title: Nucleophosmin contains amyloidogenic regions that are able to form toxic aggregates under physiological conditions.
abstract: Nucleophosmin (NPM)-1 is a multifunctional protein involved in a variety of biologic processes and has been implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments in NPM1 activities, we dissected the C-terminal domain (CTD) into its helical fragments. In this study, we observed the unexpected structural behavior of the peptide fragment corresponding to helix (H)2 (residues 264-277). This peptide has a strong tendency to form amyloidlike assemblies endowed with fibrillar morphology and β-sheet structure, under physiologic conditions, as shown by circular dichroism, thioflavin T, and Congo red binding assays; dynamic light scattering; and atomic force microscopy. The aggregates are also toxic to neuroblastoma cells, as determined using 3-(4;5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and Ca(2+) influx assays. We also found that the extension of the H2 sequence beyond its N terminus, comprising the connecting loop with H1, delayed aggregation and its associated cytotoxicity, suggesting that contiguous regions of H2 have a protective role in preventing aggregation. Our findings and those in the literature suggest that the helical structures present in the CTD are important in preventing harmful aggregation. These findings could elucidate the pathogenesis of acute myeloid leukemia (AML) caused by NPM1 mutants. Because the CTD is not properly folded in these mutants, we hypothesize that the aggregation propensity of this NPM1 region is involved in the pathogenesis of AML. Preliminary assays on NPM1-Cter-MutA, the most frequent AML-CTD mutation, revealed its significant propensity for aggregation. Thus, the aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology.
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Di Natale
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Pasqualina Liana
authors.initials: PL
authors.lastname: Scognamiglio
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Cascella
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Cecchi
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Russo
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Marilisa
authors.initials: M
authors.lastname: Leone
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Amanda
authors.initials: A
authors.lastname: Penco
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Relini
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Federici
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Adele
authors.initials: A
authors.lastname: Di Matteo
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Chiti
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Vitagliano
authors.affiliation: *Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy daniela.marasco@unina.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Marasco
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000090243:Nucleophosmin; D066263:Protein Aggregation, Pathological; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D000682:Amyloid; C000716967:NPM1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000090243:Nucleophosmin
keywords: AFM; MTT assay; acute myeloid leukemia; circular dichroism spectroscopy; β aggregate