LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

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.

Actin Enables the Antimicrobial Action of LL-37 Peptide in the Presence of Microbial Proteases

“…7E). As we have shown recently (39), the bundling of actin filaments by s-LL-37, especially in the presence of 100 mM or higher concentrations of NaCl, is significantly lower compared with unmodified LL-37. This again points to the importance of the specific LL-37 sequence in the formation of strong hydrophobic bonds at the actin LL-37-actin interface.…”

“…8) and bundling experiments (39) indicates that LL-37 binds to the DNase I binding loop (D-loop) of actin (52). This conclusion was supported by the results of subtilisin cleavage of actin, which showed that the integrity of the D-loop is a prerequisite to the tight binding of LL-37 to actin (Fig.…”

“…Cofilin has an extended binding site on actin, which includes the D-loop that has a secondary role in cofilin binding (48). Recently, we found that both these proteins dissociate LL-37-induced F-actin bundles and that the dissociating effect of DNase I is much stronger than that of cofilin (39). On the basis of these results, we assumed that the DNase-binding site of actin has an important role in LL-37 binding.…”

“…1C). Actin was previously shown to interact with LL-37 (39,40) suggesting that it might mask the sites cleaved by the microbial proteases. Indeed, actin was found to prevent proteolysis of LL-37 by the proteases of P. gingivalis and P. aeruginosa (Fig.…”

“…These bundling factors act by nonspecific electrostatic interactions, which eliminate repulsion between negatively charged actin filaments (37). LL-37, which is a polycationic polypeptide, also acts as an actin-bundling factor (38,39). The LL-37-induced actin bundles contribute to the accumulation of sputum (40), which is a primary cause of bacterial infections and death in patients with cystic fibrosis (41).…”