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Usage and Biology
Farnesoid X receptor (FXR) is a member of metabolism related nuclear receptor family. FXR agonists have been reported to improve blood glucose levels by increasing insulin sensitivity. However, there are some side effects such as lowering the level of high density lipoprotein (HDL) and aggravating obesity after long-term administration of FXR. The antagonistic FXR can reduce body weight and blood glucose, especially can better control postprandial blood glucose. Therefore, the research of FXR antagonists is also one of the hot spots of anti-metabolic diseases. At present, the effect and mechanism of FXR on glucose metabolism are not clear. Therefore, it is important to screen a small molecule FXR antagonist as a probe to explore the mechanism of FXR regulating glucose metabolism.
Recombinant FXR ligand binding domain production and purification
DNA sequence of the FXR ligand binding domain (FXR-LBD) was cloned to the pET-15b vector and transferred to the BL21 (DE3) competent cells. The transformed cells were inoculated into fresh LB medium for recombinant FXR-LBD production. When the optical density of the culture reached 0.6, IPTG at final concentration of 0.2 mM was added. The expression cells were harvested by centrifugation and broken by sonication on ice. Proteins in supernatant was purified by Ni-NTA column using an AKTA FPLC instrument according to the protocol. The protein production and purification were analyzed by SDS-PAGE. As shown in Figure 1, recombinant FXR-LBD was purified to homogeneity.
Figure 1. SDS-PAGE analysis of recombinant FXR-LBD production and purification. Land 1, 2, 9: protein molecular weight standards. Lane 3: the insoluble proteins in expression cells. Lane 4: the soluble proteins in expression cells. Lane 5: the proteins in the flow through of Ni-NTA column. Lane 6 and 7: eluting fractions of FXR-LBD.
Application of recombinant FXR-LBD for FXR antagonist screening
FXR antagonists screened in Lab in-house compound library by AlphaScreen technology-based assay.
Alphascreen technology is used for in vitro high-throughput detection of protein interactions. In the system we constructed, we used the ability of 6×His-FXR-LBD protein and Biotin-SRC1 small peptide to bind to each other, and added Nickel chelate acceptor beads and Streptavidin donor beads. When protein and small peptide are combined with each other, the distance between the two magnetic beads is close. Under the irradiation of excitation light with wavelength of 680 nm, the photosensitizer on the donor magnetic beads converts the surrounding oxygen into monomer oxygen, and the monomer oxygen diffuses to the receptor. The bulk magnetic beads activate the fluorescent groups on the acceptor magnetic beads. Furthermore, the intensity of the interaction between the protein and the small peptide was characterized by detecting the fluorescence value at the wavelength of 520-620 nm. During the operation, we dissolve 6×His-FXR-LBD protein and SRC1 small peptide on ice, and dilute to a certain concentration with assay buffer (25 mM Hepes, pH 7.4, 100 mM NaCl and 0.1% BSA). Take a white 384 ELISA plate, add 100 nM 6×His-FXR-LBD protein, 30 nM SRC1, 0.5 μM GW4064 (FXR agonist), the selected compound, and 10 μg/mL acceptor beads to each well. Afterwards, shake the plate at 300 rpm for 10 min and incubate at room temperature for 30 min. Then add the final concentration of 10 μg/mL Donor beads 300 rpm shaker for 10 minutes, and incubate for 2 hours at room temperature in the dark. Then use PerkinElmer machine to check the fluorescence value.
As indicated in Figure 2A and 2B, compound H7 antagonized the GW4064-induced promotion of FXR-LBD binding to its coactivator SRC-1. These results thus implied the antagonistic feature of H7 against FXR.
Figure 2. H7 as an FXR antagonist inhibited FXR activity. The effect of compounds (20μM) on the combination of FXR-LBD and SRC-1 was detected by AlphaScreen-based protein-peptide interaction assay. A. Among the compounds, H7 was finally selected for its highly antipathogenic activity against FXR. B. H7 antagonized GW4064-induced AlphaScreen signal. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).
Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. Its typical functional domains include DNA binding domain and ligand binding domain. After binding its ligand, FXR forms a heterodimer with another nuclear receptor retinoid X receptor, and then the receptor dimer binds to the FXR response element (FXRE) located in the promoter region of the FXR target gene, thereby, regulating the transcription of these genes.
Transactivation of FXRE by FXR-RXRα complex
Part BBa_K3522013 consists of a FXRE fragment and luciferase gene. When presence of FXR and RXRα, the FXRE is activated, subsequently, the luciferase gene is transcribed. Such a transactivation assay cam be used to verify the antagonistic effect of H7 on FXR.
Finally, transactivation assay was further carried out to verify the antagonistic effect of H7 on FXR. As shown in Figure 3, FXR agonist GW4064 efficiently activated reporter gene expression, and H7 antagonized GW4064-induced reporter gene stimulation in transactivation assay. Thus, these results confirmed the antagonism of H7 against FXR transactivation activity.
Figure 1. H7 as an FXR antagonist inhibited transactivation activity.
HEK293T cells were transiently transfected with pcDNA3.1a-FXR, pcDNA3.1a-RXRα, pGL3-FXRE-Luc and pRL-SV40. After 6 h, cells were treated with different concentrations of H7 with GW4064 for 24 h. Then transactivation activity of FXR was detected by luciferase reporter assay. GW4064: FXR agonist, GS: FXR antagonist. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).
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