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, 7 (11), e50078

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

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

Asaf Sol et al. PLoS One.

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 MgCl(2) 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Polymerization of MgATP-G-actin by LL-37 and lysozyme at low ionic strength MgATP-G-buffer.
LL-37 (A), or lysozyme (B) were added to pyrene labeled (10% labeling ratio) 2 µM MgATP-G-actin and the polymerization was followed by increase in pyrene fluorescence. Presented data are representative of three independent experiments.
Figure 2
Figure 2. Effect of LL-37and lysozyme, on the polymerization of MgATP-G-actin induced by 100 mM NaCl or 2 mM MgCl2.
4 µM LL-37 (A) or lysozyme (B) were added (simultaneously with NaCl or MgCl2) to pyrene labeled (10% labeling ratio) 2 µM MgATP-G-actin and the polymerization was followed by an increase in pyrene fluorescence. Presented data are representative of three independent experiments.
Figure 3
Figure 3. LL-37, scrambled LL-37 and lysozyme induced bundle formation of Mg-F-actin at low and physiological (100 mM NaCl) ionic strength.
(A) Kinetics of LL-37-induced bundle formation: LL-37 (2–8 µM) was added to MgF-actin (2 µM) and bundle formation was followed as an increase in light scattering at 350 nm. Presented data are representative of three independent experiments. (B) Extent of LL-37 and lysozyme induced bundle formation: LL-37 or lysozyme (0.5–9 µM) were added to Mg-F-actin (4 µM). Samples were centrifuged at 20,800×g, for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. (C) Extent of LL-37 and scrambled LL-37 induced bundle formation: LL-37 or scrambled LL-37 (2–14 µM) were added to MgF-actin (4 µM) and bundle formation was measured as in (B). The presented data are mean and standard deviation of three independent experiments in (B) and (C).
Figure 4
Figure 4. NaCl concentration dependent bundling of MgF-actin by LL-37, lysozyme and sLL-37.
(A) 6 µM lysozyme or LL-37 were added to Mg-F-actin (4 µM). (B) 10 µM LL-37 or sLL-37 were added to 4 µM MgF-actin After 10 min incubation at room temperature samples were centrifuged at 20,800×g for 8 min. The supernatants were analyzed by SDS-PAGE and densitometry. The presented data are mean and standard deviation of three independent experiments.
Figure 5
Figure 5. Effect of 6
µM LL-37 on subtilisin digestion of CaATP-G-actin, MgATP-G-actin and Mg-F-actin. 8 µM CaATP-G-actin or 8 µM MgATP-G-actin were digested by 4 µg/ml subtilisin for 2 min, MgF-actin (8 µM) was digested by 20 µg/ml subtilisin for 30 min in the presence or absence of 6 µM LL-37. Digestions were carried out at 22°C and quenched by 1 mM PMSF. Samples were analyzed by 12% SDS-PAGE and densitometry. Gel insert shows representative actin bands: (A), actin only; (B), CaATP-G-actin, subtilisin, no LL-37; (C), CaATP-G-actin,subtilisin and LL-37, (D) MgATP-G-actin, subtilisin, no LL-37, (E) MgATP-G-actin, subtilisin and LL-37, (F) Mg-F-actin, subtilisin, no LL-37, (G) Mg-F-actin, subtilisin and LL-37, (H) Mg-F-actin, 200 mM NaCl, subtilisin, no LL-37, (I) Mg-F-actin, 200 mM NaCl, subtilisin and LL-37. The presented quantitation data are mean and standard deviation of three independent experiments.
Figure 6
Figure 6. Binding measurements of LL-37 and of scrambled LL-37 to G-actin at high ionic strength.
Biacore sensorgram [fit (solid) and experimental (dashed)] showing the interactions between LL-37 (A) or scrambled LL-37 (B) and actin, using series of concentrations of 0–500 nM (A), 0–2500 nM (B). Calculation for sLL-37 (B) were performed using a steady state affinity model based on the sensorgram shown as an insert in B. Presented data are representative of three independent experiments.
Figure 7
Figure 7. Dissociation of LL-37-induced Mg-F-actin bundles by DNase1 and cofilin at low and physiological ionic strength.
5 µM DNase1 or cofilin were added to 4 µM Mg-F-actin bundles (bundled by 4–9 µM LL-37) in the presence or absence of 100 mM NaCl. Following 30 min incubation the samples were centrifuged at 20,800×g for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. (A) Dissociation by DNase1. (B) Dissociation by cofilin. The presented data are mean and standard deviation of three independent experiments.
Figure 8
Figure 8. Kinetics of dissociation of MgF-actin bundles by DNase 1, cofilin and NaCl.
LL-37 (9 µM) was added to 4 µM MgF-actin (thin arrow), inducing immediate bundling. This was followed by the addition of 6 µM DNase1, 6 µM cofilin, or 200 mM NaCl (thick arrow). Bundling and dissociation were followed as an increase and decrease in light scattering, respectively, measured at 450 nm. Presented data are representative of three independent experiments.
Figure 9
Figure 9. Mechanism of dissociation of LL-37-induced Mg-F-actin bundles by DNase1 and cofilin.
LL-37 (9 µM) was added to 2 µM Mg-F-actin. Following a 10 minute incubation, 6 µM DNase1 or 9 µM cofilin were added. After 30 min incubation the samples were divided into two pools and centrifuged at 20,800×g or 352,271×g for 8 and 60 min, respectively. The supernatants were analyzed by SDS-PAGE and densitometry. Monomer (G-actin) separated from actin filaments (F-actin) by one hour 352,271×g centrifugation. Bundled F-actin was separated from unbundled F-actin by low speed (20,800×g for 8 min) centrifugation. Thus, the three actin forms (G-, unbundled F- and bundled F-actin) were separated from each other by low and high speed centrifugations. Addition of 6 µM DNase1 and 9 µM cofilin to 4 µM Mg-F-actin (bundled by 9 µM LL-37) promoted disappearance of bundles at 100% and 73%, respectively. The presented data are mean and standard deviation of three independent experiments. Gel insert shows representative actin bands: (A) MgF-actin, pre-spin; (B) MgF-actin low speed; (C) MgF-actin high speed centrifugation; (D) MgF-actin, LL-37 pre-spin; (E) MgF-actin, LL-37 low speed; (F) MgF-actin, LL-37 high speed centrifugation; (G) MgF-actin, LL-37, DNase1 pre-spin; (H) MgF-actin, LL-37, DNase1 low speed; (I) MgF-actin, LL-37, DNase1 high speed centrifugation; (J) MgF-actin, LL-37, cofilin pre-spin; (K) MgF-actin, LL-37, cofilin low speed; (L) MgF-actin, LL-37, cofilin high speed centrifugation.
Figure 10
Figure 10. Comparison of DNase1 and cofilin dissociation of LL-37- and lysozyme- induced Mg-F-actin bundles.
Mg-F-actin (4 µM) was bundled by 9 µM LL-37 or lysozyme in the presence of 100 mM NaCl. After 10 min of incubation, DNase1 or cofilin were added in increasing concentrations to bundled actin. After 30 min of incubation, the samples were centrifuged at 20,800×g for 8 min and the supernatants were analyzed by SDS-PAGE and densitometry. (A) Unbundling of LL-37-induced Mg-F-actin bundles. (B) Unbundling of lysozyme-induced Mg-F-actin bundles. The presented data are mean and standard deviation of three independent experiments.

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This work was supported by the Israel Science Foundation (grant No. 208/10). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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