Part:BBa_K4990001
FK-13
TO KNOW ABOUT IT!
Antimicrobial peptides, which are first discovered in the immune systems of insects, are widely found in living organisms. Subsequently, similar peptides have been extracted from bacteria, fungi, amphibians, higher plants, and mammals. These active peptides possess natural immune functions and serve as essential molecular barriers for host defense against invading pathogens, hence referred to as antimicrobial peptides[1-2].
The tissue proteinase inhibitor family is unique to mammals and constitutes a type of antimicrobial peptide.LL37 is derived from one of the tissue proteinase inhibitors, human cationic antimicrobial protein 18 (hCAP18). Among them, LL-37 is the sole antimicrobial peptide found in the human body. It possesses various functions such as antimicrobial, antitoxin, immunomodulatory, and wound healing effects. Additionally, LL-37 holds potential for treating viral diseases and neoplastic conditions. Numerous research teams have investigated the activity of truncated peptides to identify the minimal antimicrobial region of LL-37. The results indicate that the smallest region of LL-37 is within amino acid positions 17 to 29 or 18 to 29[3-4].
This suggests that the C-terminal helix, consisting of 12 or 13 amino acids of LL-37, plays a role in antimicrobial, antiviral, and antitumor activities.
What is it?
This part is the truncated peptide of LL37, representing the minimal functional segment of LL-37. It consists of only 13 AA and exhibits antimicrobial activity.As a basic part,it functions as a warhead, capable of simultaneously killing both bacteria and colorectal cancer cells.
What can it do?
As a basic part,it functions as a warhead, capable of simultaneously killing both bacteria and colorectal cancer cells.Later, building upon it as a foundation, we incorporated domains targeting Fusobacterium nucleatum (Fn) and colorectal cancer (CRC) on its head and tail. This resulted in a dual-targeting cytotoxic peptide, which we call the "Double-Edged Harpoon".
How does it work?
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[5].
We also performed an exhaustive wet experimental characterization
We rationalized truncation and mutation of LL-37 and considered the effect of possible application of linker on its activity as in Fig. xa. In order to further characterize the anticancer activity of our rationally-modified FK-13, we carried out a preliminary anticancer pre-experiment. As can be seen from Fig. xa~b, the inhibitory effect of FK-13 on sw480 was also significantly dose-dependent and time-dependent. And as can be seen from Fig. xc~f, the cancer inhibitory effect of FK-13 was significantly different between the groups administered with different doses.
Detection of anticancer activity of LL-37 and it induces cell death mode | a.Absorbance at 450 nm versus time of administration and concentration of administration. b. Heat map of absorbance at 450 nm versus time of administration and concentration of administration. c. Absorbance at 450 nm of 2 hours after FK-13 administration. d. Absorbance at 450 nm of 4 hours after FK-13 administration. e. Absorbance at 450 nm of 6 hours after FK-13 administration. f. Absorbance at 450 nm of 8 hours after FK-13 administration. (The data were analyzed using student’s T-test; *: P<0.05; **: P<0.01; ***: P<0.001).
According to the preexperiment, we further purified and prepared FK-13 with higher purity and utilized it for more detailed experiments. As can be seen from Figure e~f, the inhibitory effect of FK-13 on sw480 was significantly dose-dependent and time-dependent. And as can be seen from Figure a~b, the inhibition rate after 2 hours of administration showed a curve similar to S shape. While the group with further prolongation of the administration time was closer to linear at the same time, which might be caused by the bias caused by the CCK-8 assay itself.According to the flow cytometric analysis of the single administration of Fig. g and the two administration groups of Fig. h, it was known that LL-37 could cause severe apoptotic cell death. As shown in Figure c~d, we plotted the inhibition rate-concentration curves logarithmically and performed a linear fit to the 2-hour dosing group, which had a better linear relationship of the inhibition rate-concentration logarithmic value, and calculated the IC50 of FK-13=124.03667 μg/mL. Meanwhile, according to the flow cytometric analysis of the single administration of Fig. g and the two administration groups of Fig. 5h, it was known that FK-13 could cause severe apoptosis. According to Figure 5i~j, it can be seen that the anticancer activity of FK-13 was mainly realized by inducing apoptosis, which of course was accompanied by the occurrence of cell necrosis. This indicates that our modification work did not affect the anticancer mechanism of FK-13. Similarly, since FK-13 can cause sw480 necrosis, the percentage of necrotic cells was slightly lower because they tend to flaunt in the culture medium and are not easy to collect. In addition we also detected the apoptosis of adherent cells using TUNEL staining, and those stained green were apoptotic cells (Figure k).
The above experiments proved that FK-13, which was rationalized by us, has good anticancer activity, can effectively inhibit the activity of sw480, and can also induce apoptosis and cell necrosis.
Detection of anticancer activity of FK-13 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 FK-13 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).
Here are the results of all our flow cytometry assays.
While designing FK-13, we also performed extensive wet experimental characterization of LL-37, which we complemented in the parts of our predecessors, https://parts.igem.org/Part:BBa_K1162006
Here is a screenshot of the page
Reference
［1］de Breij, A., Riool, M., Cordfunke, R. A., Malanovic, N., de Boer, L., Koning, R. I., Ravensbergen, E., Franken, M., van der Heijde, T., Boekema, B. K., Kwakman, P. H. S., Kamp, N., El Ghalbzouri, A., Lohner, K., Zaat, S. A. J., Drijfhout, J. W., & Nibbering, P. H. (2018). The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms. Science translational medicine, 10(423), eaan4044.
［2］Zhang, M., Liang, W., Gong, W., Yoshimura, T., Chen, K., & Wang, J. M. (2019). The Critical Role of the Antimicrobial Peptide LL-37/ CRAMP in Protection of Colon Microbiota Balance, Mucosal Homeostasis, Anti-Inflammatory Responses, and Resistance to Carcinogenesis. Critical reviews in immunology, 39(2), 83–92.
［3］Fabisiak, A., Murawska, N., & Fichna, J. (2016). LL-37: Cathelicidin-related antimicrobial peptide with pleiotropic activity. Pharmacological reports : PR, 68(4), 802–808.
［4］Chieosilapatham, P., Ikeda, S., Ogawa, H., & Niyonsaba, F. (2018). Tissue-specific Regulation of Innate Immune Responses by Human Cathelicidin LL-37. Current pharmaceutical design, 24(10), 1079–1091.
［5］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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |