Santofimia-Castaño Patricia, Rizzuti Bruno, Pey Ángel L, Soubeyran Philippe, Vidal Miguel, Urrutia Raúl, Iovanna Juan L, Neira José L
Centre de Recherche en Cancérologie de Marseille, INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, 13288 Marseille, France.
Liquid Crystal Laboratory, Consiglio Nazionale delle Ricerche-NANOTEC, Università della Calabria, 87036 Rende, Italy.
Proc Natl Acad Sci U S A. 2017 Aug 1;114(31):E6332-E6341. doi: 10.1073/pnas.1619932114. Epub 2017 Jul 18.
Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 μM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins.
内在无序蛋白（IDP）在真核生物中普遍存在，并且它们常常与人类疾病相关。蛋白NUPR1是一种多功能IDP，参与染色质重塑以及胰腺癌的发生和发展；然而，此类功能的细节尚不清楚。多梳蛋白参与特定的转录级联反应和基因沉默。多梳复合体的一种蛋白是指环蛋白1（RING1）。RING1与多种癌症类型中的侵袭性肿瘤特征相关。在这项工作中，我们在体外、计算机模拟和细胞内对NUPR1与旁系同源物RING1B之间的相互作用进行了表征。这种相互作用通过RING1B的C末端区域（C-RING1B）发生，亲和力在低微摩尔范围内（约10 μM）。通过核磁共振（NMR）确定的NUPR1的结合区域是其序列30s区域的一个疏水多肽片段，这一点通过计算结果和Ala33位点的定点诱变得到证实。如通过蛋白质连接试验所检测，C-RING1B与野生型NUPR1之间的结合在细胞内也会发生；这种相互作用受到三氟拉嗪的抑制，三氟拉嗪是一种已知会阻碍野生型NUPR1与其他蛋白结合的药物。此外，体外、计算机模拟和细胞内研究表明，Thr68Gln和Ala33Gln/Thr68Gln突变体与C-RING1B的结合减少。这是NUPR1的一个折叠良好的相互作用伙伴的例子，因为它的其他相互作用蛋白也是未折叠的。我们推测，NUPR1与多梳蛋白一起在染色质重塑和致癌过程中发挥积极作用。
