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

Nucleophosmin 1 (NPM1) undergoes liquid-liquid phase separation (LLPS) and can form amyloid-like aggregates when structural elements within its C-terminal domain become destabilized. The helical region spanning residues 264-277 represents a key amyloidogenic segment, and its conformational balance is involved in NPM1 misfolding in acute myeloid leukemia (AML). Here, we examined how N- and C-terminal sequence extensions modulate the structural and aggregative behavior of two C-terminal fragments, NPM1259-280 and NPM1263-280. Although the two fragments exhibit comparable theoretical charge properties, ThT kinetics revealed markedly different aggregation profiles, with the shorter NPM1263-280 fragment aggregating more rapidly and to a greater extent. Circular dichroism, Fourier transform infrared (FT-IR), and NMR analyses demonstrated that both peptides possess an intrinsic α-helical propensity, particularly across residues 267-273, and that stabilization of this helical population by 2,2,2-trifluoroethanol (TFE) reduces β-sheet formation and delays aggregation. Electron microscopy confirmed that the two fragments form fibrils with distinct morphologies and that TFE alters fibril organization by promoting more bundled architectures. Functionally, both peptides reduced OCI-AML2 cell viability, with NPM1259-280 showing the strongest effect; however, helix-stabilizing conditions reduced cytotoxicity. Together, these findings demonstrate that N-terminal extension exerts a protective effect on the aggregation-prone 264-277 region by stabilizing local α-helical structure and delaying the α → β transition that underlies NPM1 aggregation, thereby attenuating fibril formation and cytotoxicity. These results suggest that helical stabilization could limit the aggregation propensity of C-terminal regions of NPM1 and exert a potential protective mechanism against its misfolding.