G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection |
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Authors: | Pasqualina Liana Scognamiglio Concetta Di Natale Marilisa Leone Mattia Poletto Luigi Vitagliano Gianluca Tell Daniela Marasco |
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Affiliation: | 1. Department of Pharmacy, University of Naples “Federico II”, DFM-Scarl, 80134 Naples, Italy;2. CIRPEB: Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples “Federico II”, DFM-Scarl, 80134 Naples, Italy;3. Center for Advanced Biomaterials for Healthcare@CRIB, Istituto Italiano di Tecnologia (IIT), 80125 Naples, Italy;4. Institute of Biostructures and Bioimaging, CNR, 80134 Naples, Italy;5. Department of Medical and Biological Sciences, University of Udine, 33100 Udine, Italy |
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Abstract: | BackgroundNucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.MethodsThrough a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.ResultsThese results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.ConclusionsThese findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.General observationThis study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease. |
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Keywords: | TIS, Triisopropylsilane TFA, Trifluoroacetic acid DMF, Dimethylformamide DCM, Dichloromethane HBTU, 1-H-Benzotriazolium, 1-[bis(dimethylamino)methylene]-hexafluorophosphate(1-), 3 oxide HOBt, N-hydroxybenzotriazole DIEA, Di-isopropylethylamine Fmoc, Fluorenylmethoxycarbonyl TCEP, Tris(2-Carboxyethyl) phosphine HPLC, High Performance Liquid Chromatography LC&ndash MS, Liquid Chromatography&ndash Mass Spectrometry EDC, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride NHS, N-hydroxy-succinimide NOESY, Nuclear Overhauser Enhancement Spectroscopy RMSD, Root Mean Square Deviation TOCSY, Total Correlation Spectroscopy C_53 NPM1, NPM1 242&ndash 294 C_70 NPM1, NPM1 225&ndash 294 C_107 NPM1, NPM1 188&ndash 294 |
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