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Unveiling the effect of phosphorylation and phosphomimetics on the structural and aggregation properties of the amyloidogenic intrinsically disordered protein DPF3a

par Leyder, Tanguy;Mignon, Julien;Bongiovanni, Emma;Machiels, Quentin ;Waeytens, Jehan ;Raussens, Vincent ;Monari, Antonio;Mottet, Denis;Michaux, Catherine
Référence International journal of biological macromolecules, 335, page (149393)
Publication Publié, 2026-01-01

Article révisé par les pairs

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Résumé :

DPF3a is a human epigenetic regulator involved in chromatin remodelling, cell division, and ciliogenesis. This protein is deregulated in various cancers and neurodegenerative diseases. In our previous work, DPF3a has been described as an amyloidogenic intrinsically disordered protein (IDP). While the casein kinase 2 (CK2) can phosphorylate DPF3a at S138 (pS138), phosphorylation of DPF3a at S348 (pS348) by CK2 has been linked to cardiac hypertrophy. However, no structural information is available on phosphorylated DPF3a. In this study, we investigated the effect of phosphorylation on DPF3a structural and aggregation properties. Two single-mutated phosphomimetics (S138E and S348E) were characterised in vitro and compared to DPF3a WT, while in silico analyses were performed on pS138 and pS348 to assess structural changes at the molecular level. Circular dichroism and fluorescence spectroscopy revealed that both phosphomimetics are hybrid IDPs, with increased turn and antiparallel β-sheet content as well as more buried aromatic residues compared to DPF3a WT, suggesting conformational rearrangements and a more folded N-terminal region. In silico characterisation supported these results, showing that phosphorylation of S138 and S348 induce extended conformation, especially the C-terminal extremity, due to electrostatic repulsion, while local folding occurs due to a proximity with arginine and lysine residues. Complementarily, MD simulations were also performed on phosphomimetics. The resulting analyses revealed trends similar to those observed for pDPF3a, confirming that phosphomimetics faithfully reproduce the structural effects of DPF3a phosphorylation. Furthermore, spectroscopic and microscopic analyses unveiled that S138E and S348E exhibit slower fibrillation kinetics compared to DPF3a WT involving distinct aggregation mechanisms.