Abstract

The Cholesterol Recognition/Interaction Amino acid Consensus (CRAC) motif is a critical structural element in many membrane proteins, mediating their interaction with cholesterol and regulating diverse cellular functions. This study investigates the effect of structural modifications within CRAC-containing peptides on the cholesterol-dependent activity of the mouse macrophage cell line, IC-21. Specifically, we synthesized a series of CRAC peptides with targeted substitutions in the motif-forming amino acids to probe the structure-activity relationship. Our primary objective was to determine how these subtle changes influence the peptide's ability to modulate key macrophage functions, such as phagocytosis and lipid metabolism, which are intrinsically linked to membrane cholesterol levels. The results demonstrate that the integrity of the CRAC motif's core residues is essential for the peptide's modulatory effect, with specific substitutions leading to a significant alteration or complete loss of activity. These findings provide crucial insights into the molecular mechanism of CRAC-cholesterol interaction and offer a rational basis for designing novel peptide-based tools to selectively regulate cholesterol-dependent cellular processes in macrophages, with potential implications for understanding and treating lipid-related pathologies.