FXR interaction protein analysis strategy based on double-ligand driving and application of FXR interaction protein analysis strategy

Abstract

The invention discloses discovery of more FXR protein interaction information in primary cells on the basis of protein interaction driven by double ligands in combination with a co-immunoprecipitation Co-IP and nano LC-MS combined method. By researching other protein molecules interacting with the FXR, the FXR can be used for analyzing new biological functions.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to analyzing the interacting proteins of FXR under dual ligand drive. Background Art

[0002] Proteins in organisms can exert their biological functions by interacting with other protein molecules. Therefore, studying protein-protein interactions is of great significance for clarifying the biological functions of proteins and their mechanisms of action at the molecular and cellular levels, and helps to understand the regulatory mechanisms of the body's health and disease states.

[0003] Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. It is closely related to bile acid homeostasis, glucose and lipid metabolism, and inflammatory response. It is one of the important drug targets for many metabolic diseases. Therefore, in-depth research on the transcriptional regulation mode and rules of FXR is crucial. Nuclear receptors generally function in the form of homodimers or heterodimers. For example, FXR forms a heterodimer with retinal X receptor RXR and binds to DNA response elements FXREs. The selective recruitment of cofactors by different ligands is the main mechanism of FXR. In the absence of ligand, nuclear receptor dimers may bind to corepressors, thereby inhibiting their transactivation properties. In the presence of ligand, there may be another activation pathway, such as phosphorylation, the corepressor is released, and the coactivator is recruited, allowing interaction with the transcription initiation complex and local histone modifications, ultimately triggering or enhancing transcription. These cofactors are shared by many transcription factors, including nuclear receptors, and they may play a key role in the integration and coordination of the responses of multiple genes to various signals.

[0004] FXR is a receptor for bile acids, which are a family of atypical steroids produced by the liver. There are two main bile acid families in the human body, including the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) produced from cholesterol, and the secondary bile acids lithocholic acid (LCA) and deoxycholic acid (DCA) produced from CA and CDCA. The ability of bile acids to activate FXR is ranked as follows: CDCA>DCA>LCA>CA. Bile acids act as signaling molecules to regulate their own synthesis and affect various pathophysiological processes, such as liver regeneration, pro-apoptosis, and pro-inflammatory effects. α- and β-Muricholic acid (MCA) are produced by CDCA in the liver and are antagonists of FXR. Increased tauro-β-MCA inhibits FXR signaling, FGF15 expression, and ceramide synthesis. Obeticholic acid (OCA) is a semisynthetic derivative of CDCA that is being developed for the treatment of a variety of liver diseases, including biliary atresia, PBC, NASH, and primary sclerosing cholangitis. Since the US FDA approved OCA as a second-line treatment for primary biliary cholangitis in 2016, more and more small molecule drugs targeting FXR have been developed and entered clinical trials. Therefore, the discovery and identification of FXR interacting proteins induced by ligands is of great significance for studying the function of FXR and drug development. The function of FXR is regulated by multiple ligands, and dual ligand induction can simulate the synergistic effects of multiple signaling pathways under physiological conditions, thereby more comprehensively revealing the complex regulatory mechanism between FXR and interacting proteins. Summary of the invention

[0005] The purpose of the present invention is to provide potential interacting protein types that are unique to FXR in a dual-ligand environment. The present invention combines immunoprecipitation Co-IP with proteomic detection methods, and with the advantages of high sensitivity and high-throughput detection of mass spectrometry, can be used to analyze the protein interaction information of FXR driven by dual ligands, compare the differences in protein interaction information driven by a single ligand, and provide a reference for the study of FXR function and mechanism of action.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a method based on the combination of co-immunoprecipitation (Co-IP) and nano-liquid chromatography tandem mass spectrometry (nanoLC-MS), comprising the following steps:

[0008] (1) Pre-treating the cell sample;

[0009] (2) incubating the magnetic beads with the antibody and then incubating with the sample pretreated in step (1);

[0010] (3) SDS-PAGE analysis after incubation;

[0011] (4) The sample from step (3) is subjected to disulfide bond reduction and alkylation, as well as in-gel digestion, and the interacting proteins are analyzed by nanoLC-MS.

[0012] In step (1), the cells are pretreated by blowing off the cells after trypsin digestion, centrifuging at 8000 rpm for 5 min to obtain a cell precipitate, and lysing to obtain a cell lysate protein;

[0013] In step (2), the magnetic beads and the antibody are incubated overnight at 4°C with a flip-over incubation;

[0014] In step (2), the incubation method with the sample is to incubate overnight at 4°C with a flip-over;

[0015] In step (3), SDS-PAGE analysis was performed using Coomassie Brilliant Blue staining solution.

[0016] The second object of the present invention is to provide the application of the aforementioned method in analyzing single-ligand and / or dual-ligand driven FXR interacting proteins.

[0017] Beneficial effects:

[0018] (1) The present invention obtains the FXR interacting protein group under different ligand conditions by combining Co-IP and nanoLC-MS technology.

[0019] (2) The present invention compares the results of single-ligand and dual-ligand interactions to analyze and obtain the protein types unique to the dual-ligand conditions, and conducts biological function analysis on this basis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Heat map of non-labeled quantitative analysis for protein identification. Figure 1 A is the heat map of non-labeled quantitative analysis of FXR interacting proteins identified under single ligand (OCA, CDCA, T-β-MCA) conditions; Figure 1 B is the heat map of non-labeled quantitative analysis of FXR interacting proteins identified under dual ligand (OCA:T-β-MCA and CDCA:T-β-MCA) conditions.

[0021] Figure 2 This is a statistical chart of GO functional annotation of FXR-specific interacting proteins under dual ligand conditions versus single ligand conditions. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0023] The experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the raw material or product manufacturers. The reagents without specific sources are conventional reagents purchased from the market.

[0024] Example 1 Results of single ligand-driven FXR interacting protein Co-IP

[0025] (1) Mouse primary hepatocytes were cultured in DMEM medium containing OCA (5 μM), CDCA (100 μM), and T-β-MCA (100 μM) at 37°C and 5% CO2 in a cell culture incubator. They were lysed on ice for 30 min using IP lysis buffer (purchased from ThermoFisher Scientific) containing 1× protease inhibitor (obtained by diluting 50× protease inhibitor with PBS) and phosphatase inhibitor (50× protease inhibitor and phosphatase inhibitor purchased from Bio-Tech Biotech Co., Ltd.). The cells were inverted every 10 min to obtain mouse primary hepatocyte lysate, and then centrifuged at 13,000 g for 10 min to remove cell debris. The supernatant was obtained to obtain the cell lysate protein. The total protein concentration was determined using the BCA method. The protein concentration was adjusted to 1.5 mg / ml, and 400-500 μg of protein was taken for the experiment.

[0026] (2) Add the diluted antibody to the pretreated magnetic beads (purchased from ThermoFisher Scientific), suspend thoroughly, incubate overnight at 4°C using a flip mixer, and magnetically separate and discard the supernatant to obtain the antibody-magnetic bead complex. Add the lysate protein to the antibody-magnetic bead complex, suspend thoroughly, incubate the sample overnight at 4°C using a flip mixer, magnetically separate and discard the supernatant, and wash the magnetic beads with 200μL Washing buffer. Magnetic separation and discard the supernatant, add 15μL Elutionbuffer and 5μL 4×SDS-PAGE Loading buffer, suspend thoroughly, boil for 10 minutes, magnetically separate, and collect the supernatant for SDS-PAGE analysis.

[0027] (3) After the separation gel strips are washed with ultrapure water, an appropriate amount of Coomassie Brilliant Blue staining solution is added and incubated on a shaker for 2 h until the color of the gel strips is close to that of the staining solution. Ultrapure water is used for decolorization until the blue background of the gel strips is mostly removed.

[0028] (4) Cut the strip into 1mm pieces 3The size of the micelles was washed with ultrapure water and then decolorized with acetonitrile: water (50:50, volume ratio). 10mM DTT and 55mM IAA were added in turn for disulfide bond reduction and alkylation, and then enzymatically digested in 25mM NH4HCO3 system overnight. The micelles after in-gel enzymatic digestion were added with extracting solution (5% FA:ACN=1:2) to extract peptides. After desalting, the peptide mixture was detected and analyzed by nanoLC-MS. The enzymatic products after Co-IP were separated by Easy-nLC 1200 (Thermo Scientific) liquid phase system. The chromatographic column used was C18 column (Acclaim PepMapTM RSLC, 75μm×25cm); mobile phase A was water containing 0.1% formic acid, and mobile phase B was 80% acetonitrile; the flow rate was 0.3μL / min. The gradient elution program was 3-8% B (2 min), 8-28% B (2-28 min), 28-38% B (28-58 min), 38 -100% B (58-61 min), and finally maintained at 100% B (61-71 min). Orbitrap Eclipse Tribrid mass spectrometry was used to detect the enzymatic hydrolysis products, and DDA mode was used for data acquisition. The main mass spectrometry parameters were: primary m / z acquisition range of 350-2000; RFLens of 50%; minimum acquisition signal intensity of 5e 4 ; Peptide charge range is 1-6; primary resolution is 120000; secondary resolution is 30000; fragmentation mode is HCD; Data Dependent mode is Cycle Time; scanning interval is 3s. Mass spectrometry data were identified using PEAKS Studio software (version 8.0), and the parameters were set as follows: the mass error range of the parent ion was set to 10ppm; the mass error range of the HCD fragments was set to 0.02Da; Trypsin was selected for the enzyme setting. The post-translational modification types of proteins were set to include: fixed modification at the cysteine ​​site (C, Carbamidomethylation, +57.02Da), and variable oxidation modification at the methionine site (M, Oxidation, +15.995Da). The Nuclear Receptor FASTA library downloaded from the Uniprot proteome database was searched. The screened proteins were subjected to non-standard quantitative analysis, and the results are shown below. Figure 1 As shown in A.

[0029] Example 2 Results of dual ligand-driven FXR interacting protein Co-IP

[0030] Mouse primary hepatocytes were cultured in DMEM medium containing dual ligands OCA (5 μM) and T-β-MCA (100 μM), CDCA (100 μM) and T-β-MCA (100 μM) respectively, at 37°C and 5% CO2 in a cell culture incubator. The same experimental conditions were used as in Example 1, and the same nanoLC-MS method was used for enzymatic product separation and data acquisition as in Example 1. The PEAKS Studio software parameter settings were the same as those for protein identification in Example 1. The screened proteins were subjected to non-standard quantitative analysis, and the results were as follows: Figure 1 As shown in B.

[0031] The results of Example 1 and Example 2 were combined to analyze the unique interacting proteins of FXR in the dual ligand group, as shown in Table 1.

[0032] Table 1 Basic information of unique FXR interacting proteins detected under dual ligand conditions

[0033]

[0034] Figure 2 The GO functional annotation results of the unique FXR interacting proteins under dual ligand conditions were obtained through the DAVID website. The analysis results showed that the unique FXR interacting proteins under dual ligand conditions were mainly involved in biological processes such as RNA polymerase II transcriptional regulation, adipose tissue development regulation, and estrogen receptor signaling pathway; cellular component enrichment showed that they mainly constituted chromatin, transcriptional repressor complexes, and protein-containing complexes; molecular function enrichment was mainly involved in protein binding, protein-arginine methyltransferase activity, and histone H3R2 and H3R8 methyltransferase activity.

[0035] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. The Co-IP and nanoLC-MS detection technology includes the following steps: (1) Pre-treating the cell sample; (2) incubating the magnetic beads with the antibody and then incubating with the sample pretreated in step (1); (3) SDS-PAGE analysis after incubation; (4) The sample from step (3) is subjected to disulfide bond reduction and alkylation, as well as in-gel digestion, and the interacting proteins are analyzed by nanoLC-MS.

2. The method according to claim 1, characterized in that In step (1), the cells are pretreated by blowing off the cells after trypsin digestion, centrifuging at 8000 rpm for 5 minutes to obtain a cell precipitate, and then lysing to obtain a cell lysate protein.

3. The method according to claim 1, characterized in that In step (2), the magnetic beads and antibodies are incubated overnight at 4°C with a flip-over incubation.

4. The method according to claim 1, characterized in that In step (2), the incubation method with the sample is to incubate overnight at 4°C with a flip.

5. The method according to claim 1, characterized in that: In step (3), the SDS-PAGE analysis is processed using Coomassie Brilliant Blue staining solution.

6. Use of the method of claim 1 in analyzing FXR interacting proteins driven by single ligand and / or dual ligands.

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