Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France.
CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF, Cal, Department of Physics, University of Calabria, Rende, Italy.
Biochim Biophys Acta Gen Subj. 2018 Jun;1862(6):1283-1295. doi: 10.1016/j.bbagen.2018.03.009. Epub 2018 Mar 10.
NUPR1 is a multifunctional intrinsically disordered protein (IDP) involved, among other functions, in chromatin remodelling, and development of pancreatic ductal adenocarcinoma (PDAC). It interacts with several biomolecules through hydrophobic patches around residues Ala33 and Thr68. The drug trifluoperazine (TFP), which hampers PDAC development in xenografted mice, also binds to those regions. Because of the large size of the hot-spot interface of NUPR1, small molecules could not be adequate to modulate its functions.
We explored how amphipathic helical-designed peptides were capable of interacting with wild-type NUPR1 and the Thr68Gln mutant, inhibiting the interaction with NUPR1 protein partners. We used in vitro biophysical techniques (fluorescence, circular dichroism (CD), nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC)), in silico studies (docking and molecular dynamics (MD)), and in cellulo protein ligation assays (PLAs) to study the interaction.
Peptide dissociation constants towards wild-type NUPR1 were ~ 3 μM, whereas no interaction was observed with the Thr68Gln mutant. Peptides interacted with wild-type NUPR1 residues around Ala33 and residues at the C terminus, as shown by NMR. The computational results clarified the main determinants of the interactions, providing a mechanism for the ligand-capture that explains why peptide binding was not observed for Thr68Gln mutant. Finally, the in cellulo assays indicated that two out of four peptides inhibited the interaction of NUPR1 with the C-terminal region of the Polycomb RING protein 1 (C-RING1B).
Designed peptides can be used as lead compounds to inhibit NUPR1 interactions.
Peptides may be exploited as drugs to target IDPs.
NUPR1 是一种多功能的无规卷曲蛋白（IDP），除其他功能外，还参与染色质重塑和胰腺导管腺癌（PDAC）的发展。它通过残基 Ala33 和 Thr68 周围的疏水区与几种生物分子相互作用。药物三氟拉嗪（TFP）可阻止异种移植小鼠中 PDAC 的发展，也与这些区域结合。由于 NUPR1 热点界面的尺寸较大，小分子不足以调节其功能。
我们探索了两亲性螺旋设计肽如何与野生型 NUPR1 和 Thr68Gln 突变体相互作用，抑制与 NUPR1 蛋白伴侣的相互作用。我们使用体外生物物理技术（荧光、圆二色性（CD）、核磁共振（NMR）和等温热力学滴定（ITC））、计算研究（对接和分子动力学（MD））和细胞内蛋白质连接测定（PLAs）来研究相互作用。
肽与野生型 NUPR1 的解离常数约为 3µM，而与 Thr68Gln 突变体则没有相互作用。NMR 显示肽与野生型 NUPR1 残基周围的 Ala33 和 C 末端的残基相互作用。计算结果阐明了相互作用的主要决定因素，提供了配体捕获的机制，解释了为什么未观察到 Thr68Gln 突变体的肽结合。最后，细胞内测定表明，四个肽中的两个抑制了 NUPR1 与 Polycomb RING 蛋白 1（C-RING1B）C 末端区域的相互作用。
设计肽可用作抑制 NUPR1 相互作用的先导化合物。
肽可被开发为针对 IDP 的药物。
