Difference between revisions of "Part:BBa K3522013"

Latest revision as of 10:30, 12 October 2022

FXRa-RXRa-F luc-R luc：a platform to screen a small molecule FXR antagonist

This composite part is a platform to screen a small molecule FXR antagonist.

Sequence and Features

Contribution

Biology 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.

Engineering Success

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

                            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).

Characterization by 2022 iGEM Team Jiangsu_United

Profile

Name: hFoxO1-F.luciferase-R.luciferase

Base Pairs: 6187 bp


Origin: HepG2 cell genome

Properties: expression plasmid that can duplicate both in E.coli and HepG2 cells

Contribution

FoxO1, as a transcription factor, plays an important role in the regulation of blood glucose balance. FoxO1 in the liver can promote the expression of key gluconeogenesis enzyme genes, and an important mechanism of insulin regulation of blood sugar is to increase the phosphorylation of FoxO1, promote its nuclear export, and then reduce its transcriptional activation activity. According to this theory, we constructed a human FoxO1 full-length plasmid, used Firefly luciferase and Renilla luciferase as a reporter gene, established a FoxO1 transcriptional activation screening platform, and used this platform to screen out active compounds that inhibit FoxO1 transcriptional activation activity.

In order to construct a FoxO1 expression plasmid that can shuttle both in E.coli and HepG2 cells, we designed the DNA sequences of hFoxO1 to be inserted into the XhoI and KpnI sites of the pcDNA3.1 vector (Fig.1), and transfected the HepG2 cells with the recombinant plasmid to set up our experiment platform.

Engineering Success

How we design our plasmid

In order to construct a FoxO1 expression plasmid that can duplicate both in E.coli and HepG2 cells, we designed the DNA sequences of hFoxO1 to be inserted into the XhoI and KpnI sites of the pcDNA3.1 vector (Fig.1), and transfect the HepG2 cells with the recombinant plasmid and set up our experiment platform.

Fig. 1 The map of recombinant plasmid pcDNA3.1-hFoxO1..

How we build our plasmid

To build the plasmid, we use PCR to amplify the hFoxO1 gene from template DNA (HepG2 cell genome), and extract the target fragment (Fig.2). At the same time, we did the plasmid extraction to obtain the plasmid pcDNA3.1. The second step was double-enzyme digestion with XhoI and KpnI. The goal of digestion was to get the linearized pcDNA3.1 vector and inserted DNA fragments of hFoxO1. The third step was to ligate the inserts and linearized vector and transfer the ligation product into DH5α competent.

Fig.2 Agarose gel electrophoresis of PCR product...

(A)Lane 1 is the hFoxO1 target band.

We send the constructed recombinant plasmid to a sequencing company for sanger sequencing. The returned sequencing comparison results showed that the plasmid was successfully constructed (Fig.3). Then we extract the recombinant plasmid from E.coli DH5α and transfect it into HepG2 cells to express hFoxO1 proteins.

Fig.3 Agarose gel electrophoresis diagram of the clone. (A) Verify the colony in lanes 1-6 (B) Sequence comparison results of successful gene editing..

How we test our hFoxO1

a) Protein expression and verification

In order to verify if hFoxO1 protein was successfully expressed in HepG2 cells which were transfected with the correct recombinant plasmid after 12 hours, we did Western blot (Fig.4). The result shows that the protein expression level of hFoxO1 increased accompanied by the increase of plasmid concentration, indicating that hFoxO1 was expressed successfully in the cell.

Fig.4 Protein determination of HepG2 cells after transfection. (A) GAPDH was used as control; The numbers on the X-axis represent the different concentrations of hFoxO1 plasmids...

improvement of an existing part

Our composite component, BBa_K4516019, is a platform to screen small molecule FoxO1 antagonists that can be used to treat type 2 diabetes. As early as 2020, the iGEM20_XDFYZ team was committed to developing a drug screening platform (BBa_K3522013) for the treatment of type 2 diabetes. They found candidate drug molecules by screening FXR antagonists. Based on their work, by reading literature and consulting experts in related fields, we found that in addition to the FXR target, there are also a FoxO1 target that plays an important role in the regulation of blood glucose balance. Therefore, we further optimized the diabetes drug screening platform and constructed a new composite part BBa_K4516019 to screen more kinds of drug molecules that can be used to treat type 2 diabetes. In order to prove the function of our new composite part hFoxO1-F. luciferase-R. luciferase, we transferred the recombinant plasmid into HepG2 to establish a transcriptional activation screening platform, and screened a totally new compound 355, which has an inhibitory effect on the transcriptional activation activity of FoxO1. Then, by detecting the effect of 355 active compounds on hepatic gluconeogenesis and the mRNA levels of key enzymes in hepatic gluconeogenesis, it was further confirmed that 355 compounds have the effect of inhibiting hepatic gluconeogenesis, thereby achieving the effect of lowering blood sugar.

Fig5. The blast results about the nucleotide sequence of our new part BBa_K4516019 and the old one BBa_K3522013. ..

Results of Compounds Screening Using this Platform

To regulate the expression of hFoxO1, we used small molecular compounds from the database to examine their effects on gluconeogenesis (Table1). Compound 335 was found to have a significant role in reducing glucose concentrations in HepG2 cells. Table 1. The effects of different small molecular compounds on gluconeogenesis.

Table 1. The effects of different small molecular compounds on gluconeogenesis...

In Fig.6, in the third column of the histogram, the luciferase activity level was low in the presence of the hFoxO1 transcriptional activator under the action of compound 355, so it could be concluded that compound 355 has an obvious inhibitory effect on hFoxO1 transcriptional activation.

Figure 6. Compound 355 inhibits hFoxO1 transcription activation...

c) Detection of the 355 compounds on hepatic gluconeogenesis

After screening for compound 355 by luciferase activity detection (Fig.6), compound 355 was also used to inhibit both glucose concentration in HepG2 cells and mRNA abundance of key enzymes of gluconeogenesis. In Fig7A, we can find that with the gradual increase of the concentration of compound 355, the glucose level showed a counter gradient change and was concentration-dependent, indicating that compound 355 could significantly inhibit gluconeogenesis in hepatocytes.

Figure 7. Detection of the 355 compounds on hepatic gluconeogenesis. (A) Effect of compound 355 on gluconeogenesis in hepatocytes (B) Effect of compound 355 on reducing the mRNA level of key gluconeogenesis enzymes G6Pase (C) Effect of compound 355 on reducing the mRNA level of key gluconeogenesis enzymes PEPCK..

G6Pase is an enzyme that releases glucose into the blood by hydrolyzing glucose-6-phosphate in liver tissue. PEPCK is a gluconeogenic enzyme that allows hepatic parenchymal cells to produce glucose from pyruvate derived from amino acid metabolism. In Fig.7 B and 7C, when the volume of compound 355 increases gradually, the mRNA levels of G6pase and PEPCK gradually decreased in a counter gradient and were also concentration-dependent, indicating that compound 355 could significantly reduce the mRNA levels of key enzymes in gluconeogenesis.

Future plan

We have already collected the data and figures from our experiments. hFoxO1 plays a role in regulating downstream genes, especially in gluconeogenesis. And we find out that within a certain range, we could see a gradient of luciferin activity and concentration dependence. After expressing hFoxO1 protein in HepG2 cells, we can easily detect the downstream gene expression through a fluorescence reporting system. By the way, when we compared these data with the positive control group, we find that the activity of the hFoxO1 is easily regulated by special small components. Because of the great effect of the hFoxO1, we believe that if we can regulate the expression of hFoxO1 in the future and promote it in the market, it will become a great power to fight against type 2 diabetes, and it may be applied to clinical disease treatment, improve the quality of life of patients, and even reduce the number of diabetes patients.

Reference

1.IDF., Diabetes Atlas. 10th Edition, 2021.

2.Choi, H.E., et al., Novel FoxO1 inhibitor, JY-2, ameliorates palmitic acid-induced lipotoxicity and gluconeogenesis in a murine model. Eur J Pharmacol, 2021. 899: p. 174011.

3.Oh, K.J., et al., CREB and FoxO1: two transcription factors for the regulation of hepatic gluconeogenesis. BMB Rep, 2013. 46(12): p. 567-74.

4.Zhao, Y., Y. Wang, and W.G. Zhu, Applications of post-translational modifications of FoxO family proteins in biological functions. J Mol Cell Biol, 2011. 3(5): p. 276-82.

5.Nagashima, T., et al., Discovery of novel forkhead box O1 inhibitors for treating type 2 diabetes: improvement of fasting glycemia in diabetic db/db mice. Mol Pharmacol, 2010. 78(5): p. 961-70.

6.Ohtake, F., et al., Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature, 2003. 423(6939): p. 545-50.