Abstract
Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an α-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization.
Highlights
Nucleophosmin 1 (NPM1, known as B23.1, No38 and numatrin) is an abundant multifunctional protein belonging to the nucleoplasmin family of nuclear chaperones [1, 2]
The classification of acute myeloid leukemia (AML) into prognostic subgroups depending on the specific gene mutations, the so-called ‘‘AML with gene mutations’’, includes mutations in Fms-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1) and chloramphenicol acetyltransferase-box enhancer-binding protein alpha (CEBPA) [40]
This is probably necessary for the survival of leukaemic cells [44], since NPM1 mutations in AML are always heterozygous and knock-out mice with complete deletion of the NPM1 gene die during early embryogenesis [10]
Summary
Nucleophosmin 1 (NPM1, known as B23.1, No38 and numatrin) is an abundant multifunctional protein belonging to the nucleoplasmin family of nuclear chaperones [1, 2]. This protein is present in high amounts in the granular region of nucleoli, taking part in rRNA maturation processes and it is essential for embryonic development. The N-terminal domain is the oligomerization domain mainly involved in its chaperone activity, it extends for approximately 100 residues and displays an eight-stranded β-barrel fold. NMR analysis showed that these three helices span over residues 243–259 (H1), 264–277 (H2), 280–294 (H3) [6]
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