified, and GO functional enrichment analysis was used to screen intracellular proteins with significantly increased phosphorylation levels; S3, based on the screening of intracellular proteins with significantly increased phosphorylation levels, the phosphorylation levels of intracellular kinases after treatment were detected using human phosphorylation kinase solid phase antibody chip, and the kinases involved in human intestinal α-defensin 5 signal transduction and its related signal pathways were screened through grayscale analysis; S4, based on screening kinases involved in human intestinal α-defensin 5 signal transduction and their related signaling pathways, cells were treated with inhibitors of kinase-related signaling pathways, the effects of inhibitors on cell filopodia were detected, and the signaling pathways involved in human intestinal α-defensin 5-induced cytoskeletal rearrangement were verified; S5, based on the validation of the signaling pathway involved in the cytoskeletal rearrangement induced by human intestinal α-defensin 5, cells were treated with specific inhibitors of upstream proteins in the signaling pathway that promotes cytoskeletal rearrangement by human intestinal α-defensin 5, the effects of the inhibitors on cell filopodia were detected, and the types of receptor proteins associated with human intestinal α-defensin 5 signaling were determined; S6, based on the identification of receptor protein types associated with human intestinal α-defensin 5 signaling, human intestinal α-defensin 5 receptor P2Y11 was screened out through cell transcriptome sequencing analysis.

2. A method for screening human intestinal α-defensin 5 receptor P2Y11 according to claim 1, characterized in that: In S1, the automatic characterization and quantification of filopodia produced by treated cells was performed using an open source algorithm of ImageJ.

3. A method for screening human intestinal α-defensin 5 receptor P2Y11 according to claim 1, characterized in that S2 include: S21, cells were treated with human intestinal α-defensin 5 and a broad-spectrum inhibitor of G protein-coupled receptors to obtain treated cells; S22, cells after lysis treatment, centrifugation to obtain supernatant, protein alkylation treatment, protein precipitation, protein enzyme digestion, desalting and enrichment to obtain lysed cells; S23, liquid chromatography-mass spectrometry analysis of protein phosphorylation levels in cells after lysis, identification of phosphorylated proteins and phosphorylation sites, and cell component enrichment analysis using the GO database to screen intracellular proteins with significantly increased phosphorylation levels.

4. A method for screening human intestinal α-defensin 5 receptor P2Y11 according to claim 1, characterized in that: In S3, the human phosphorylation kinase solid phase antibody chip is an R&D SystemsTM human phosphorylation kinase solid phase antibody chip.

5. A method for screening human intestinal α-defensin 5 receptor P2Y11 according to claim 1, characterized in that: In S5, the specific inhibitor of the upstream protein of the pathway in which human intestinal α-defensin 5 promotes cytoskeleton rearrangement is NF449 or PTX.

6. A method for screening human intestinal α-defensin 5 receptor P2Y11 according to claim 1, characterized in that: In S6, the human intestinal α-defensin 5 receptor P2Y11 was screened out by cell transcriptome sequencing analysis, TPM was used to measure the gene expression level, genes with TPM>1 were screened, and the intersection was integrated with the genes with cell nTPM>1 in the Human Protein Atlas database to screen out the human intestinal α-defensin 5 receptor P2Y11.

7. The method for screening human intestinal α-defensin 5 receptor P2Y11 according to claim 1, characterized in that: The method also includes the step of identifying the human intestinal alpha defensin 5 receptor P2Y11.

8. The method for screening human intestinal α-defensin 5 receptor P2Y11 according to claim 7, characterized in that: The identification of the human intestinal α-defensin 5 receptor P2Y11 is carried out by using a bioinformatics integrated biochemical method, including: constructing biotin-labeled human intestinal α-defensin 5, pulling down human intestinal α-defensin 5 with streptavidin magnetic beads to bind human intestinal α-defensin 5 to its potential receptor, and verifying it with a specific antibody for P2Y11, and simultaneously searching the library by mass spectrometry to find the peptide fragment of the human intestinal α-defensin 5 receptor P2Y11, confirming that P2Y11 is the human intestinal α-defensin 5 receptor.

9. A platform for implementing the method for screening human intestinal α-defensin 5 receptor P2Y11 according to any one of claims 1 to 8, characterized in that: The platform includes: The cell culture and processing analysis module is used to provide and maintain optimal cell growth conditions, process cells and observe cell responses, and preliminarily screen the receptor types of human intestinal α-defensin 5; Protein phosphorylation analysis module, used to detect the phosphorylation level of intracellular proteins and identify phosphorylated proteins and phosphorylation sites; Kinase phosphorylation level detection module, used to screen kinases involved in defensin receptor signal transduction and their related signal pathways; Signaling pathway and receptor protein verification module, which is used to verify the involvement of upstream proteins and signaling pathways in the cytoskeleton rearrangement induced by human intestinal α-defensin 5, and to determine the type of receptor protein associated with it; The transcriptome sequencing and analysis module is used for transcriptome sequencing, analyzing changes in gene expression levels, and screening out human intestinal alpha defensin 5 receptor.

10. Use of the platform according to claim 9 in screening defensin receptors and drug targets, characterized in that: The defensin receptor includes any one of an intestinal defensin receptor, a respiratory defensin receptor and a pathogen-associated defensin receptor, and the drug target is a defensin receptor as a drug target.

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