Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, 60438, Frankfurt, Germany.
Department of Biochemistry and Molecular Biology I, University of Würzburg, 97074, Würzburg, Germany.
Cell Death Dis. 2023 Oct 12;14(10):674. doi: 10.1038/s41419-023-06213-0.
The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p63/p73 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p63/p73 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.
p53 的两个同源物 p63 和 p73 调节上皮组织中的转录程序，并且这些组织中的几种细胞类型都表达这两种蛋白。p53 家族的所有成员在其活性状态下通过专门的寡聚化结构域形成四聚体，该结构域作为二聚体的二聚体组装。p63 和 p73 的寡聚化结构域具有很高的序列同一性，但 p53 寡聚化结构域更为多样化，并且缺乏其他两个家族成员中存在的功能重要的 C 端螺旋。基于这些结构差异，p53 不会与 p63 或 p73 异源寡聚化。相比之下，p63 和 p73 形成所有可能化学计量的异源寡聚体，由 p63 二聚体和 p73 二聚体构建的异源四聚体在热力学上比两个同源四聚体更稳定。这表明在表达这两种蛋白的细胞中形成了 p63/p73 异源四聚体。到目前为止，还缺乏研究这种异源四聚体生物学功能的工具。在这里，我们报告了生成和表征与 p63/p73 异源四聚体具有高亲和力和选择性结合的设计锚蛋白重复蛋白（DARPin）。使用这些 DARPin，我们能够在实验上证实这种异源四聚体在上皮的小鼠和人类组织中的存在，并表明其水平在鳞状细胞癌中增加。
