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.

中文翻译：

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具有取代基序形成氨基酸的 CRAC 肽调节巨噬细胞 IC-21 的胆固醇依赖性活性


摘要 许多膜蛋白的活性，如受体、离子通道、转运蛋白和酶，都是胆固醇依赖性的。然而，胆固醇依赖性蛋白质功能调节机制仍不清楚。最近的研究表明，由于胆固醇识别氨基酸共有（CRAC）基序的存在，膜蛋白可以直接与胆固醇相互作用。验证和进一步发展这一概念的方法之一是设计含 CRAC 的肽并研究它们对胆固醇依赖性细胞功能的影响。之前我们表明，新构建的肽 R TKL W EML V ELGNMDKA V KL W RKL KR（肽 P4）含有两个 CRAC 基序，可调节培养巨噬细胞 IC-21 与 2 μm 颗粒的胆固醇依赖性相互作用。在这项工作中，为了阐明CRAC形成氨基酸的作用，我们采用相同的实验系统来测试与P4密切相关但具有修饰的CRAC基序的肽的活性。我们发现肽S TKL S EML S ELGNMDKA S KL S RKL SR (Mut2)与P4类似，除了所有CRAC形成氨基酸(V、W、K/R)被丝氨酸取代外，在浓度范围 0.5–50 μM 对应于 P4 活性范围。 R TKL S EML V ELGNMDKA V KL S RKL KR (Mut3) 都不是有效的肽，其中仅取代了 CRAC 基序的芳香族氨基酸 (W)。仅改变CRAC基序中的阳离子氨基酸(R/K)的肽S TKL W EML V ELGNMDKA V KL W RKL SR (Mut4)，在钟形剂量下产生与肽P4几乎相同的效果–响应曲线。 在低浓度 (1–4 μM) 下，Mut4 显着增加了每个细胞的珠子数量，在较高浓度下，该参数减少，而在 50 μM 下，Mut4 产生了强大的毒性作用。最后，肽 EWGMA VLWER NRKLKKDLKVLKMLRT (Mut1) 由与 P4 相同的氨基酸残基组成，但以随机顺序（“乱序”）组成，并具有一个与 P4 不同的 CRAC 基序，在 4-10 μM 下产生中等刺激，但50 μM 时无毒性。与肽 P4 一样，Mut4 和 Mut1 的作用取决于细胞膜中的胆固醇含量：将细胞与胆固醇提取剂甲基-β-环糊精一起孵育后，低剂量的 Mut4 和 Mut1 产生的刺激作用被抑制。压制。我们的结果表明，CRAC 基序在所用实验系统中肽诱导的胆固醇依赖性细胞功能调节机制中发挥着重要作用，并且在三种基序形成氨基酸中，关键是芳香族氨基酸的存在（W）。需要进一步的研究来理解含 CRAC 的肽与细胞膜成分相互作用的分子机制，从而调节细胞功能。我们预计，含有 CRAC 的肽可能为开发新工具提供基础，用于直接调节目标胆固醇依赖性膜蛋白的活性，特别是设计新的抗菌和免疫调节药物。