Modulation of the Cholesterol-Dependent Activity of Macrophages IC-21 by CRAC Peptides with Substituted Motif-Forming Amino Acids.

The activity of many membrane proteins, such as receptors, ionic channels, transporters, and enzymes, is cholesterol dependent; however, mechanisms of the cholesterol-dependent regulation of protein functions remain obscure. Recent studies suggest that membrane proteins can directly interact with cholesterol owing to the presence of the cholesterol-recognizing amino-acid consensus (CRAC) motifs. One of the ways to verify and further develop this notion is a design of CRAC-containing peptides and investigation of their effects on cholesterol-dependent cell functions. Previously we showed that a newly constructed peptide RTKLWEMLVELGNMDKAVKLWRKLKR (peptide P4) containing two CRAC motifs modulates cholesterol-dependent interactions of cultured macrophages IC-21 with 2-µm particles. In this work, in order to clarify the role of CRAC-forming amino acids, we employed the same experimental system to test the activity of peptides closely related to P4 but with modified CRAC motifs. We found that peptide STKLSEMLSELGNMDKASKLSRKLSR (Mut2) analogous to P4, except that all CRAC-forming amino acids (V, W, K/R) were substituted by serine, did not produce any effect in the concentration range 0.5-50 µM corresponding to the range of the P4 activity. Neither was effective peptide RTKLSEMLVELGNMDKAVKLSRKLKR (Mut3), in which only aromatic amino acids (W) of the CRAC motifs were substituted. Peptide STKLWEMLVELGNMDKAVKLWRKLSR (Mut4), in which only cationic amino acids (R/K) in the CRAC motifs were changed, produced almost the same effect as that of peptide P4 with a bell-shape dose-response curve. At low concentrations (1-4 µM) Mut4 notably increased the number of beads per cell, at higher concentrations this parameter diminished, and at 50 µM Mut4 produced a robust toxic effect. Finally, peptide EWGMAVLWERNRKLKKDLKVLKMLRT (Mut1) composed of the same amino acid residues as P4 but in a random order ("scramble") and possessing one CRAC motif, different from that in P4, produced a moderate stimulation at 4-10 µM but was not toxic at 50 µM. As in the case of peptide P4, the effects of Mut4 and Mut1 depended on the cholesterol content in the cell membrane: after the incubation of cells with cholesterol-extracting agent methyl-ß-cyclodextrin stimulatory effects produced by Mut4 and Mut1 at low doses were suppressed. Our results indicate that CRAC motifs play an important role in the mechanisms of the peptide-induced modulations of cholesterol-dependent cell functions in the experimental system used and that of the three motif-forming amino acids, critical is the presence of the aromatic amino acid (W). Further research is required to comprehend the molecular mechanisms of interactions of CRAC-containing peptides with cell membrane components that lead to modulation of cell functions. We anticipate that CRAC-containing peptides may provide a basis for the development of new tools for directed regulation of the activity of target cholesterol-dependent membrane proteins and for the design of new antimicrobial and immunomodulating drugs in particular.

Erratum to: "Amphipathic CRAC-Containing Peptides Derived from the Influenza Virus A M1 Protein Modulate Cholesterol-Dependent Activity of Cultured IC-21 Macrophages" [Biochemistry (Moscow), 83, 982 (2018)].

This corrects the article DOI: 10.1134/S0006297918080096.

Amphipathic CRAC-Containing Peptides Derived from the Influenza Virus A M1 Protein Modulate Cholesterol-Dependent Activity of Cultured IC-21 Macrophages.

Entry of many viral and bacterial pathogens into host cells depends on cholesterol and/or cholesterol-enriched domains (lipid rafts) in the cell membrane. Earlier, we showed that influenza virus A matrix protein M1 contains amphipathic α-helices with exposed cholesterol-recognizing amino acid consensus (CRAC) motifs. In order to test possible functional activity of these motifs, we studied the effects of three synthetic peptides corresponding to the CRAC-containing α-helices of the viral M1 protein on the phagocytic activity of cultured mouse IC-21 macrophages. The following peptides were used: LEVLMEWLKTR (M1 α-helix 3, a.a. 39-49; further referred to as peptide 1), NNMDKAVKLYRKLK (M1 α-helix 6, a.a. 91-105; peptide 2), and GLKNDLLENLQAYQKR (M1 α-helix 13, a.a. 228-243; peptide 3). We found that all three peptides modulated interactions of IC-21 macrophages with non-opsonized 2-µm target particles. The greatest effect was demonstrated by peptide 2: in the presence of 35 µM peptide 2, the phagocytic index of IC-21 macrophages exceeded the control value by 60%; 10-11 mM methyl-ß-cyclodextrin abolished this effect. Peptides 1 and 3 exerted weak inhibitory effect in a narrow concentration range of 5-10 µM. The dose-response curves could be approximated by a sum of two (stimulatory and inhibitory) components with different Hill coefficients, suggesting existence of at least two peptide-binding sites with different affinities on the cell surface. CD spectroscopy confirmed that the peptides exhibit structural flexibility in solutions. Altogether, our data indicate that amphipathic CRAC-containing peptides derived from the viral M1 protein modulate lipid raft-dependent processes in IC-21 macrophages.

Modification of human fibroblast properties in microtransplant.

We studied the properties of human skin fibroblast in filamentous polyglycolic microtransplant. Fibroblast adhesion to the microtransplant filaments is followed by the formation of a network cross-linked with fibroblasts. The cells rapidly proliferate during the first few days; after transfer of the microtransplant to the standard culture flask, the cells migrate to the plastic and continue proliferation. The cells are uniform and exhibit high colony-formation capacity. The bundles of microtransplant filaments persist in the culture for several days and through the cells completely leave them, the area around these filaments remains the most populated for 40 days. Mitotic cells are seen in the immediate proximity to the degrading filaments of the transplant. The effect of cell "rejuvenation" in the microtransplant can be explained by selection of cells by their adhesion to relatively thin (about 15 µ) filaments, which excludes large old cells.