UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS

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2024-11-23 18:29

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Conference: Bucharest University Faculty of Physics 2023 Meeting


Section: Biophysics; Medical Physics


Title:
Fluorescence studies on the influence of synthetic antimicrobial peptide HRWWRWWRH-NH2 on the cell membrane fluidity


Authors:
Francesca-Giulia GUARNERI(1), Marcela-Elisabeta BĂRBÎNȚĂ-PĂTRAȘCU(1), Bogdan ZORILĂ(2), Mina RĂILEANU(2), Mihaela BACALUM(2)


Affiliation:
1) University of Bucharest, Faculty of Physics, Department of Electricity, Solid-State Physics and Biophysics, Atomistilor Street 405, 077125 Măgurele, Ilfov, Romania;

2) “Horia Hulubei” National Institute for Physics and Nuclear Engineering, Department of Life and Environmental Physics, Reactorului 30, 077125 Măgurele, Ilfov Romania;



E-mail
giuliafrancesca22@gmail.com


Keywords:
Antimicrobial peptides, Laurdan, GP, membrane fluidity, model membranes, methyl-β-cyclodextrin, cholesterol


Abstract:
Antimicrobial peptides (AMPs) are short peptides found in humans, animals, and plants with a wide spectrum of bio-activities (antibacterial, antifungal, antiparasitic, and antiviral properties) [1], [2]. In this study, the Laurdan fluorescence with key-parameter GP (Generalized Polarization), was used to investigate the changes in the membrane fluidity of two cell lines (normal human skin cells-BJ and melanoma murine cells-B16), at two different temperatures (20°C and 37°C), induced by the gradual addition of the synthetic antimicrobial peptide P8 (HRWWRWWRH-NH2). Additionally, as a positive control test, we used the compound methyl-β-cyclodextrin (MβCD) in the same conditions to further verify the results obtained for P8. Due to the existence of its hydrophobic core, MβCD has a strong affinity to cholesterol and is used to deplete the cholesterol from the cells [3], [4]. Extending the study, we tested the effects of MβCD on LUV model membranes made from phosphatidylcholine and cholesterol at relevant temperatures for the experiment and at different concentrations of this compound, ranging from 0.33 mM to 1.66 mM. The results showed that the synthetic antimicrobial peptide P8 increased the membrane fluidity of both cell lines at both temperatures. This was also confirmed by the addition of MβCD on LUV model membranes at the same two temperatures, resulting in the decreasing of the GP values. The evaluation of the LUV model membranes between 10°C and 40°C with MβCD showed a decrease in GP values with the temperature for all four experimental conditions that have been tested, corresponding to 0, 0.33, 1 and 1.66 mM MβCD. However, adding the MβCD on cells, particularly on the B16 cell line, resulted in the increasing of the GP values at 20°C, therefore a more rigid condition of the membrane. At 37°C, GP values were observed to increase slightly (at 0.33 mM MβCD), immediately followed by decreasing values, indicating a more fluid cell membrane. This effect can be associated with the presence of cholesterol in the eukaryotic cell membrane and its complex role in maintaining the fluidity of the cell membrane.


References:

[1] J. Li, S. Hu, W. Jian, C. Xie, and X. Yang, “Plant antimicrobial peptides: structures, functions, and applications,” Bot. Stud. 2021 621, vol. 62, no. 1, pp. 1–15, Apr. 2021.

[2] Q. Y. Zhang et al., “Antimicrobial peptides: mechanism of action, activity and clinical potential,” Mil. Med. Res. 2021 81, vol. 8, no. 1, pp. 1–25, Sep. 2021.

[3] S. K. Rodal, G. Skretting, Ø. Garred, F. Vilhardt, B. Van Deurs, and K. Sandvig, “Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles,” Mol. Biol. Cell, vol. 10, no. 4, p. 961, 1999.

[4] R. Zidovetzki and I. Levitan, “Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies,” Biochim. Biophys. Acta, vol. 1768, no. 6, pp. 1311–1324, Jun. 2007.