Modulation of Cholesterol-Dependent Activity of Macrophages IC-21 by a Peptide Containing Two CRAC-Motifs from Protein M1 of Influenza Virus

Dunina-Barkovskaya, Antonina; Vishnyakova, Khava S.; Baratova, Ludmila A.; Radyukhin, V. A.

doi:10.1134/s1990747819030139

Cited by 7 publications

(32 citation statements)

References 39 publications

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“…5b, 5c), similar to what was observed in the case of peptide P4 ( Fig. 5a; [28]). As was noted above, the mβCD-induced inhibition of the peptide stimulating effect cannot be explained by the toxic effect of mβCD, since in our system mβCD at concentrations below 5 mM did not suppress but stimulated the cell activity (Fig.…”

Section: Discussionsupporting

confidence: 86%

“…Quantification of the peptide effects on the macrophage activity. The activity of macrophages was assessed by their ability to bind non-opsonized latex 2-μm particles and was measured as the number of particles associated with the cells ("phagocytic index") according to the protocol described previously [27][28][29][30][31] with minor modifications. Cells in 6-well plates were preincubated for 1 h at 37°C, 5% CO 2 in serumfree DMEM in order to minimize the inhibitory effect of serum on particle adhesion and phagocytosis [30].…”

Section: Methodsmentioning

confidence: 99%

“…Particle adhesion on the surface of the phagocyte precedes phagocytosis, depends on the availability of phagocytic receptors, including integrins, and is a necessary condition for phagocytosis [32][33][34][35][36]. The number of particles per cell was determined according to the procedure described previously [27][28][29][30][31]. Microphotographs of cells were taken in phase contrast and fluorescence mode (excitation/emission 490/520 and 520/590 nm for fluorescence of particles and autofluorescence of glutaraldehyde, respectively) using fluorescent microscope Zeiss Axiovert 200M (Carl Zeiss, Germany niya) equipped with a digital video camera ORCAII-ERG2 (Hamamatsu, Japan) and corresponding software (Axiovision 4.5, Carl Zeiss Imaging).…”

Section: Methodsmentioning

confidence: 99%

“…All peptides (purity ≥95%) were synthesized in Syneuro Co. Ltd., Russia. Peptide P4 (“parental”) was constructed earlier on the basis of two α-helical CRAC-containing fragments of the M1 protein of the influenza virus [ 27 , 28 ]. Peptide Mut1 consisted of the same 26 amino acids as P4 but in a random order (“scramble”) and contained one incidentally generated CRAC motif, different from the CRAC motifs of peptide P4.…”

Section: Methodsmentioning

confidence: 99%

“…Earlier, we showed that a new peptide RTKLWEMLVELGNMDKAVKLWRKLKR (hereinafter P4), constructed from two CRAC-containing peptides corresponding to α-helices 3 and 6 of the influenza virus M1 protein, dose-dependently modulates cholesterol-dependent interactions of cultured macrophages IC-21 with 2-μm particles [ 27 , 28 ]. In this work, in order to determine the role of CRAC motifs and motif-forming amino acids in the observed effects of P4, we constructed mutant peptides corresponding to P4 in amino acid composition, but with modified CRAC motifs, and investigated the effects of the new peptides in the same experimental system.…”

Section: Introductionmentioning

confidence: 99%

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

Dunina-Barkovskaya

Vishnyakova

2020

Biochem. Moscow Suppl. Ser. A

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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 cholesteroldependent 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-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 par...

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Section: Discussionsupporting

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Dunina-Barkovskaya

Vishnyakova

2020

Biochem. Moscow Suppl. Ser. A

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Interaction of Peptides Containing CRAC Motifs with Lipids in Membranes of Various Composition

Volynsky

Galimzyanov

Akimov

2021

Biochem. Moscow Suppl. Ser. A

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The lateral distribution of integral and peripheral proteins, as well as lipids in the plasma membranes of mammalian cells is extremely heterogeneous. It is believed that various lipid-protein domains are formed in membranes. Domains enriched in sphingomyelin and cholesterol are called rafts. It is assumed that the distribution of proteins into rafts is largely related to the presence in their primary sequence of a specific amino acid region called the CRAC motif, which is responsible for cholesterol binding. In this work, the interaction of two peptides containing CRAC motifs in their structure with membranes of different compositions was studied by means of molecular dynamics. It has been shown that the average number of lipid molecules in contact with each peptide is proportional to the mole fraction of lipid in the membrane. The predominant interaction of peptides with cholesterol was not observed. In addition, cholesterol did not form long-lived contacts with any amino acid or amino acid sequence. We suppose that in some cases the predominant lateral distribution of peptides and proteins containing CRAC motifs into rafts may be due to amphipathicity of the CRAC motif rather than due to specific strong binding of cholesterol.

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Cell Membrane Cholesterol and Regulation of Cellular Processes: New and the Same Old Thing

Dunina-Barkovskaya

2024

Biochem. Moscow Suppl. Ser. A

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Modulation of Cholesterol-Dependent Activity of Macrophages IC-21 by a Peptide Containing Two CRAC-Motifs from Protein M1 of Influenza Virus

Cited by 7 publications

References 39 publications

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

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

Interaction of Peptides Containing CRAC Motifs with Lipids in Membranes of Various Composition

Cell Membrane Cholesterol and Regulation of Cellular Processes: New and the Same Old Thing

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