Laboratory of Molecular Function of Food, Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Laboratory of Bio-organic Chemistry, Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Nutrients. 2020 Sep 18;12(9):2859. doi: 10.3390/nu12092859.
IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel cholesterol-lowering pentapeptide derived from bovine milk β-lactoglobulin. However, the molecular mechanisms underlying the IIAEK-mediated suppression of intestinal cholesterol absorption are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in a human intestinal model using Caco-2 cells. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1). Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Through photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered the common IIAEK-binding amino acid sequence, GFYLFVEGGR. IIAEK significantly increased IAP mRNA and protein levels while decreasing ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway involving the specific activation of IAP and downregulation of intestinal ABCA1.
IIAEK（异亮氨酰-异亮氨酰-丙氨酰-谷氨酸-赖氨酸，乳白蛋白抑制素）是一种新型的降胆固醇五肽，来源于牛乳β-乳球蛋白。然而，IIAEK 介导的抑制肠道胆固醇吸收的分子机制尚不清楚。因此，我们使用 Caco-2 细胞在人肠道模型中评估了 IIAEK 对肠道胆固醇代谢的影响。我们发现 IIAEK 可显著降低肠道胆固醇代谢相关基因的表达，尤其是 ATP 结合盒转运体 A1（ABCA1）的表达。随后，我们通过化学合成了一种新型分子探针 IIXEK，该探针可通过荧光物质可视化与光亲和标记的 IIAEK 相互作用的靶蛋白复合物。通过 IIXEK 对大鼠小肠黏膜和 Caco-2 细胞肠脂筏部分的光亲和标记和 MS 分析，我们鉴定出肠碱性磷酸酶（IAP）是与 IIAEK 相互作用的特异性分子，并发现了共同的 IIAEK 结合氨基酸序列 GFYLFVEGGR。IIAEK 可显著增加 Caco-2 细胞中的 IAP mRNA 和蛋白水平，同时降低 ABCA1 mRNA 和蛋白水平。综上所述，我们发现 IIAEK 通过一种涉及 IAP 特异性激活和肠道 ABCA1 下调的新型信号通路靶向 IAP，从而改善胆固醇代谢。
