Helix-Aggregation Interplay in Nucleophosmin 1: Structural, Morphological, and Cytotoxic Consequences of Fragment...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Nucleophosmin 1 (NPM1) is implicated in acute myeloid leukemia (AML) pathogenesis.
• NPM1 misfolding involves liquid-liquid phase separation (LLPS) and amyloid-like aggregate formation.
• The C-terminal helical region (residues 264-277) is key to NPM1 amyloidogenicity.

Purpose of the Study:

• To investigate how N- and C-terminal sequence extensions affect the aggregation of NPM1 C-terminal fragments.
• To understand the structural basis of NPM1 aggregation and its modulation by sequence context.
• To explore the potential of helical stabilization as a therapeutic strategy against NPM1 misfolding.

Main Methods:

• Thioflavin T (ThT) kinetics to assess aggregation rates.
• Circular dichroism (CD), Fourier transform infrared (FT-IR), and NMR spectroscopy to analyze secondary structure.
• Electron microscopy (EM) to visualize fibril morphology.
• Cell viability assays (OCI-AML2 cells) to determine cytotoxicity.

Main Results:

• NPM1 fragments (NPM1_259-280 and NPM1_263-280) showed distinct aggregation profiles, with the shorter fragment aggregating faster.
• Both fragments exhibited intrinsic α-helical propensity, which was stabilized by 2,2,2-trifluoroethanol (TFE).
• TFE reduced β-sheet formation, delayed aggregation, altered fibril morphology, and decreased cytotoxicity.
• N-terminal extension of NPM1_259-280 stabilized α-helical structure, delaying aggregation and reducing cytotoxicity.

Conclusions:

• N-terminal extension provides a protective effect by stabilizing α-helical structure in the aggregation-prone NPM1 C-terminal region.
• This stabilization delays the α → β transition, inhibiting fibril formation and associated cytotoxicity.
• Helical stabilization emerges as a potential strategy to mitigate NPM1 aggregation and its role in AML.