Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a.

Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically disordered proteins. DPF3 isoforms differ from their C-terminal region (C-TERb and C-TERa), containing zinc fingers and disordered domains. Herein, we investigated the disorder aggregation properties of C-TER isoforms. In agreement with the predictions, spectroscopy highlighted a lack of a highly ordered structure, especially for C-TERa. Over a few days, both C-TERs were shown to spontaneously assemble into similar antiparallel and parallel ß-sheet-rich fibrils. Altered metal homeostasis being a neurodegeneration hallmark, we also assessed the influence of divalent metal cations, namely Cu2+, Mg2+, Ni2+, and Zn2+, on the C-TER aggregation pathway. Circular dichroism revealed that metal binding does not impair the formation of ß-sheets, though metal-specific tertiary structure modifications were observed. Through intrinsic and extrinsic fluorescence, we found that metal cations differently affect C-TERb and C-TERa. Cu2+ and Ni2+ have a strong inhibitory effect on the aggregation of both isoforms, whereas Mg2+ impedes C-TERb fibrillation and, on the contrary, enhances that of C-TERa. Upon Zn2+ binding, C-TERb aggregation is also hindered, and the amyloid autofluorescence of C-TERa is remarkably red-shifted. Using electron microscopy, we confirmed that the metal-induced spectral changes are related to the morphological diversity of the aggregates. While metal-treated C-TERb formed breakable and fragmented filaments, C-TERa fibrils retained their flexibility and packing properties in the presence of Mg2+ and Zn2+ cations.

In-depth investigation of the effect of pH on the autofluorescence properties of DPF3b and DPF3a amyloid fibrils.

Double PHD fingers 3 (DPF3) protein exists as two splicing variants, DPF3b and DPF3a, the involvement of which in human cancer and neurodegeneration is beginning to be increasingly recognised. Both isoforms have recently been identified as intrinsically disordered proteins able to undergo amyloid fibrillation. Upon their aggregation, DPF3 proteins exhibit an intrinsic fluorescence in the visible range, referred to as deep-blue autofluorescence (dbAF). Comprehension of such phenomenon remaining elusive, we investigated in the present study the influence of pH on the optical properties of DPF3b and DPF3a fibrils. By varying the excitation wavelength and the pH condition, the two isoforms were revealed to display several autofluorescence modes that were defined as violet, deep-blue, and blue-green according to their emission range. Complementarily, analysis of excitation spectra and red edge shift plots allowed to better decipher their photoselection mechanism and to highlight isoform-specific excitation-emission features. Furthermore, the observed violation to Kasha-Vavilov's rule was attributed to red edge excitation shift effects, which were impacted by pH-mediated H-bond disruption, leading to changes in intramolecular charge and proton transfer, or π-electrons delocalisation. Finally, emergence of different autofluorescence emitters was likely related to structurally distinct fibrillar assemblies between isoforms, as well as to discrepancies in the amino acid composition of their aggregation prone regions.