Difference between revisions of "Part:BBa K4839008"

Latest revision as of 15:50, 12 October 2023

IRF4 Binding protein

Our modeling work also contribute to our part collection. In order to directly target to the IRF4 protein (which is also reffered as PU.1) (Figure 1.), we synthesis an IRF4 binding protein through modeling. The detailed process of our modeling work can be found on the model page.

Figure1. The function of anti-IRF4 BioPROTAC

With the help of computer modeling including molecular dynamics, we succeedly synthesis the Anti-IRF4 protein, which is called IRF4 binding protein here. The IRF4 binding protein can specifically binds to the IRF4 protein, and thus the BioPROTAC will trigger the degradation of the IRF4 and furher lead to the changing of the phenotype of the macrophage.

We have successfully constructed the IRF4 Binding protein (anti-IRF4 BioPROTAC) and assembled it into the pLVX-TREG3S-FLAG-TetOne-Puro vector regulated by doxycycline-controlled Tet-on system. We have also completed the construction of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and the sequencing results are shown in Figure 2.

Figure2. Sequencing results confirm the successful construction of IRF4 Binding protein (PU.1-SPOP).

We have performed structure simulation using Alphafold and conducted molecular docking using our pipeline. The results are shown in Figure 3.

Among all the conformations generated, Piper clustered the first 1000 rotational conformations based on the RMSD between each atom, and the representative conformations in each class were selected from the conformations with the most neighbors in this group. Piper sorts the generated conformations based on the number of clusters in each category. The conformation with the largest number of clusters ranks first, and the first conformation is selected for follow-up analysis.

Figure 3. The interface between PU.1 and IRF4

The experimental verification of anti-IRF4 BioPROTAC

After finishing the screening and modelling siitimulation of IRF4 binding protein (Pu.1), we soon conduct experimental verification of this protein region.

To demonstrate the effectiveness of anti-IRF4 BioPROTAC in degrading IRF4, we constructed expression vectors for multiple IRF4 proteins. Initially, we attempted to construct pEGFP-(EAAAK)3-IRF4 to visually observe the degradation effect of IRF4 under a microscope. However, due to time constraints, we have not completed the construction of this vector (see Engineering section for details). Therefore, we only completed the construction of pcDNA3.1-CMV-HA-IRF4 and pcDNA3.1-CMV-FLAG-IRF4. Additionally, we synthesized PU.1-(SSG)3-SPOP through Sangon Biotech and constructed the plasmid pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro.

Next, we transfected HEK293T cells in a 12-well plate with 1 μg of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and 1 μg of pcDNA3.1-CMV-FLAG-IRF4/pcDNA3.1-CMV-HA-IRF4, induced expression with 1 μg/mL of doxycycline for 48h, and collected the protein for western blot analysis. The results are shown in Figure 4, where we observed significant degradation of both HA-IRF4 and FLAG-IRF4.

Figure 4. Western blot signing degradation of IRF4. "D" represents doxycycline.

Due to time constraints, we have not been able to conduct this experiment in the THP-1 cell line. However, in the HEK293T cell experiment, we have observed a certain degree of IRF4 degradation, which is a breakthrough that has not been achieved in previous research. We are very excited about these results. We will then continue to optimize the transfection/infection efficiency for THP-1 cells and have also contacted Professor Chong Wu, a macrophage expert from Sun Yat-sen University School of Life Sciences, to explore ways to optimize related experiments for THP-1 cells. Additionally, we will conduct further experimental validation for the designed anti-IRF5 BioPROTAC and optimize anti-IRF4 BioPROTAC using bioinformatics methods to enhance its degradation efficiency.

The overall desgn of this part in our project (SYN-MACRO)

In our design, the engineered macrophages, SYN-MACRO could recognize GPC3 via SNIPR. Through Notch signaling pathway, SNIPR will be cleaved 3 times by γ-secretase and release transcription factor Gal4VP64 to activate AntiIRF4-BioPROTAC expression, effectively degrading the key protein IRF4 that regulates macrophage M2 polarization. Thereby promoting macrophage M1 polarization (Figure 5). Further SYN-MACRO will release pro-inflammatory cytokines and recruit CD8 T cells for effective tumor eradication.

Figure 5. GPC3 induced M1 polarization by activation of anti-IRF4 BioPROTAC.

After effective tumor eradication, doxycycline is used to induce downstream AntiIRF5-BioPROTAC expression to restore the anti-inflammatory phenotype in the tumor microenvironment also as a safety module, while inhibiting chronic inflammation caused by triglyceride accumulation in liver tissues of many HCC patients, thereby reducing further damage to liver tissue caused by chronic inflammation, also for inhibition of M1 over-polarization (Figure 6)

Figure 6. Doxycycline induced M1 polarization by activation of anti-IRF5 BioPROTAC.

Figure 7. Schematic of the SYN-MACRO M1/M2 polarization.

So now we have the complete gene circuit design of SYN-MACRO (Figure 7). We can control the polarization process of SYN-MACRO in vivo. We hope to precisely target GPC3-positive liver cancer cells with SYN-MACRO and effectively polarize them towards the M1 phenotype, thereby restructuring the tumor microenvironment, promoting the occurrence of inflammation, and inhibiting the development of tumors.

Reference

[1] Lim S , Khoo R , Peh K M ,et al.bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA)[J].Proceedings of the National Academy of Sciences, 2020(11).DOI:10.1073/PNAS.1920251117.

[2] Békés, M., Langley, D.R. & Crews, C.M. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 21, 181–200 (2022). https://doi.org/10.1038/s41573-021-00371-6

Sequence and Features