CHARACTERIZATION OF PRION PROTEIN CONDENSATE AND AGGREGATE FORMATION IN THE PRESENCE OF COPPER IONS AND DNA APTAMERS

The prion protein (PrP) is highly expressed in neurons and can misfold into beta-sheet enriched aggregates, leading to uncurable prionopathies. Although there are different pathogenic mechanisms proposed for amyloidogenic proteins, such as PrP, they are not yet fully understood. Besides, the role of physio/pathological PrP ligands in the PrP misfolding and conversion process is still under debate. Cellular PrP can coordinate up to 6 Cu(II) ions in its flexible N-terminal domain and can bind nucleic acids in adjacent domains. We have previously demonstrated that PrP can form liquid-like or gel-like condensates upon binding to high-affinity structured nucleic acids or copper ions, which act as modulators of phase transitions in vitro. Likewise, we have also shown that these oligonucleotides modify the secondary structure content of PrP90-231 condensates by circular dichroism (CD) spectroscopy and that they can evolve into gel-like and solid-like assemblies overtime. Our main goal is to fully characterize phase transitions of full-length recombinant PrP (rPrP23-231) and N-terminally truncated PrP constructions upon simultaneous exposure to Cu(II) and to 25-mer DNA aptamers (A1 and A1_mut). We will characterize rPrP23-231 interactions with both high-affinity ligands (DNA and copper ions) by using complementary biophysical techniques, such as microscopy, Fluorescence Recovery After Photobleaching (FRAP), X-ray photon correlation spectroscopy, Isothermal Titration Calorimetry and CD. Our fluorescence microscopy preliminary results revealed that the aptamers A1 and A1_mut induced phase separation of PrP into viscous condensates in the presence of copper. Furthermore, FRAP analysis revealed that the addition of Cu(II) (provided as CuCl2) on top of PrP-aptamer treatment further reduced the fluorescence recovery, by using Cy5-labeled aptamers. This result indicates that the PrP condensates formed in the presence of both ligands present different viscoelastic properties than PrP-aptamer or PrP-Cu(II) condensates individually. Next, we will quantify PrP and nucleic acids in the condensates, investigate PrP structural changes upon interaction with both ligands, and refine previously obtained results. In summary, we expect to contribute to the comprehension of nucleic acids’ and Cu(II) role in PrP phase separation and to decipher new mechanisms behind aggregate formation.