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22 pags., 4 figs., 2 tabs. -- This article belongs to the Special Issue The Amazing World of IDPs in Human Diseases ; Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impα3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impα3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impα3 with low micromolar affinity (1.7–27 μM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure. ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI2018-097991-B-I00 to J.L.N. and BFU2016-78232-P to A.V.C.); La Ligue Contre le Cancer, INCa, Canceropole PACA, and INSERM to J.L.I.; Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to OA); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future") (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to A.V.C. and Digestive Pathology Group B25_17R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). The NMR equipment used in this work was funded by the Generalitat Valenciana and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020). ; Peer reviewed

3 pags, 1 fig ; Intrinsically disordered proteins (IDPs) do not have a well-defined structure, but they have key biological tasks in cancer development. By using the disordered cancer-related protein NUPR1 as a proof-of-concept, we have developed a new multidisciplinary approach to tackle drug-design against IDPs, using it to repurpose drugs for treating pancreatic adenocarcinoma (PDAC). ; This work in the authors' laboratories was supported by [La Ligue Contre le Cancer], INCa, [Canceropole PACA] and [INSERM] to JLI and LP; [Miguel Servet Program from Instituto de Salud Carlos III] under grant [CPII13/00017] to OA; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') under grants [PI15/00663 and PI18/00349] to OA; Ministerio de Economia, Industria y Competitividad, Gobierno de Espana under grants [BFU2016-78232-P to AVC, CTQ2015-64445-R to JLN]; [Diputacion General de Aragon] under grants [Protein Targets and Bioactive Compound Group to AVC, and Digestive Pathology Group to OA]; [Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas] (CIBERehd) toOAand AVC; [Programme XUGUANGQI] to YX and JLI; and [National Natural Science Foundation of China (81502920), the Fundamental Research Funds for the Central Universities] under grant [106112017CDJQJ468823] to YX. The Fundacion Alfonso Martin-Escudero and Fondation de France supported to PSC. ; Peer reviewed

14 pags., 5 figs., 1 tab. ; The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity. ; This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/ FEDER, EU) (RTI 2018-097991-B-I00 to JN and PGC 2018- 094548-B-I00 to AA and PM), and the Basque Government (IT1175-19 to AA and PM).

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs. ; This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to J.L.N.]; La Ligue Contre le Cancer, INCa, Canceropole PACA and INSERM to JLI; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI18/00349 to O.A.); Diputacion General de Aragon ('Protein Targets and Bioactive Compounds Group' E45_20R to A.V.-C., 'Digestive Pathology Group' B25_20R to O.A.); Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd) (O.A. and A.V.-C.); CAI YUANPEI Scholarship (201906050187) to Y.X.; and Jeunes Talents France-Chine Program (JTFC) to Y.X. P.S.-C. was supported by Fondation de France; and W.L. by China Scholarship Council. ; Peer reviewed

Patient stratification has been instrumental for the success of targeted therapies in breast cancer. However, the molecular basis of metastatic breast cancer and its therapeutic vulnerabilities remain poorly understood. Here we show that PML is a novel target in aggressive breast cancer. The acquisition of aggressiveness and metastatic features in breast tumours is accompanied by the elevated PML expression and enhanced sensitivity to its inhibition. Interestingly, we find that STAT3 is responsible, at least in part, for the transcriptional upregulation of PML in breast cancer. Moreover, PML targeting hampers breast cancer initiation and metastatic seeding. Mechanistically, this biological activity relies on the regulation of the stem cell gene SOX9 through interaction of PML with its promoter region. Altogether, we identify a novel pathway sustaining breast cancer aggressiveness that can be therapeutically exploited in combination with PML-based stratification. ; The work of A.C. is supported by the Ramón y Cajal award, the Basque Department of Industry, Tourism and Trade (Etortek), Health (2012111086) and Education (PI2012-03), Marie Curie (277043), Movember Foundation (GAP1), ISCIII (PI10/01484, PI13/00031), FERO (VIII Fellowship) and ERC (336343). N.M.-M. and P.A. are supported by the Spanish Association Against Cancer (AECC), AECC JP Vizcaya and Guipuzcoa, respectively. J.U. and F.S. are Juan de la Cierva Researchers (MINECO). L.A., A.A.-A. and L.V.-J. are supported by the Basque Government of education. M.L.-M.C. acknowledges SAF2014-54658-R and Asociación Española contra el Cancer. R.B. acknowledges Spanish MINECO (BFU2014-52282-P, Consolider BFU2014-57703-REDC), the Departments of Education and Industry of the Basque Government (PI2012/42) and the Bizkaia County. M.S., V.S. and J.B. acknowledge Banco Bilbao Vizcaya Argentaria (BBVA) Foundation (Tumour Biomarker Research Program). M.S. and J.B. are supported by NIH grant P30 CA008748. M.dM.V. is supported by the Institute of Health Carlos III (PI11/02251, PI14/01328) and Basque Government, Health Department (2014111145). A.M. is supported by ISCIII (CP10/00539, PI13/02277) and Marie Curie CIG 2012/712404. V.S. is supported by the SCIII (PI13/01714, CP14/00228), the FERO Foundation and the Catalan Agency AGAUR (2014 SGR 1331). R.R.G. research support is provided by the Spanish Ministry of Science and Innovation grant SAF2013-46196, BBVA Foundation, the Generalitat de Catalunya (2014 SGR 535), Institució Catalana de Recerca i Estudis Avançats, the Spanish Ministerio de Economia y Competitividad (MINECO) and FEDER funds (SAF2013-46196). ; This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms12595