Difference between revisions of "Part:BBa K4990010"
 
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===Usage in short===
 
===Usage in short===
−
You can use it to target and kill Fn.
+
You can use it to target and kill CRC.
 
===What is it===
 
===What is it===
−
This is the structure of (Bacteria-Targeting Peptide)BTP, which is consited of B-domain, Linker A, FK-13.
+
This is the structure of (Tumor-Targeting Peptide)TTP, which is consited of HlpA, GPNG ,Linker B, FK-13.
  
−
B-domain targets the pilus of Fn, Linker A is a rigid and cleavage linker, FK-13 has antimicrobial activity.
+
HlpA targets HSPG on CRC, Linker A is a rigid and cleavage linker, FK-13 has antimicrobial activity.
 
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===How does it work?===
 +
In 2006, Tjalsma employed the Highly accurate tandem MS method to identify a protein named Histone-like protein A (HlpA). This discovery highlighted a bridge between Streptococcus bovis and colorectal cancer. It is postulated that S. bovis establishes a bond with colorectal cancer cell surfaces via heparan sulfate-proteoglycans (HSPG) mediated by HlpA [1].
 +
 +
By 2009, Boleij confirmed that Streptococcus gallolyticus binds to the surface of colorectal cancer cells through HlpA interacting with HSPG [2].
 +
 +
In 2016, O'Neil unveiled the crystal structure of Hlp, revealing its crab-claw-like architecture. The claw section's basic amino acids can bind with DNA and simultaneously with heparin [3].
 +
 +
In 2018, Chun Loong Ho harnessed the binding of HlpA to HSPG to construct an engineered Escherichia coli that specifically targets colorectal cancer, thus marking the commencement of the HlpA targeting system [4].
 +
 +
In 2022, the iGEM22_LZU-CHINA team, utilizing synthetic biology, developed an Escherichia coli that targets colorectal cancer, employing the method of HlpA targeting tumor cell surface HSPG [5].
 +
 +
By 2023, Tang engineered a probiotic using HlpA targeting and Azurin-induced cytotoxicity, demonstrating promising therapeutic efficacy in colorectal cancer mice models [6].
 +
 +
 +
<html>
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<figure style="text-align:middle;">
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                <img style="max-width:550px;" src="https://static.igem.wiki/teams/4990/wiki/registry/1618.png" alt="control">
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                <figcaption><b>Figure A:</b>Crystal structure of HlpA(O'Neil 2016) <b>Figure B:</b>Engineered EcN treat CRC(Chun 2018)</figcaption>
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              </figure>
 +
</html>
 +
HlpA, as suggested by its name "Histone-like protein A", binds to DNA similar to histones. Its overall morphology resembles a crab claw, allowing it to non-specifically and tightly bind to the minor groove of DNA using its ribbon-like β-fold region. This results in significant DNA bending, DNA compaction, and negative supercoiling. S. bovis, through an unknown mechanism, secretes it extracellularly, acting as an anchorless protein. It becomes a target for the humoral immune system during infections. By linking bacterial lipoteichoic acid (LTA) and HSPG on colon epithelial cells, it mediates bacterial adhesion to colon tumor cells.
 +
 +
Wu et al. [7] found that through their research on chitosan-fused rigid linker peptides, that rigid linker peptides exhibit self-cleaving properties under specific conditions. At pH=6-7, cleavage occurs. It's hypothesized that the EAAAK amino acid arrangement can form a stable hydrophilic α-helical structure. The forces at play can be attributed to the Glu-...Lys+ salt bridges between Glu and Lys. Hydrogen bonds are pivotal in forming these salt bridges, so pH greatly influences the stability of the salt bridges. At neutral pH, the strength of salt bridges is at its weakest, making cleavage more likely. Conversely, in high salt environments, the ionic strength can stabilize these bridges, preventing cleavage [8].
 +
 +
 +
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<img src="https://static.igem.wiki/teams/4990/wiki/registry/bridge.png" width="650" >
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</html>
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<img src="https://static.igem.wiki/teams/4990/wiki/registry/cleavage.png" width="750" >
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</html>
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FK-13 exhibits antibacterial effects against both Gram-positive and Gram-negative bacteria. Through electrostatic interactions, the positively charged FK-13 connects with negatively charged bacteria, inserting into bacterial cell membranes and leading to the leakage of their contents, resulting in cell death.
 +
 +
<html>
 +
<img src="https://static.igem.wiki/teams/4990/wiki/registry/ll37character.png" width="600" >
 +
</html>
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 +
Currently, two hypotheses support the mechanism by which FK-13 disrupts cell membranes: the carpet model, based on FK-13's cationic amino acids interacting with the phospholipid head groups in the cell membrane, which can form a carpet-like structure on the membrane surface, ultimately compromising membrane integrity. Changes in helical structure, charge, and hydrophobicity influence its antibacterial activity. On the other hand, the toroidal pore model suggests that due to membrane surface tension and curvature, FK-13 induces the formation of toroidal pores in bacterial membranes, causing leakage of bacterial contents and leading to bacterial death.
 +
 +
<html>
 +
<img src="https://static.igem.wiki/teams/4990/wiki/registry/ll37func.png" width="600" >
 +
</html>
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 +
For mammalian cells, whose membranes are mostly neutral, FK-13's interaction with membranes is comparatively weak, and cells are less susceptible to damage. Furthermore, FK-13 can enhance the rigidity of epithelial cell membranes and reduce their permeability, thus minimizing bacterial attacks. Additionally, FK-13 possesses an amphipathic helical structure, which under hydrophobic conditions promotes oligomerization. The potency of the immune response is directly proportional to the α-helical structure of the oligomers. When there are more α-helices present, the antibacterial activity is stronger[9].
 +
 +
===Wetlab Characterization===
 +
<html>
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<img src="https://static.igem.wiki/teams/4990/wiki/registry/s7.png" width="600" >
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</html>
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Interaction of TTP and heparin.
 +
 +
A.Pull down to enrich TTP and prove the interaction ability of TTP and heparin.
 +
 +
B.The structure of TTP.
 +
 +
C.The interaction of TTP and heparin.
 +
 +
We enrich TTP with utilized magnetic, further proving the interaction ability of TTP and heparin. It was observed that TTP heparin interaction bands exhibited higher molecular weight and wider bands, likely due to the different polymerization levels of heparin when compared with pure TTP. This illustrates the capability of DEH to bind to the sugar chains of HSPG glycoproteins expressed abundantly on tumour cell surfaces, thus effectively targeting them.
 +
 +
 +
Based on the existing modification of FK-13 and linker, we combined the results of a computational structural biology-based screen to construct the fusion protein TTP by binding HlpA monomer to FK-13.TTP (TUMOR TARGETING PEPTIDE) is a protein capable of binding to HSPG glycan chains on the surface of CRC cells via HlpA. We utilized the conditional break linker to connect FK-13 with HlpA to achieve the intention of targeting and killing CRC cells. To verify the killing of sw480 by TTP, we performed an exhaustive characterization of the anticancer activity of TTP.
 +
The by Figure e~f, the inhibitory effect of TTP on sw480 had a significant dose-dependent and time-dependent effect. And as can be seen from Figure a~b, the inhibition rate after 2 hours of administration showed a curve similar to S shape. And the TTP concentration-inhibition rate of the group with further prolongation of the administration time was closer to linearity. As shown in Figs. 6c~d, we plotted the inhibition rate-concentration curve logarithmically and performed a linear fit for the 2 h administration group with a better linear relationship of the inhibition rate-concentration logarithmic value, and calculated the IC50 of TTP = 136.03 μg/mL. the IC50 of TTP was slightly higher than that of FK-13, which was in accordance with the theoretical model. According to the flow cytometric analysis of Fig. g single administration and Fig. 6h two administration groups, it is known that TTP can cause severe apoptosis.
 +
According to Figs. i~j, it can be seen that the anticancer activity of TTP is mainly realized by inducing apoptosis. And there was a significant correlation between the number of late apoptotic cells and the number of administration. Similar to the previously mentioned FK-13 and LL-37, similarly, the percentage of necrotic cells was slightly lower because TTP could lead to sw480 necrosis, but the percentage of necrotic cells was also slightly lower because the necrotic cells tended to be suspended in the culture medium, which was not easy to collect.
 +
In addition ,we also detected the apoptosis of adherent cells using TUNEL staining, and those that were stained green were apoptotic cells (Figure k).
 +
The above experiments proved that our new targeted killing anticancer peptide, TTP, has good anticancer activity, which can effectively inhibit the activity of sw480 and also induce apoptosis and cell necrosis.
 +
 +
 +
 +
<html>
 +
<img src="https://static.igem.wiki/teams/4990/wiki/registry/part2-4-1-fig6.jpg" width="750" >
 +
</html>
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 +
Detection of anticancer activity of TTP and it induces cell death mode | a.Absorbance at 450 nm versus time of administration and concentration of administration. b. Inhibition ratio versus time of administration and concentration of administration. c. Inhibition ratio versus concentration of administration logarithmically. d. Inhibition ratio versus concentration of administration logarithmically of 2 hours after TTP administration and its fitted straight line. e. Heat map of absorbance at 450 nm versus time of administration and concentration of administration. f. Heat map of Inhibition ratio versus time of administration and concentration of administration. g. Flow cytometric analysis of the single-dose administration group. h. Flow cytometric analysis of the two-dose administration group. i. Comparison of the percentage of necrotic, late apoptotic, early apoptotic, and normal cells in the flow cytometric analysis results of the single-dose administration group. j. Comparison of the percentage of necrotic, late apoptotic, early apoptotic, and normal cells in the flow cytometric analysis results of the two-dose administration group. k. TUNEL staining detected apoptosis in adherent cells. (The data were analyzed using student’s T-test; *: P<0.05; **: P<0.01; ***: P<0.001).
 +
 +
 +
 +
 +
 +
 +
===Reference===
 +
[1] Tao, L., Gao, M., & Zhou, H. (2015). Research progress on novel antibody-chemotherapy drug conjugates. Journal of Pharmaceutical Biotechnology, 22(3), 253-258.
 +
 +
[2] Gustavsson M，Lehtio J，Denman S，et al． Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in pichia pastoris［J］． Protein Eng，2001，14（9）:711-715.
 +
 +
[3] Wang SH，Zheng CJ，Liu Y，et al． Construction of multiform scFv antibodies using linker peptide［J］． J Genetics and Genomics，2008，35:313-316.
 +
 +
[4] Zhang JH，Yun J，Shang ZG，et al． Design and optimization of a linker for fusion protein construction［J］． Progr Natur Sci，2009，19: 1197-1200.
 +
 +
[5] Shan D，Press OW，Tsu TT，et al． Characterization of scFv-Ig constructs generated from the Anti-CD20 mAb 1F5 using linker peptides of varying lengths［J］.J Immunology，2014，162:6589-6595.
 +
 +
[6]Tang, H., Zhou, T., Jin, W., Zong, S., Mamtimin, T., Salama, E. S., ... & Li, X. (2023). Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice. Life Sciences, 324, 121709.
 +
 +
[7]Guo N，Zheng J，Wu L，et al. Engineered bifunctional enzymes of endo-1，4-β-xylanase/endo-1，4-β-mannanase were constructed for synergistically hydrolyzing hemicellulose[J]. Journal of Molecular Catalysis B：Enzymatic，2013，97：311-318.
 +
 +
[8]Arai R，Wriggers W，Nishikawa Y，et al. Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering[J]. Proteins：Structure，Function，and Bioinformatics，2004，57（4）：829-838.
 +
 +
[9]Wang, X., Junior, J. C. B., Mishra, B., Lushnikova, T., Epand, R. M., & Wang, G. (2017). Arginine-lysine positional swap of the LL-37 peptides reveals evolutional advantages of the native sequence and leads to bacterial probes. Biochimica et biophysica acta. Biomembranes, 1859(8), 1350–1361.
 +
 +
Sequence and Features
 +
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 14:21, 12 October 2023


Tumour-Targeting Peptide

TO KNOW ABOUT IT!

In the wake of advancements in molecular biology, numerous proteins and peptides with active functions have been discovered or invented for pharmaceutical applications. Recombinant technology, characterized by its high expression levels and ease of operation, has been extensively applied in biomedicine and related fields. To obtain multi-target, multifunctional active proteins, it is requisite to link and fuse two or more proteins with known functions. This method of obtaining bifunctional or multifunctional fusion proteins has become one of the new approaches for developing new drugs and researching bioproducts, especially widely used in the preparation of bispecific single-chain variable fragments (scFv) or antibody-drug conjugates[1-5].

Fusion proteins are principally composed of two parts: the functional protein and the linker peptide. The functional protein is the original protein intended for fusion, typically with known structure and function, posing no issues in selection. However, due to the significance of the linker peptide in the overall structure of the fusion protein, its selection and design require thoughtful research to ensure the overall activity of the fusion protein remains unchanged[2]. Consequently, research on linker peptides has gradually come into focus.

Usage in short

You can use it to target and kill CRC.

What is it

This is the structure of (Tumor-Targeting Peptide)TTP, which is consited of HlpA, GPNG ,Linker B, FK-13.

HlpA targets HSPG on CRC, Linker A is a rigid and cleavage linker, FK-13 has antimicrobial activity.

How does it work?

In 2006, Tjalsma employed the Highly accurate tandem MS method to identify a protein named Histone-like protein A (HlpA). This discovery highlighted a bridge between Streptococcus bovis and colorectal cancer. It is postulated that S. bovis establishes a bond with colorectal cancer cell surfaces via heparan sulfate-proteoglycans (HSPG) mediated by HlpA [1].

By 2009, Boleij confirmed that Streptococcus gallolyticus binds to the surface of colorectal cancer cells through HlpA interacting with HSPG [2].

In 2016, O'Neil unveiled the crystal structure of Hlp, revealing its crab-claw-like architecture. The claw section's basic amino acids can bind with DNA and simultaneously with heparin [3].

In 2018, Chun Loong Ho harnessed the binding of HlpA to HSPG to construct an engineered Escherichia coli that specifically targets colorectal cancer, thus marking the commencement of the HlpA targeting system [4].

In 2022, the iGEM22_LZU-CHINA team, utilizing synthetic biology, developed an Escherichia coli that targets colorectal cancer, employing the method of HlpA targeting tumor cell surface HSPG [5].

By 2023, Tang engineered a probiotic using HlpA targeting and Azurin-induced cytotoxicity, demonstrating promising therapeutic efficacy in colorectal cancer mice models [6].


control
Figure A:Crystal structure of HlpA(O'Neil 2016) Figure B:Engineered EcN treat CRC(Chun 2018)
HlpA, as suggested by its name "Histone-like protein A", binds to DNA similar to histones. Its overall morphology resembles a crab claw, allowing it to non-specifically and tightly bind to the minor groove of DNA using its ribbon-like β-fold region. This results in significant DNA bending, DNA compaction, and negative supercoiling. S. bovis, through an unknown mechanism, secretes it extracellularly, acting as an anchorless protein. It becomes a target for the humoral immune system during infections. By linking bacterial lipoteichoic acid (LTA) and HSPG on colon epithelial cells, it mediates bacterial adhesion to colon tumor cells.

Wu et al. [7] found that through their research on chitosan-fused rigid linker peptides, that rigid linker peptides exhibit self-cleaving properties under specific conditions. At pH=6-7, cleavage occurs. It's hypothesized that the EAAAK amino acid arrangement can form a stable hydrophilic α-helical structure. The forces at play can be attributed to the Glu-...Lys+ salt bridges between Glu and Lys. Hydrogen bonds are pivotal in forming these salt bridges, so pH greatly influences the stability of the salt bridges. At neutral pH, the strength of salt bridges is at its weakest, making cleavage more likely. Conversely, in high salt environments, the ionic strength can stabilize these bridges, preventing cleavage [8].


FK-13 exhibits antibacterial effects against both Gram-positive and Gram-negative bacteria. Through electrostatic interactions, the positively charged FK-13 connects with negatively charged bacteria, inserting into bacterial cell membranes and leading to the leakage of their contents, resulting in cell death.

Currently, two hypotheses support the mechanism by which FK-13 disrupts cell membranes: the carpet model, based on FK-13's cationic amino acids interacting with the phospholipid head groups in the cell membrane, which can form a carpet-like structure on the membrane surface, ultimately compromising membrane integrity. Changes in helical structure, charge, and hydrophobicity influence its antibacterial activity. On the other hand, the toroidal pore model suggests that due to membrane surface tension and curvature, FK-13 induces the formation of toroidal pores in bacterial membranes, causing leakage of bacterial contents and leading to bacterial death.

For mammalian cells, whose membranes are mostly neutral, FK-13's interaction with membranes is comparatively weak, and cells are less susceptible to damage. Furthermore, FK-13 can enhance the rigidity of epithelial cell membranes and reduce their permeability, thus minimizing bacterial attacks. Additionally, FK-13 possesses an amphipathic helical structure, which under hydrophobic conditions promotes oligomerization. The potency of the immune response is directly proportional to the α-helical structure of the oligomers. When there are more α-helices present, the antibacterial activity is stronger[9].

Wetlab Characterization

Interaction of TTP and heparin.

A.Pull down to enrich TTP and prove the interaction ability of TTP and heparin.

B.The structure of TTP.

C.The interaction of TTP and heparin.

We enrich TTP with utilized magnetic, further proving the interaction ability of TTP and heparin. It was observed that TTP heparin interaction bands exhibited higher molecular weight and wider bands, likely due to the different polymerization levels of heparin when compared with pure TTP. This illustrates the capability of DEH to bind to the sugar chains of HSPG glycoproteins expressed abundantly on tumour cell surfaces, thus effectively targeting them.


Based on the existing modification of FK-13 and linker, we combined the results of a computational structural biology-based screen to construct the fusion protein TTP by binding HlpA monomer to FK-13.TTP (TUMOR TARGETING PEPTIDE) is a protein capable of binding to HSPG glycan chains on the surface of CRC cells via HlpA. We utilized the conditional break linker to connect FK-13 with HlpA to achieve the intention of targeting and killing CRC cells. To verify the killing of sw480 by TTP, we performed an exhaustive characterization of the anticancer activity of TTP. The by Figure e~f, the inhibitory effect of TTP on sw480 had a significant dose-dependent and time-dependent effect. And as can be seen from Figure a~b, the inhibition rate after 2 hours of administration showed a curve similar to S shape. And the TTP concentration-inhibition rate of the group with further prolongation of the administration time was closer to linearity. As shown in Figs. 6c~d, we plotted the inhibition rate-concentration curve logarithmically and performed a linear fit for the 2 h administration group with a better linear relationship of the inhibition rate-concentration logarithmic value, and calculated the IC50 of TTP = 136.03 μg/mL. the IC50 of TTP was slightly higher than that of FK-13, which was in accordance with the theoretical model. According to the flow cytometric analysis of Fig. g single administration and Fig. 6h two administration groups, it is known that TTP can cause severe apoptosis. According to Figs. i~j, it can be seen that the anticancer activity of TTP is mainly realized by inducing apoptosis. And there was a significant correlation between the number of late apoptotic cells and the number of administration. Similar to the previously mentioned FK-13 and LL-37, similarly, the percentage of necrotic cells was slightly lower because TTP could lead to sw480 necrosis, but the percentage of necrotic cells was also slightly lower because the necrotic cells tended to be suspended in the culture medium, which was not easy to collect. In addition ,we also detected the apoptosis of adherent cells using TUNEL staining, and those that were stained green were apoptotic cells (Figure k). The above experiments proved that our new targeted killing anticancer peptide, TTP, has good anticancer activity, which can effectively inhibit the activity of sw480 and also induce apoptosis and cell necrosis.



Detection of anticancer activity of TTP and it induces cell death mode | a.Absorbance at 450 nm versus time of administration and concentration of administration. b. Inhibition ratio versus time of administration and concentration of administration. c. Inhibition ratio versus concentration of administration logarithmically. d. Inhibition ratio versus concentration of administration logarithmically of 2 hours after TTP administration and its fitted straight line. e. Heat map of absorbance at 450 nm versus time of administration and concentration of administration. f. Heat map of Inhibition ratio versus time of administration and concentration of administration. g. Flow cytometric analysis of the single-dose administration group. h. Flow cytometric analysis of the two-dose administration group. i. Comparison of the percentage of necrotic, late apoptotic, early apoptotic, and normal cells in the flow cytometric analysis results of the single-dose administration group. j. Comparison of the percentage of necrotic, late apoptotic, early apoptotic, and normal cells in the flow cytometric analysis results of the two-dose administration group. k. TUNEL staining detected apoptosis in adherent cells. (The data were analyzed using student’s T-test; *: P<0.05; **: P<0.01; ***: P<0.001).




Reference

[1] Tao, L., Gao, M., & Zhou, H. (2015). Research progress on novel antibody-chemotherapy drug conjugates. Journal of Pharmaceutical Biotechnology, 22(3), 253-258.

[2] Gustavsson M，Lehtio J，Denman S，et al． Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in pichia pastoris［J］． Protein Eng，2001，14（9）:711-715.

[3] Wang SH，Zheng CJ，Liu Y，et al． Construction of multiform scFv antibodies using linker peptide［J］． J Genetics and Genomics，2008，35:313-316.

[4] Zhang JH，Yun J，Shang ZG，et al． Design and optimization of a linker for fusion protein construction［J］． Progr Natur Sci，2009，19: 1197-1200.

[5] Shan D，Press OW，Tsu TT，et al． Characterization of scFv-Ig constructs generated from the Anti-CD20 mAb 1F5 using linker peptides of varying lengths［J］.J Immunology，2014，162:6589-6595.

[6]Tang, H., Zhou, T., Jin, W., Zong, S., Mamtimin, T., Salama, E. S., ... & Li, X. (2023). Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice. Life Sciences, 324, 121709.

[7]Guo N，Zheng J，Wu L，et al. Engineered bifunctional enzymes of endo-1，4-β-xylanase/endo-1，4-β-mannanase were constructed for synergistically hydrolyzing hemicellulose[J]. Journal of Molecular Catalysis B：Enzymatic，2013，97：311-318.

[8]Arai R，Wriggers W，Nishikawa Y，et al. Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering[J]. Proteins：Structure，Function，and Bioinformatics，2004，57（4）：829-838.

[9]Wang, X., Junior, J. C. B., Mishra, B., Lushnikova, T., Epand, R. M., & Wang, G. (2017). Arginine-lysine positional swap of the LL-37 peptides reveals evolutional advantages of the native sequence and leads to bacterial probes. Biochimica et biophysica acta. Biomembranes, 1859(8), 1350–1361.

Sequence and Features


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 282
  • 1000
    COMPATIBLE WITH RFC[1000]