Screening identification method, platform and application of a receptor P2Y11 of human intestinal alpha defensin 5

Abstract

The application discloses a screening method, platform and application of human intestinal alpha defensin 5 (HD5) receptor P2Y11, and belongs to the field of biological medicines. The method fuses multiple omics and experimental strategies, effectively solves the problems of complex receptor proteins and difficult independent identification of interactions, significantly improves the accuracy and reliability of defensin receptor identification, and provides a method system with strong universality and wide applicability for receptor research. The method successfully identifies the first cell receptor P2Y11 of HD5. The discovery of the P2Y11 receptor not only opens up a new perspective for in-depth understanding of the biological function and mechanism of HD5, but also provides a new potential target for the treatment of related diseases. In particular, for infectious diseases such as bacterial dysentery caused by Shigella and the like in which HD5 is involved, the P2Y11 receptor as a drug target provides a possibility for developing a new treatment scheme, and is expected to bring more effective treatment options for patients, and significantly improve the treatment effect and the quality of life.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to a screening and identification method, platform and application of a receptor P2Y11 of human enteric alpha defensin 5. BACKGROUND

[0002] Defensins are a class of cationic small molecule polypeptides rich in beta-sheet structure, belong to antimicrobial peptide molecules existing in all biological categories, and are a conservative component in the innate immune system of organisms. In the human body, human alpha-defensin 5 (HD5) belongs to enteric defensins. In recent years, studies have shown that, contrary to its immune effects of resisting viruses and bacteria, part of human alpha-defensin can promote the infection of viruses and pathogenic bacteria to host cells under certain physiological conditions, and plays a "double-edged sword" role in the immune process of the host. For the study of human alpha-defensin 5 participating in the process of Shigella infection of intestinal epithelial cells, it is found that HD5 promotes the invasion and infection process of Shigella by inducing rapid rearrangement of the host cell skeleton network to produce a large number of pseudopod-like structures to participate in the capture of Shigella. This is a new mode of host cell regulation of Shigella infection. However, the cell receptor to which HD5 is combined behind this mode is not clear.

[0003] Most of the receptors of the defensin family known so far belong to G protein-coupled receptors (GPCRs), such as the receptors of human beta-defensin are CCR6 and CCR2, and the receptor of mouse beta-defensin is Mrgpra2, and there is no report on the receptor of alpha-defensin. The reason is that the defensin receptor is difficult to identify by traditional biological methods, the types of proteins combined with human alpha-defensin are complex, and it is difficult to determine which one of them plays a role. Secondly, the reported identification methods of beta-defensin receptors and other receptors are based on molecular biology experiments, and the use of inhibitors for screening has strong randomness, and it is difficult to form a universal identification system for application to other receptor screening and identification research. Thirdly, many ligand receptors belong to GPCRs, and GPCRs are a complex and diverse family with complexity in structure, signal transduction and interaction, and often interact with other proteins such as chaperones, other receptors, etc., making it more difficult to identify the specific types and functions of GPCRs alone. SUMMARY

[0004] In view of the complicated identification method of defensin receptors, the strong randomness and the identification difficulty caused by the diversity of GPCR receptor family, especially the current technical situation of unknown alpha-defensin receptors, the application aims to provide a screening and identification method, platform and application of the receptor P2Y11 of human intestinal alpha-defensin 5, and innovatively establishes a defensin receptor screening method and screening platform, first discloses the alpha-defensin 5 receptor P2Y11, provides new insights for the recognition of pathogens and immune response mechanism of the body, and is expected to develop a new strategy for the treatment of bacterial dysentery and highly infectious bacterial infection, which has great scientific significance and application prospect.

[0005] The application solves the above technical problems by the following technical means:

[0006] The application provides a screening method of the receptor P2Y11 of human intestinal alpha-defensin 5, which comprises the following steps:

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

[0008] S2, based on the preliminary screening of the receptor type of human intestinal alpha-defensin 5, detecting the phosphorylation level of intracellular proteins in the treated cells by liquid chromatography-mass spectrometry, identifying the phosphorylated proteins and phosphorylation sites, and screening intracellular proteins with significantly improved phosphorylation levels by GO function enrichment analysis;

[0009] S3, based on the screening of intracellular proteins with significantly improved phosphorylation levels, detecting the phosphorylation level of kinases in the treated cells by using a human phosphorylated kinase solid-phase antibody chip, screening the kinases involved in the signal transduction of human intestinal alpha-defensin 5 and the signal pathways related to the kinases by gray scale analysis;

[0010] S4, based on the screening of the kinases involved in the signal transduction of human intestinal alpha-defensin 5 and the signal pathways related to the kinases, treating cells with inhibitors of the kinases-related signal pathways, detecting the effect of the inhibitors on the filopodia of the cells, and verifying the signal pathways involved in the cytoskeleton rearrangement induced by human intestinal alpha-defensin 5;

[0011] S5, based on the verification of the signal pathways involved in the cytoskeleton rearrangement induced by human intestinal alpha-defensin 5, treating cells with a specific inhibitor of the upstream protein of the signal pathway promoting the cytoskeleton rearrangement of human intestinal alpha-defensin 5, detecting the influence of the inhibitor on the filopodia of the cells, and determining the receptor protein type related to the signal transduction of human intestinal alpha-defensin 5;

[0012] S6, based on the determination of the receptor protein type related to the signal transduction of human intestinal alpha-defensin 5, screening the receptor P2Y11 of human intestinal alpha-defensin 5 by cell transcriptome sequencing analysis.

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

[0014] Further, the length, number and perimeter of the filopodia of the cells are automatically quantified; the cells are adherent cells or suspension cells.

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

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

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

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

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

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

[0021] S2 comprises:

[0022] S21, treating the cells with human intestinal alpha defensin 5 and G protein-coupled receptor broad-spectrum inhibitor to obtain treated cells;

[0023] S22, lysing the treated cells, centrifuging to obtain supernatant, protein alkylation treatment, protein precipitation, protein digestion, desalting and enrichment to obtain lysed cells;

[0024] S23, analyzing the protein phosphorylation level in the lysed cells by liquid chromatography-mass spectrometry, identifying the phosphorylated proteins and phosphorylation sites, and using GO database for cellular component enrichment analysis to screen intracellular proteins with significantly increased phosphorylation level.

[0025] Further, in S22, the protein alkylation treatment is incubated with dithiothreitol (DTT), iodoacetamide (IAA) and dithiothreitol (DTT) at room temperature for 45-60 minutes.

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

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

[0028] In S3, the gray scale analysis adopts ImageJ software, and the kinase involved in the signal transduction of human intestinal alpha defensin 5 is a kinase with a significantly increased phosphorylation level; and the related signal pathway is a cAMP / PKA signal pathway.

[0029] In S4, the signal pathway involved in the cytoskeleton rearrangement induced by HD5 is a corresponding signal pathway of an inhibitor capable of significantly inhibiting the formation of cell filopodia promoted by human intestinal alpha defensin 5.

[0030] In S5, the specific inhibitor of the upstream protein of the human intestinal alpha defensin 5 cell cytoskeleton rearrangement pathway is NF449 or PTX.

[0031] In S6, the human intestinal alpha defensin 5 receptor P2Y11 is screened by cell transcriptome sequencing, the gene expression level is measured by TPM, the gene with TPM>1 is screened, and the genes with nTPM>1 in the Human Protein Atlas database are integrated to obtain the intersection, and the human intestinal alpha defensin 5 receptor P2Y11 is screened.

[0032] The method further comprises a step of identifying the human intestinal alpha defensin 5 receptor P2Y11.

[0033] The identification of the human intestinal alpha defensin 5 receptor P2Y11 adopts bioinformatics integrated biochemical methods, comprising: constructing biotin-labeled human intestinal alpha defensin 5, using streptavidin magnetic beads to pull down the human intestinal alpha defensin 5 to combine with its potential receptor, and verifying with a specific antibody of P2Y11, while mass spectrometry target search library is used to find the peptide segment of the human intestinal alpha defensin 5 receptor P2Y11, to confirm that P2Y11 is the human intestinal alpha defensin 5 receptor.

[0034] The present application provides a platform for realizing the screening method of the human intestinal alpha defensin 5 receptor P2Y11, and the platform comprises:

[0035] A cell culture and processing analysis module is used to provide and maintain the optimal growth conditions of cells, process the cells and observe the cell response, and preliminarily screen the receptor type of human intestinal alpha defensin 5;

[0036] A protein phosphorylation analysis module is used to detect the phosphorylation level of proteins in cells, identify phosphorylated proteins and phosphorylation sites;

[0037] A kinase phosphorylation level detection module is used to screen the kinase involved in the defensin receptor signal transduction and the signal pathway related thereto;

[0038] A signal pathway and receptor protein verification module is used for verifying upstream proteins and signal pathways involved in human intestinal alpha defensin 5 induced cytoskeleton rearrangement process, and determining the types of receptor proteins related thereto;

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

[0040] The above platform is applied to screening of defensin receptors and drug targets, the defensin receptors including any one of intestinal defensin receptors, respiratory tract defensin receptors and pathogen associated defensin receptors, and the drug targets being defensin receptors as drug targets.

[0041] Compared with the prior art, the present application has the following technical effects:

[0042] The screening method of the human intestinal alpha defensin 5 receptor P2Y11 provided by the present application combines multi-omics analysis and molecular experimental strategies to form a set of receptor screening and identification method from “function-protein-gene” reverse protein translation. The method combines multi-omics and experimental strategies, and through a variety of technical means such as cell processing, phosphorylated protein detection, kinase phosphorylation level analysis, signal pathway verification and transcriptome sequencing, the accuracy and reliability of the screening are ensured, the problems of complex receptor proteins and difficult independent identification of interactions are avoided, and compared with the existing defensin receptor identification method, the screening method has higher accuracy and reliability, and provides a method system that can be widely used for receptor identification. Through the method of the present application, the first HD5 cell receptor P2Y11 is successfully found, which is a major breakthrough in the field of defensin receptor research.

[0043] Further, the open-source algorithm of ImageJ is used to automatically characterize and quantify the cell filopodia, realizing high-throughput and high-precision preliminary screening, which not only improves the screening efficiency, but also ensures the accuracy and consistency of the data; through liquid chromatography-mass spectrometry technology, the phosphorylation level of the intracellular protein after treatment can be accurately detected, and the phosphorylated protein and phosphorylation site can be identified, providing reliable data for subsequent GO function enrichment analysis; the R&D SystemsTM human phosphorylated kinase solid-phase antibody chip is used for detecting the kinase phosphorylation level, ensuring the consistency and repeatability of the experiment; the human phosphorylated kinase solid-phase antibody chip and gray scale analysis are used to screen the kinase involved in human intestinal alpha defensin 5 signal transduction and the signal pathway related thereto, which not only reveals the molecular mechanism of HD5 signal transduction, but also provides potential targets for subsequent drug research and development; through the cell transcriptome sequencing technology, the HD5 receptor P2Y11 is screened out, and the TPM is used to measure the gene expression level, and the gene is compared with the gene in the Human Protein Atlas database, thereby improving the accuracy and reliability of the screening; the identification steps of the receptor P2Y11 ensure the accuracy and reliability of the screened receptor protein, and provide a solid foundation for subsequent functional research and drug research and development.

[0044] The platform provided by the application integrates cell culture and processing, protein phosphorylation analysis, kinase phosphorylation level detection, signal pathway and receptor protein verification, and transcriptome sequencing and analysis, realizes whole-process coverage from cell processing to gene expression analysis, and greatly improves the 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, which can accurately detect the phosphorylation level of intracellular protein and the activity of kinase; 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, which can accurately detect the phosphorylation level of intracellular protein and the activity of kinase; the design of the platform allows flexible adjustment according to experimental requirements, such as replacing processing reagents and adjusting detection conditions, to adapt to 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 possible new defensin receptors and signal pathways in the future; the platform is equipped with a powerful data management system and analysis software, which can integrate data from different functional modules, perform comprehensive analysis, and improve the accuracy and reliability of the screening results.

[0045] The application provided by the application can be used for screening not only human intestinal alpha defensin 5 receptor P2Y11, but also other intestinal defensin receptors, respiratory tract defensin receptors and pathogen-related defensin receptors, and greatly widens the screening range. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Figure 1 is the Filoquant algorithm quantification results of HeLa cell filopodia under the action of HD5, wherein A is the Filoquant algorithm quantification results of the filopodia density, average length and filopodia generation ability of HeLa cells treated with HD5 and the control group, B is the HeLa cell skeleton immunofluorescence images treated with HD5 and Suramin, and C is the Filoquant algorithm quantification results of the cell filopodia generation ability of the HD5 treatment group, Suramin treatment group and control group;

[0047] Figure 2 Figure 2 is the HeLa cell phosphoproteomics results of the application, wherein A is the HeLa cell phosphoprotein results of the HD5 treatment group, Suramin treatment group and control group, B is the cell component bubble chart of GO function enrichment analysis, and C is the molecular function bubble chart of GO function enrichment analysis;

[0048] Figure 3 Figure 3 is the HD5-treated HeLa cell phosphokinase chip result of the application, wherein A is the HD5-treated epithelial cell phosphokinase chip, and B is the kinase with increased phosphorylation level after HD5 treatment;

[0049] Figure 4 Figure 4 is the Filopodia algorithm quantification results of the filopodia growth ability of different signal pathway inhibitor treatment groups of the application;

[0050] Figure 5 Figure 5 is the laser confocal fluorescence photos and cell filopodia generation ability Filoquant algorithm quantification of HeLa cells treated with Gi and Gs protein inhibitors of the application, wherein A is the laser confocal fluorescence photo, and B is the cell filopodia generation ability Filoquant algorithm quantification;

[0051] Figure 6 Figure 6 is the transcriptome analysis of GPCR expression in the cell line of the application, wherein A is the nTPM value of Gs-GPCR gene in the HPA database, B is the TPM value of Gs-GPCR gene in the test cell line, and C is the process diagram of screening P2Y11 using transcriptome data and Reactome (human protein atlas database);

[0052] Figure 7 For the P2Y11 receptor identified in the present application, A is the immunoblot analysis of the protein sample pulled down by the P2Y11 antibody against Biotin-HD5, and B is the mass spectrometry-based P2Y11 targeting peptide segment search;

[0053] Figure 8 The Filopodia algorithm quantification result of the ability of the NF157 of the present application to inhibit filopodia growth. DETAILED DESCRIPTION

[0054] To make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0055] The test materials and reagents used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0056] The present application provides a multi-omics screening and experimental identification method of human intestinal alpha defensin 5 receptor P2Y11. In the present application, it is considered that the defensin receptors reported so far all belong to GPCRs, therefore Suramin (a broad-spectrum inhibitor of GPCRs) is used to initially explore the receptor of HD5, and the Filoquant algorithm is used to provide the statistical difference of the pseudopod generation ability, so as to prove that the HD5 receptor belongs to GPCR. The process of HD5 promoting cytoskeleton rearrangement is very rapid, and the protein phosphorylation modification of cells is a way of short-time scale signal transmission, therefore a part of proteins with large difference in protein phosphorylation level in the presence of HD5 signal is screened through the phosphorylation proteome screening, and the molecular functions and component elements of these proteins are analyzed by GO enrichment. Combined with the phosphorylation level screening of protein kinases, the cAMP-PKA signal pathway activated by HD5 is screened out. The inhibitors are used to screen the Gs-GPCR or Gi-GPCR coupled upstream of the cAMP-PKA pathway, so as to narrow the range to Gs-GPCR. Finally, through the transcriptome level of different cells after HD5 treatment, combined with the Human Protein Atlas database, a GPCR coupled with Gs protein, P2Y11, is screened out. It is verified by the immuno-precipitation-mass spectrometry combination that there is P2Y11 peptide segment in the protein pulled down after the interaction of HD5 and cells. After identifying the receptor, a specific drug NF157 targeting HD5 to promote bacterial invasion of the host can be found, and the NF157 can eliminate the generation of the filopodia promoted by HD5.

[0057] 1. Morphological characterization of cell filopodia

[0058] (1) Adherent and suspension cell culture

[0059] Culture condition of adherent cells (HeLa, Hep-G2, MS751, SiHa): DMEM low glucose medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin double antibiotic was used, and the cells were cultured in a 37°C, 5% carbon dioxide incubator. When cell plating was performed, 24-well plates were used, 4x10 5 cells / well, and the culture conditions were the same as above. When cell starvation treatment was performed, DMEM low glucose medium without serum and double antibiotic was used, and the cells were cultured in a 37°C, 5% carbon dioxide incubator. Culture condition of suspension cells (THP-1, Jurkat, K562, U937): RPMI medium containing 10% fetal bovine serum, 1% penicillin-streptomycin double antibiotic was used, and the cells were cultured in a 37°C, 5% carbon dioxide incubator. When cell plating was performed, no special requirements were needed, and the culture conditions were the same as above. When cell starvation treatment was performed, RPMI medium without serum and double antibiotic was used, and the cells were cultured in a 37°C, 5% carbon dioxide incubator.

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

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

[0062] (3) Cell skeleton immunofluorescence staining

[0063] After a certain period of treatment, the culture medium was removed, 4% paraformaldehyde was added to fix the cells at room temperature for 20 minutes, and PBS was used for washing 3 times. 1% Triton X-100 diluted with PBS solution was used for room temperature punching for 5 minutes, and PBS was used for washing 3 times. Rhodamine fluorescently labeled phalloidin was used for light-avoiding staining of the microfilament structure of the cells for 20 minutes. After rinsing in deionized water, DAPI-containing anti-fluorescence quenching mounting medium was used for mounting. The sample was placed at 4°C for air drying and storage, and a single cell image with complete edges was taken under a laser confocal microscope.

[0064] (4) Filoquant quantification

[0065] From the initial cell micrograph library, select the single cell and the pseudopod-like structure as a candidate for analysis, import the original 16-bit color depth photo into ImageJ for initial processing, adjust the color depth to 8-bit to meet the accuracy requirements of FiloQuant, manually frame the single cell without interference from other cell images, and use "Single Image FiloQuant" for automatic analysis and processing. Set the cell edge threshold to 6 pixels, the pseudopod length range threshold to 0.5-25 pixels, the processing iteration number to 5, and select the automatic filling of cell edge curve. After processing, the number of pseudopods, the length of individual pseudopods, and the total length of the cell edge of the selected cell can be obtained. The pseudopod generation capacity is the length of the pseudopod that can be generated within the unit length of the cell. The calculation formula is: the sum of the length of individual cell pseudopods / the perimeter of individual cells = the pseudopod generation capacity of individual cells. Randomly select 30 cells and compare the average pseudopod generation capacity of the HD5 group and the Suramin + HD5 group. See Figure 6 for details. Figure 1

[0066] As shown in Figure 6. Figure 1 As shown in Figure 6. As shown in Figure 6.

[0067] In summary, HD5 can stimulate HeLa cells to produce more filopodia and promote the growth of filopodia. Suramin can inhibit the promoting effect of HD5 on filopodia, and the receptor of HD5 is GPCR.

[0068] 2. Signal pathway screening of HD5 regulating host cell cytoskeleton rearrangement

[0069] (1) Phosphorylated protein mass spectrometry

[0070] Cell lysate preparation: The second / third generation of adherent or suspension cells were seeded in six-well plates the day before the experiment. The next day, when the cells were 70% confluent, the medium was removed and the cells were washed 2-3 times with pre-warmed serum-free medium to remove serum. After treatment of the cells with HD5, the medium was removed and the cells were washed with ice-cold PBS. Lysis buffer was added and the cells were lysed by repeated pipetting. The supernatant was collected by centrifugation at 4°C.

[0071] Protein alkylation: Dithiothreitol (DTT) stock solution was added to the supernatant sample to a final concentration of 10 μΜ, and the sample was vortexed and incubated at room temperature for 45-60 minutes on a rotating incubator. Iodoacetamide (IAA) stock solution was added to a final concentration of 40 μΜ, and the sample was vortexed and incubated at room temperature for 45-60 minutes on a rotating incubator. DTT stock solution was added again to a final concentration of 40 μΜ to neutralize the remaining unreacted IAA and terminate the alkylation reaction, and the sample was vortexed and incubated at room temperature for 45-60 minutes on a rotating incubator. The sample was centrifuged at 4°C and the supernatant was collected in a clean tube.

[0072] Protein precipitation: Acetone (precipitation solution) was added to the supernatant at 5 times the original volume, and the sample was incubated at -20°C overnight. After a large amount of flocculent precipitate appeared, the sample was centrifuged at 4°C and the supernatant was discarded. The protein precipitate was resuspended and washed with ice-cold acetone. The sample was centrifuged at 4°C and the supernatant was discarded. The protein precipitate was left to dry at room temperature with the lid open, and the remaining solution was allowed to evaporate. The precipitate was completely dissolved by incubation at 4°C on a rotating incubator for 10-30 minutes using 8 μΜ urea buffer. The urea concentration in the protein extract was diluted to less than 2 M using 100 mM NH4HCO3solution. After the protein was completely dissolved, the sample was centrifuged at 4°C and the supernatant was collected. The protein concentration was determined using the BCA (bicinchoninic acid) method.

[0073] Protein digestion, desalting and enrichment: The amount of protein was 50 times the amount of enzyme, and the protein was digested with the enzyme at 37°C for 16-18 hours. Desalting was performed using 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. In this experiment, the TiO2enrichment method was used to enrich serine and threonine phosphorylated peptides. After enrichment, the sample was desalted again and the polypeptides were collected. 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 Suramin and HD5 co-treatment group. The proteins selected in the above were subjected to GO (Gene Ontology) functional enrichment analysis, as shown in the accompanying Figure 2 .

[0075] By the attached Figure 2 From the data, the protein phosphorylation level of the HD5 treatment group was significantly higher than that of the control group and the Suramin + HD5 treatment group, confirming the significant effect of HD5 on the protein phosphorylation of HeLa cells; the enrichment results of cell components: multiple cell components such as "focal adhesion" (adhesion plaque), "cell-substrate junction" (cell-substrate junction), "Actin cytoskeleton" (actin cytoskeleton), lameliipodium (lameliipodium) and the like were significantly enriched, which are closely related to cell skeleton, cell movement and pseudopod extension and the like, suggesting that HD5 treatment affects the functions of these cell components; "ATP-dependent chromatin remodeler activity" (ATP-dependent chromatin remodeler activity), "protein kinase A regulatory subunit binding" (protein kinase A regulatory subunit binding) and "protein kinase A binding" (protein kinase A binding) and the like were significantly enriched, especially the functions related to "protein kinase A" (PKA) pathway were mentioned multiple times, indicating that the PKA pathway played a dominant role in the rearrangement of the cytoskeleton regulated by HD5.

[0076] In summary, through the GO function enrichment analysis of the screened proteins, the PKA pathway occupies a dominant position in the phosphorylated proteins of HeLa cells treated with HD5; in addition, multiple cell components and molecular functions related to cell skeleton, cell movement and chromosome structure and the like are also significantly enriched, which provides important clues and basis for further understanding the effect of HD5 on the phosphorylated proteins of HeLa cells.

[0077] 3. High-throughput protein chip screening

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

[0079] (2) Assay for cellular phosphorylated kinase content: The human phosphorylated kinase solid-phase antibody chip was placed in a well plate and blocked at room temperature for 1 hour using the kit blocking buffer. The blocking buffer was then removed. Diluted cell lysis buffer was added, and the chip was incubated overnight at 4°C. After incubation, the chip was gently vortexed three times with 1× washing buffer and incubated for 2 hours with diluted detection antibody mixture. The chip was washed again and incubated for 30 minutes at room temperature in the dark with diluted streptavidin-HRP. The chip was then washed, and any remaining buffer at the chip edges was removed with absorbent paper. 1 mL of horseradish peroxidase chemiluminescent solution was evenly dropped onto the chip surface, and the reaction was allowed to proceed for 1 minute before the chemiluminescence level was detected using a chemiluminescence analyzer.

[0080] (3) Gray-scale analysis: ImageJ software was used to perform gray-scale analysis on the chemiluminescence photographs and to quantify and plot the data. See Appendix for details. Figure 3 As shown.

[0081] From the appendix Figure 3 Data shows that by identifying kinases with significantly increased phosphorylation levels after HD5 treatment, Akt 1 / 2 / 3, Erk1 / 2, CREB, and c-Jun were found to be downstream kinase effector molecules of the cAMP / PKA signaling pathway.

[0082] In summary, this experiment successfully determined the content of phosphorylated kinases in cell lysates using high-throughput protein chip technology, and identified kinases whose phosphorylation levels significantly increased after HD5 treatment through grayscale analysis. These kinases were found to be downstream effector molecules 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 cell plating and adhesion, cells were treated with a variety of cell pathway inhibitors and HD5, and the cytoskeleton was stained with immunofluorescence, using the same method as described above.

[0085] Laser confocal microscopy was used to capture fluorescence images of the cytoskeleton, and Filoquant was applied for quantification, using the same methods as described above. See the appendix for specific results. Figure 4 As shown.

[0086] From the appendix Figure 4 Data shows that only cAMP-PKA pathway inhibitors PKI 14-22 and KH7 have significant antagonistic effects, confirming that HD5 promotes cytoskeleton rearrangement by activating the host cell cAMP-PKA pathway.

[0087] 4. Receptor selection for HD5 regulation of host cytoskeleton rearrangement

[0088] (1) Screening of GPCR inhibitors

[0089] The known cellular pathway by which HD5 regulates cytoskeleton rearrangement is cAMP-PKA, upstream of which is a Gs- or Gi-coupled GPCR. Therefore, inhibitors of these two GPCRs were used for screening. After cell plating and adhesion, cells were treated with known Gs and Gi protein inhibitors NF449 and PTX, as well as HD5. Immunofluorescence staining of the cytoskeleton was performed using the same methods as described above.

[0090] Laser confocal microscopy was used to capture fluorescence images of the cytoskeleton, and Filoquant was applied for quantification, using the same methods as described above. See the appendix for specific results. Figure 5 As shown.

[0091] From the appendix Figure 5 Data show that cytopod quantification revealed that only NF449 exhibited significant antagonistic activity, indicating that NF449, as an inhibitor of Gs protein, can effectively block the influence of HD5 on the cytoskeleton through the Gs-GPCR-mediated signaling pathway. In contrast, PTX, as an inhibitor of Gi protein, did not show significant antagonistic activity, suggesting that HD5 does not function through the Gi-GPCR-mediated signaling pathway. Based on these results, it can be concluded that the HD5 receptor is a Gs-coupled GPCR, meaning that HD5 activates the downstream cAMP-PKA signaling pathway by binding to Gs-GPCR, thereby promoting cytoskeleton rearrangement.

[0092] (2) Cell transcriptome sequencing screening

[0093] Cell culture, collection, and transcriptome sequencing: Eight cell lines that responded to HD5 and produced significant pseudopodia 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 after trypsin digestion, centrifugation, and discarding the supernatant. Suspended cells were collected directly after centrifugation and discarding the supernatant, and stored using TRizol reagent. RNA extraction, library construction, and sequencing were then performed sequentially.

[0094] Public database data download and screening: 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 integrated analysis was performed on the transcriptome data of 8 cells cultured, collected and sequenced by ourselves and 10 cells in the database. TPM (Transcripts Per Kilobase Million) was used to measure gene expression level, which was calculated by dividing the read count of each gene by the length of the gene (unit: kilobase), and then dividing the value by the sum of all gene per kilobase read count, and multiplying by one million, so that the TPM value of the gene was obtained. As a result, TPM can make the expression level of genes of different lengths comparable, and it is easier to compare between different samples. For specific results, see FIG. 1. Figure 6

[0095] As shown in FIG. 2, by screening Gs-GPCR genes with TPM > 1 and relatively high gene expression level in transcriptome level, and combining with Gs-GPCR genes with nTPM > 1 in 14 cells (Jurkat, HEK293, SiHa, HeLa, PC-3, A-549, MS751, CACO-2, MCF-7, HT-29, K-562, Hep-G2, THP-1, U-937) in Human Protein Atlas database, only one GPCR coupled with Gs— P2Y11 was finally screened out. Figure 6

[0096] Example 2

[0097] In this embodiment, biotin-HD5 pull-down and mass spectrometry were used to identify the screened HD5 receptor P2Y11 based on Example 1, which specifically included the following steps:

[0098] Lysis of cells: 1 × 10 7 Hela cells were prepared, and GPCR extraction and stabilization reagent containing protease inhibitor was used for lysis. Stable protein receptors were in the supernatant. Protein concentration was detected by Enhanced BCA Protein Assay Kit.

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

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

[0101] After elution, pull down the interacting proteins: 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: After bead reduction, alkylation, and trypsin digestion, the product was desalted using a C18 membrane-packed column and then detected by LC-MS / MS. The presence of the P2Y11 peptide was identified using a protein target search method. See the appendix for detailed test results. Figure 7 As shown.

[0103] From the appendix Figure 7 Data shows that after in vitro pull-down experiments using biotin-bound HD5 (Biotin-HD5), subsequent protein immunoblotting and proteomic analysis yielded a series of significant results. In the protein immunoblotting experiment, when tested with a specific antibody against P2Y11, P2Y11 was clearly present in the positive control group HeLaextract (input). Crucially, P2Y11 was also detected in the proteins pulled down by HD5 (IP group: HeLaextract + Botin-HD5 + SAVBeads). Simultaneously, in more in-depth proteomic targeted library search analysis, the P2Y11 peptide was successfully located. These combined experimental results strongly validate that P2Y11 is the binding receptor for HD5.

[0104] The specific inhibitor NF157 of P2Y11 can eliminate the effect of HD5 in promoting pseudopodia formation.

[0105] P2Y11, as a member of the purinergic receptor family, has significant potential applications in biomedical research. However, the lack of the P2Y11 gene in the mouse genome has led to relatively limited research on its function. This invention successfully identified the HD5 receptor as P2Y11, a discovery that opens new avenues for in-depth research into the function of P2Y11.

[0106] Cells were pretreated with NF157, a known specific inhibitor of P2Y11, and the Filopodia algorithm was used to quantify its ability to inhibit filopodia growth. (See Appendix)Figure 8 As shown. (From the appendix) Figure 8 Data showed that cells treated with HD5 exhibited a significant advantage in pseudopodia growth capacity, which was more pronounced compared to the control group and the NF157-treated group. After NF157 pretreatment, the addition of HD5 to the cell system resulted in pseudopodia generation capacity that was essentially similar to that of the control group; statistical analysis revealed no significant difference between the two. This result indicates that NF157 can eliminate the pseudopodia-promoting effect of HD5, providing a valuable tool for studying the regulatory mechanisms of pseudopodia generation. Furthermore, Filoquant was used to quantify the pseudopodia generation capacity of the NF157+HD5 group and the HD5-treated group, and statistical differences were compared, providing a reliable method for accurately assessing the role of P2Y11 in pseudopodia generation.

[0107] The results of this invention provide a widely applicable method for receptor screening and identification combining multi-omics analysis and molecular experiments from the "function-protein-gene" reverse protein translation process. Specifically, a receptor for human intestinal α-defensin 5, P2Y11, was successfully screened. Furthermore, the method of this invention can also be applied to drug development. P2Y11, as a switch for HD5-regulated cytoskeleton rearrangement, plays a crucial role in HD5's involvement in Shigella infection. By screening for novel inhibitors or agonists targeting P2Y11, drugs with specific therapeutic effects can be developed. Specifically, this provides possibilities for intervention strategies against pathogens that utilize HD5-induced filamentous extension, opening up new perspectives and fields for the application of P2Y11.

[0108] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A screening method for human intestinal alpha defensin 5 receptor P2Y11, characterized by, The method comprises: S1, treating cells with human intestinal alpha defensin 5 and G protein-coupled receptor broad-spectrum inhibitor to obtain treated cells, automatically characterizing and quantifying the filopodia generated by the treated cells, and preliminarily screening the receptor type of human intestinal alpha defensin 5; S2, based on the preliminary screening of the receptor type of human intestinal alpha defensin 5, detecting the phosphorylation level of proteins in the treated cells by liquid chromatography-mass spectrometry, identifying the phosphorylated proteins and phosphorylation sites, and screening intracellular proteins with more than 2-fold increase in phosphorylation level by GO function enrichment analysis; S3, based on screening intracellular proteins with more than 2-fold increase in phosphorylation level, detecting the phosphorylation level of kinases in the treated cells by using human phosphorylated kinase solid-phase antibody chip, screening kinases involved in human intestinal alpha defensin 5 signal transduction and signal pathways related thereto by gray scale analysis; S4, based on screening kinases involved in human intestinal alpha defensin 5 signal transduction and signal pathways related thereto, treating cells with inhibitors of the signal pathways related to the kinases, detecting the effect of the inhibitors on cell filopodia, and verifying the signal pathways involved in human intestinal alpha defensin 5-induced cytoskeleton rearrangement; S5, based on verifying the signal pathways involved in human intestinal alpha defensin 5-induced cytoskeleton rearrangement, treating cells with specific inhibitors of upstream proteins of the signal pathways promoting cytoskeleton rearrangement by human intestinal alpha defensin 5, detecting the effect of the inhibitors on cell filopodia, and determining the receptor protein type related to human intestinal alpha defensin 5 signal transduction; S6, based on determining the receptor protein type related to human intestinal alpha defensin 5 signal transduction, screening human intestinal alpha defensin 5 receptor P2Y11 by cell transcriptome sequencing analysis.

2. The method of screening for human intestinal alpha defensin 5 receptor P2Y11 according to claim 1, wherein, In S1, the automatic characterization and quantification of the filopodia generated by the treated cells uses the open-source algorithm of ImageJ.

3. The method of screening for human intestinal alpha defensin 5 receptor P2Y11 according to claim 1, wherein S2 It comprises: S21, treating cells with human intestinal alpha defensin 5 and G protein-coupled receptor broad-spectrum inhibitor to obtain treated cells; S22, lysing the treated cells, centrifuging to obtain supernatant, alkylating proteins, precipitating proteins, enzymatically digesting proteins, desalting and enriching to obtain lysed cells; S23, analyzing the protein phosphorylation level in the lysed cells by liquid chromatography-mass spectrometry, identifying phosphorylated proteins and phosphorylation sites, and performing cellular component enrichment analysis using the GO database to screen intracellular proteins with more than 2-fold increase in phosphorylation level.

4. The method of screening for human intestinal alpha defensin 5 receptor P2Y11 according to claim 1, wherein, In S3, the human phosphorylated kinase solid-phase antibody chip is an R&D SystemsTM human phosphorylated kinase solid-phase antibody chip.

5. The method of screening for human intestinal alpha defensin 5 receptor P2Y11 according to claim 1, wherein, In S5, the specific inhibitor of the upstream protein of the pathway promoting cytoskeleton rearrangement by human intestinal alpha defensin 5 is NF449 or PTX.

6. The method of screening for human intestinal alpha defensin 5 receptor P2Y11 according to claim 1, wherein, In S6, human intestinal alpha defensin 5 receptor P2Y11 is screened by cell transcriptome sequencing analysis, the gene expression level is measured by TPM, genes with TPM>1 are screened, and genes with cell nTPM>1 in the Human Protein Atlas database are integrated to obtain the intersection, and human intestinal alpha defensin 5 receptor P2Y11 is screened.

7. The screening method for human intestinal α-defensin 5 receptor P2Y11 according to claim 1, characterized in that, The method further comprises a step of identifying human intestinal alpha defensin 5 receptor P2Y11.

8. The screening method for human intestinal α-defensin 5 receptor P2Y11 according to claim 7, characterized in that, The identification of the human intestinal alpha defensin 5 receptor P2Y11 is achieved by bioinformatics and biochemical methods, including: constructing biotin-labeled human intestinal alpha defensin 5, using streptavidin magnetic beads to pull down human intestinal alpha defensin 5 and its potential receptors, and verifying with specific antibodies of P2Y11, while mass spectrometry is used to search for peptide segments of human intestinal alpha defensin 5 receptor P2Y11, confirming that P2Y11 is the human intestinal alpha defensin 5 receptor.

9. A platform for performing the screening method of any one of claims 1 to 8 for the human intestinal alpha defensin 5 receptor P2Y11, characterized in that, The platform comprises: a cell culture and processing analysis module for providing and maintaining optimal growth conditions for cells, processing cells and observing cell responses, and preliminary screening of the receptor type of human intestinal alpha defensin 5; a protein phosphorylation analysis module for detecting the phosphorylation level of intracellular proteins, identifying phosphorylated proteins and phosphorylation sites; a kinase phosphorylation level detection module for screening kinases involved in defensin receptor signaling and related signaling pathways; a signal pathway and receptor protein verification module for verifying upstream proteins and signal pathways involved in human intestinal alpha defensin 5-induced cytoskeleton rearrangement process, and determining the related receptor protein type accordingly; a transcriptome sequencing and analysis module for transcriptome sequencing, analyzing changes in gene expression levels, and screening human intestinal alpha defensin 5 receptors.

10. Use of the platform of claim 9 for screening defensin receptors and drug targets. The defensin receptor includes any one of an intestinal defensin receptor, a respiratory tract defensin receptor, and a pathogen-associated defensin receptor, and the drug target is the defensin receptor as a drug target.

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