Characterization of a New CDC73 Missense Mutation that Impairs Parafibromin Expression and Nucleolar Localization

• Published in: PloS one Vol. 9; no. 5; p. e97994
• Main Authors: Masi, Giulia, Iacobone, Maurizio, Sinigaglia, Alessandro, Mantelli, Barbara, Pennelli, Gianmaria, Castagliuolo, Ignazio, Palù, Giorgio, Barzon, Luisa
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 19.05.2014; Public Library of Science (PLoS)
• Subjects: Amino acids; Apoptosis; Base Sequence; Biology and Life Sciences; c-Myc protein; Cancer; Cell cycle; Cell division; Cell Proliferation; Confocal microscopy; DNA Mutational Analysis; DNA Primers - genetics; Family medical history; Flow Cytometry; Gene expression; Gene Expression Regulation - genetics; Genetic aspects; HeLa Cells; Humans; Hyperparathyroidism; Immunohistochemistry; Immunoreactivity; Jaw; Localization; Mandible; Medicine; Medicine and Health Sciences; Microscopy; Microscopy, Confocal; Microscopy, Fluorescence; Missense mutation; Molecular Sequence Data; Mutagenesis; Mutants; Mutation; Mutation, Missense - genetics; Myc protein; N-Terminus; Nucleoli; Parathyroid; Parathyroid hormones; Patients; Proteasome inhibitors; Proteasomes; Proteins; Real-Time Polymerase Chain Reaction; Research and Analysis Methods; Reverse Transcriptase Polymerase Chain Reaction; Saccharomyces cerevisiae; Sequence Analysis, DNA; Tumor suppressor genes; Tumor Suppressor Proteins - genetics; Tumor Suppressor Proteins - metabolism; Tumors; Uterus; Western blotting; Italy
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0097994

Mutations of the Cell Division Cycle 73 (CDC73) tumor suppressor gene (previously known as HRPT2), encoding for parafibromin, are associated with the Hyperparathyroidism-Jaw Tumor (HPT-JT) syndrome, an autosomal dominant disease whose clinical manifestations are mainly parathyroid tumors and, less frequently, ossifying fibromas of the jaws, uterine and renal tumors. Most mutations of CDC73 are nonsense or frameshift, while missense mutations are rare and generally affect the N-terminal domain of parafibromin, a region that is still poorly characterized. The aim of this study was to characterize a novel somatic CDC73 missense mutation (Ile60Asn) identified in the mandibular tumor of a HPT-JT patient carrying a germline CDC73 inactivating mutation. Immunostaining of the tumor showed reduced nuclear parafibromin immunoreactivity. Western blotting and confocal microscopy of transfected cells demonstrated that the Ile60Asn mutant parafibromin was less expressed than the wild-type protein and exhibited impaired nucleolar localization. Treatment of transfected cells with translation and proteasome inhibitors demonstrated a decreased stability of the Ile60An mutant, partially due to an increase in proteasomal degradation. Overexpression of the Ile60Asn mutant led to increased cell proliferation and to accumulation in the G2/M phase of cell cycle. Moreover, mutant parafibromin lost the ability to down-regulate c-myc expression. In conclusion, our study shows that a missense mutation in the N-terminus of parafibromin, identified in an ossifying fibroma from a HPT-JT patient, stimulated cell proliferation and impaired parafibromin expression and nucleolar localization, suggesting a relevant role of the N-terminal domain for parafibromin function.