n端修饰的CCL5趋化因子的聚类构建,用于交叉醛醇生物偶联的光亲和受体下拉

IF 3.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Afzaal Tufail, Matthew E Warnes, Nathalie Signoret, Martin A Fascione
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引用次数: 0

摘要

趋化因子如CCL5 (RANTES)通过与g蛋白偶联受体(如CCR5)相互作用介导免疫反应,CCR5也作为HIV-1进入宿主细胞的共同受体。改良的CCL5类似物已经显示出作为抗hiv策略的CCR5拮抗剂的前景,但目前的方法涉及水解不稳定的连接或费力的合成。在这里,我们展示了使用有机催化剂介导的蛋白醛醇连接(OPAL)来构建具有水解稳定碳-碳键的n端修饰CCL5类似物。利用重组CCL5 P2G突变体和选择性氧化在n端引入α-氧醛,我们实现了OPAL与多种醛供体(包括烷基和芳基乙醛)的高效生物偶联。值得注意的是,一个4-叠氮基芳基乙醛CCL5 OPAL产品被用作CCR5光亲和探针。这种修饰的趋化因子通过光交联成功地从哺乳动物细胞中捕获CCR5,使受体下拉能够进行生化分析。我们的工作表明,交叉醛醇生物偶联是稳定趋化因子功能化的一种通用和收敛策略,在趋化因子受体相互作用的治疗开发和机制研究中具有潜在的应用。该方法为调节或探测CCL5-CCR5轴提供了一个有前景的化学生物学平台,具有更高的精度和合成可及性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Convergent construction of N-terminally modified CCL5 chemokines for photoaffinity receptor pull-down using cross-aldol bioconjugations.

Chemokines such as CCL5 (RANTES) mediate immune responses via interaction with G-protein-coupled receptors like CCR5, which also serves as a co-receptor for HIV-1 entry into host cells. Modified CCL5 analogues have shown promise as CCR5 antagonists for anti-HIV strategies, but current approaches involve hydrolytically unstable linkages or laborious synthesis. Here, we demonstrate the use of an organocatalyst-mediated protein aldol ligation (OPAL) to construct N-terminally modified CCL5 analogues bearing hydrolytically stable carbon-carbon linkages. Using a recombinant CCL5 P2G mutant and selective oxidation to introduce an α-oxo aldehyde at the N-terminus, we achieved efficient OPAL bioconjugation with various aldehyde donors, including alkyl and aryl acetaldehydes. Notably, a 4-azido aryl acetaldehyde CCL5 OPAL product was utilised as a CCR5 photoaffinity probe. This modified chemokine successfully captured CCR5 from mammalian cells via photo-crosslinking, enabling receptor pull-down for biochemical analysis. Our work showcases cross-aldol bioconjugations as a versatile and convergent strategy for stable chemokine functionalisation, with potential applications in therapeutic development and mechanistic studies of chemokine-receptor interactions. This method offers a promising chemical biology platform for modulating or probing the CCL5-CCR5 axis with enhanced precision and synthetic accessibility.

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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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