Difference between revisions of "Part:BBa K4165001"

Latest revision as of 00:10, 14 October 2022

Truncated tripartite motif-containing 21 (TRIM21)

An E3 ligase from the TRIM family, which participates in the ubiquitin-proteasome degradation cascade of misfolded proteins.

Usage and Biology

Tripartite motif-containing 21 (TRIM21) is an E3 ubiquitin ligase that has a strong affinity for the Fc domain of antibodies. It is mainly composed of four domains (RING domain, B-box domain, coiled-coil domain, and PRYSPRY antibody-binding region). TRIM21 engages the ubiquitin-proteasome system to destroy antibody-bound pathogens during infection. In our project, we used the truncated version of Trim21 proposed by team NUDT 2020 (BBa_K3396007), they replaced the ‘PRYSPRY’ region with a protein pair (Cohesin 2 and Dockerin S), Cohesin will be fused to Trim21 and Dockerin to our tau binding peptide, resulting in targeting of tau upon binding of the protein pair, followed by its ubiquitination and degradation.

For the degradation to occur, a highly conserved, 76 amino acid polypeptide called ubiquitin must first be activated by an E1 enzyme in an ATP-dependent manner. The E1 binds ubiquitin's C-terminal end to a cysteine residue in its active site through a covalent connection. The E2 or ubiquitin-conjugating enzyme is the second enzyme in the cascade to receive the thioesterified ubiquitin from the E1 active site. Finally, the E3 ubiquitin ligase promotes the transfer of ubiquitin onto the substrate by binding to both the protein substrate and the E2-bound ubiquitin.

                            Figure 1.: A graphical illustration showing the domains of TRIM21. 

Figure 2.: A Graphical illustration showing mechanism of action of Snitch system to degrade hyperphosphorylated tau protein. 

Features

Ring-Domain: This part is responsible for the dimerization and activation of Trim21.

B-Box Domain: This domain exerts a regulatory role through interaction with ring domains as it drives the self-assembly of oligomers, bringing together multiple RING domains, which may facilitate RING dimerization.

Coiled-coil Domain: This domain undergoes monomer–dimer exchange to allow homodimerization of TRIM.

Dry Lab Characterization

Optimization

Firstly, we removed the last nucleotide in the original sequence from NUDT team 2020 to avoid the frameshifting, then the sequence was optimized to be expressed in E-coli BL21.

Modeling

Through our work, we modeled Trim-21 once fused to Dockerin and once again to Cohesin, to find out which one of them is more stable when it binds to Trim 21, the structures were modeled by 5 tools (Robetta-iTASSER-Alphafold-Modeller-TR Rosetta) to reach the best model.

The results showed that N-terminal attachment of DocS to Trim-21 is more preferable than that of Coh2, just like it was mentioned in literature. Our top 2 models ranked 5 out of 6 according to our QA code.

                            Figure 3.: Predicted 3D structure of truncated Trim21 model1.

                            Figure 4.: Predicted 3D structure of truncated Trim21 model2.

WetLab Results

Tripartite motif-containing 21 (TRIM21) is an E3 ubiquitin ligase that has a strong affinity for the Fc domain of antibodies. It is mainly composed of four domains (RING domain, B-box domain, coiled-coil domain, and PRYSPRY antibody-binding region). In the wet lab, we cloned the part using DH5 alpha using pJET. Then we extract the plasmid and restrict it to ligate Trim21 in pGS-21a to transform it into BL-21 and start the protein expression using IPTG.

Ligation of His Trim21 with pJET cloning vector

We used T4 ligase to ligate His Trim21 with pJET cloning vector so, we incubated His Trim21 with pJET overnight at 15°C

                  Figure 5. This figure shows the ligation between His Trim21 and pJET cloning vector.

Transformation of His Trim21 in DH-5 alpha using pJET cloning vector, and in BL-21 using pGS-21a expression vector

The transformation was performed using the TSS buffer protocol, after trying three different buffers which are: Calcium chloride, a combination between Calcium Chloride and Magnesium Chloride, and TSS buffer, we optimized our protocol to use TSS buffer protocol as it gives the best results. The transformation efficiency was calculated for both His Trim21 in the cloning vector and for His Trim21 in the expression vector and they are found to be 1280000 no. of transformants/µg and 32000 no. of transformants/µg respectively.

                                    Figure 6. Transformed plate of His Trim21 + pJET.

Miniprep of pJET cloning vector containing our His Trim21

Miniprep is a technique used to extract the plasmid containing our part so, we performed miniprep to extract the pJET containing His Trim21.

         Figure 7. This figure shows the miniprep of pJET cloning vector containing His Trim21, the band appears with 
                                      size (3943) which indicates miniprep is done.

Transformation of His Trim21 in BL-21 using pGS-21a expression vector

                                     Figure 8. Transformed plate of His Trim21 + pGS-21a.

References

1. Clift, D., McEwan, W. A., Labzin, L. I., Konieczny, V., Mogessie, B., James, L. C., & Schuh, M. (2017). A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell, 171(7), 1692-1706.e18. https://doi.org/10.1016/j.cell.2017.10.033

2. D.L. Mallery, W.A. McEwan, S.R. Bidgood, G.J. Towers, C.M. Johnson, L.C. James Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21) Proc. Natl. Acad. Sci. USA, 107 (2010), pp. 19985-19990

3. Kleiger, G., & Mayor, T. (2014). Perilous journey: a tour of the ubiquitin-proteasome system. Trends in cell biology, 24(6), 352. https://doi.org/10.1016/j.tcb.2013.12.003

4. L.C. James, A.H. Keeble, Z. Khan, D.A. Rhodes, J. Trowsdale Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function Proc. Natl. Acad. Sci. USA, 104 (2007), pp. 6200-

5. Zhang, Y., Li, L., Munir, M., & Qiu, H. (2018). RING-Domain E3 Ligase-Mediated Host–Virus Interactions: Orchestrating Immune Responses by the Host and Antagonizing Immune Defense by Viruses. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2018.01083

6. Zeng, J., Santos, A. F., Mukadam, A. S., Osswald, M., Jacques, D. A., Dickson, C. F., ... & James, L. C. (2021). Target-induced clustering activates Trim-Away of pathogens and proteins. Nature structural & molecular biology, 28(3), 278-289.