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bioRxiv · 10.1101/2025.08.17.670156

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

Leyder, T.·Mignon, J.·Bongiovanni, E.·Machiels, Q.·Waeytens, J.·Raussens, V.·Monari, A.·Mottet, D.·Michaux, C.
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Abstract

The double plant homeodomain fingers 3a (DPF3 isoform a) is a human epigenetic regulator involved in chromatin remodelling, cell division, and ciliogenesis. Most notably, this protein is deregulated in various cancer types and neurodegenerative diseases. In our previous work, the disorder nature of DPF3a, as well as its propensity to aggregate into amyloid fibrils, have been highlighted, making it an amyloidogenic intrinsically disordered protein (IDP). Due to their high chain accessibility, IDPs structure and function are modulated by phosphorylation. It has been reported that phosphorylation of DPF3a at S348 (pS348) by the casein kinase 2 (CK2) is implicated in cardiac hypertrophy. CK2 can also phosphorylate DPF3a at S138 (pS138), which is also located in an intrinsically disordered region (IDR). However, no structural information is available on phosphorylated DPF3a. In the present 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 {beta}-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. Furthermore, spectroscopic and microscopic analyses unveiled that S138E and S348E exhibit slower fibrillation kinetics compared to DPF3a WT involving distinct aggregation mechanisms.

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Leyder, T., Mignon, J., Bongiovanni, E., Machiels, Q., Waeytens, J., Raussens, V., Monari, A., Mottet, D., Michaux, C.. 2025-08-22. Unveiling the effect of phosphorylation on the structural and aggregation properties of the amyloidogenic intrinsically disordered protein DPF3a. https://doi.org/10.1101/2025.08.17.670156

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