Department of Chemistry, Gwangju Institute of Science and Technology, Korea.
FEBS J. 2022 Jun;289(11):3163-3182. doi: 10.1111/febs.16333. Epub 2022 Jan 3.
Cellular senescence is protective against external oncogenic stress, but its accumulation causes aging-related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting with each other and activating p21 transcription. Inhibition of the interaction is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the FOXO4-p53 interaction remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4's forkhead domain (FHD) and p53's transactivation domain (TAD), and between FOXO4's C-terminal transactivation domain (CR3) and p53's DNA-binding domain (DBD), mediate the FOXO4-p53 interaction. Isothermal titration calorimetry data showed that both interactions have micromolar K values, and FOXO4 FHD-p53 TAD interaction has a higher binding affinity. We also showed that the intramolecular CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and FOXO4 CR3 interacts with the DNA/p53 TAD-binding surface of p53 DBD, suggesting a network of potentially competitive and/or coordinated interactions. Based on these results, we propose that a network of intramolecular and intermolecular interactions contributes to the two transcription factors' proper localisation on the p21 promoter and consequently promotes p21 transcription and cell senescence. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence.
细胞衰老对外部致癌应激具有保护作用，但随着其积累会导致与衰老相关的疾病。叉头框蛋白 O4（FOXO4）和 p53 是人源转录因子，已知通过相互作用并激活 p21 转录来促进衰老。抑制相互作用是诱导衰老细胞凋亡的一种策略，但介导 FOXO4-p53 相互作用的结合面仍然难以捉摸。在这里，我们通过 NMR 光谱研究了两个参与 FOXO4 和 p53 相互作用的结合位点。NMR 化学位移扰动分析表明，FOXO4 的叉头结构域（FHD）与 p53 的转录激活结构域（TAD）之间，以及 FOXO4 的 C 端转录激活结构域（CR3）与 p53 的 DNA 结合结构域（DBD）之间的结合介导了 FOXO4-p53 相互作用。等温滴定量热法数据表明，这两种相互作用的 K 值均为微摩尔级，且 FOXO4 FHD-p53 TAD 相互作用具有更高的结合亲和力。我们还表明，FOXO4 FHD 的分子内 CR3 结合表面与 p53 TAD2 相互作用，FOXO4 CR3 与 p53 DBD 的 DNA/p53 TAD 结合表面相互作用，表明存在潜在的竞争性和/或协调性相互作用网络。基于这些结果，我们提出，一个分子内和分子间相互作用网络有助于两个转录因子在 p21 启动子上的适当定位，从而促进 p21 转录和细胞衰老。这项工作提供了分子水平的结构信息，这对于理解负责细胞衰老的两种蛋白质之间的相互作用至关重要。
