Insights into amyloid-like aggregation of H2 region of the C-terminal domain of nucleophosmin

- In this study the role of dissected C-terminal domain (CTD) helical fragments of Nucleophosmin protein was investigated.
- We deepened the tendency of H2 region of to form assemblies with fibrillar morphology through an alanine scan, structural and kinetic analyses.
- CD, Th-T, FT-IR experiments and birefringence of polarized optical microscopy and WAXS assays were employed in this study
- Our study pointed out that the basic amyloidogenic stretch and that Ile269 conferred packing patterns to the fiber.
- Present findings deepen the knowledge of AML molecular mechanisms and the role of cytoplasmatic aggregates of NPM1c +.

Nucleophosmin (NPM1) is a multifunctional protein involved in a variety of biological processes including the pathogenesis of several human malignancies and is the most frequently mutated gene in Acute Myeloid Leukemia (AML). To deepen the role of protein regions in its biological activities, lately we reported on the structural behavior of dissected C-terminal domain (CTD) helical fragments. Unexpectedly the H2 (residues 264-277) and H3 AML-mutated regions showed a remarkable tendency to form amyloid-like assemblies with fibrillar morphology and β-sheet structure that resulted as toxic when exposed to human neuroblastoma cells. More recently NPM1 was found to be highly expressed and toxic in neurons of mouse models of Huntington's disease (HD). Here we investigate the role of each residue in the β-strand aggregation process of H2 region of NPM1 by performing a systematic alanine scan of its sequence and structural and kinetic analyses of aggregation of derived peptides by means of Circular Dichorism (CD) and Thioflavin T (Th-T) assay. These solution state investigations pointed out the crucial role exerted by the basic amyloidogenic stretch of H2 (264-271) and to shed light on the initial and main interactions involved in fibril formation we performed studies on fibrils deriving from the related Ala peptides through the analysis of fibrils with birefringence of polarized optical microscopy and wide-angle X-ray scattering (WAXS). This analysis suggested that the presence of branched Ile269 conferred preferential packing patterns that, instead, appeared geometrically hampered by the aromatic side-chain of Phe268. Present investigations could be useful to deepen the knowledge of AML molecular mechanisms and the role of cytoplasmatic aggregates of NPM1c +.