Abstract
Actin exists as a monomer (G-actin) which can be polymerized to filaments) F-actin) that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl2 or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.
Highlights
Actin is the most abundant protein in the eukaryotic cell
Site specific hydrophobic interactions between actin and polycation might contribute to the bundling especially at high ionic strength, where the bundling effect of polycations decreases because the relatively high concentration of monovalent cations masks the electrostatic interactions between polycations and actin [18]
These results may indicate that monomeric actin forms complexes with LL-37 that at low LL-37 concentrations inhibit salt-induced actin polymerization
Summary
Actin is the most abundant protein in the eukaryotic cell. It is a highly dynamic structural protein with multiple functions in cell physiology which include cytoskeleton formation, cell division, motility, adhesion, signaling and more [1]. The other group of actin bundling factors are the polycations, including polycationic proteins or peptides and polyamines. This group induces bundle formation via non-specific electrostatic interactions by eliminating repulsion between negatively charged actin filaments [5]. LL-37 [6], lysozyme [7] MARCKS [8], [9], ENA/VASP [10], fesselin [11], [12] and calponin [13], [14] all belong to this group Polycationic polyamines such as spermine and spermidine [15], [16] and polylysine [17] bundle F-actin. The bundling process of F-actin by lysozyme, spermine and polylysine was recently studied in some detail [19], [20]
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