Clinical Systems Biology Laboratories, Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China.
Department of Biochemistry, Faculty of Life Science, Faculty of Natural Science, Imperial College London, London, SW7 2AZ, United Kingdom.
Eur J Med Chem. 2024 Mar 5;267:116203. doi: 10.1016/j.ejmech.2024.116203. Epub 2024 Feb 1.
Quercetin is widely distributed in nature and abundant in the human diet, which exhibits diverse biological activities and potential medical benefits. However, there remains a lack of comprehensive understanding about its cellular targets, impeding its in-depth mechanistic studies and clinical applications.
This study aimed to profile protein targets of quercetin at the proteome level.
A label-free CETSA-MS proteomics technique was employed for target enrichment and identification. The R package Inflect was used for melting curve fitting and target selection. D3Pocket and LiBiSco tools were used for binding pocket prediction and binding pocket analysis. Western blotting, molecular docking, site-directed mutagenesis and pull-down assays were used for target verification and validation.
We curated a library of direct binding targets of quercetin in cells. This library comprises 37 proteins that show increased thermal stability upon quercetin binding and 33 proteins that display decreased thermal stability. Through Western blotting, molecular docking, site-directed mutagenesis and pull-down assays, we validated CBR1 and GSK3A from the stabilized protein group and MAPK1 from the destabilized group as direct binding targets of quercetin. Moreover, we characterized the shared chemical properties of the binding pockets of quercetin with targets.
Our findings deepen our understanding of the proteins pivotal to the bioactivity of quercetin and lay the groundwork for further exploration into its mechanisms of action and potential clinical applications.
槲皮素在自然界中广泛分布，在人类饮食中也很丰富，具有多种生物活性和潜在的医学益处。然而，人们对其细胞靶标仍缺乏全面的了解，这阻碍了对其深入的机制研究和临床应用。
本研究旨在对槲皮素的蛋白质靶标进行蛋白质组水平的分析。
采用无标记 CETSA-MS 蛋白质组学技术进行靶标富集和鉴定。使用 R 包 Inflect 进行熔解曲线拟合和靶标选择。使用 D3Pocket 和 LiBiSco 工具进行结合口袋预测和结合口袋分析。采用 Western blot、分子对接、定点突变和 pull-down 实验进行靶标验证和确证。
我们构建了一个槲皮素在细胞内直接结合靶标的文库。该文库包含 37 个蛋白，这些蛋白与槲皮素结合后热稳定性增加，33 个蛋白与槲皮素结合后热稳定性降低。通过 Western blot、分子对接、定点突变和 pull-down 实验，我们验证了稳定蛋白组中的 CBR1 和 GSK3A 以及不稳定蛋白组中的 MAPK1 是槲皮素的直接结合靶标。此外，我们还对槲皮素与靶标的结合口袋的共享化学性质进行了表征。
我们的研究结果加深了对槲皮素生物活性相关蛋白的理解，为进一步探索其作用机制和潜在的临床应用奠定了基础。
