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Modulation of the Cholesterol-Dependent Activity of Macrophages IC-21 by CRAC Peptides with Substituted Motif-Forming Amino Acids
Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology  (IF),  Pub Date : 2020-10-01, DOI: 10.1134/s1990747820040054
A. Ya. Dunina-Barkovskaya, Kh. S. Vishnyakova

Abstract 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 R TKL W EML V ELGNMDKA V KL W RKL K R (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 S TKL S EML S ELGNMDKA S KL S RKL S R (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 R TKL S EML V ELGNMDKA V KL S RKL K R (Mut3), in which only aromatic amino acids (W) of the CRAC motifs were substituted. Peptide S TKL W EML V ELGNMDKA V KL W RKL S R (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 EWGMA V L W E R NRKLKKDLKVLKMLRT (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-β-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.