Liquid‐liquid phase separation and fibrillation of the prion protein modulated by a high‐affinity DNA aptamer

Abstract

Structural conversion of cellular prion protein (PrP C ) into scrapie PrP (PrP Sc ) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies (TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid-liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25-mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP 90-231 ) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP 90-231 and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration-dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs. |

Phase separation of p53 precedes aggregation and is affected by oncogenic mutations and ligands

“…The samples (20 μL) in buffer A (50 mM Tris–Cl, pH 7.4, 150 mM NaCl and 5 mM DTT) were loaded onto a glass slide and cover slip system prepared as described previously. 66 The images were acquired using a Leica TCS-SPE confocal microscope with a 63×/1.4 oil objective and a 488 nm laser. For fluorescence analysis, the samples were excited at 405 nm, and fluorescence signals were acquired at 450–500 nm.…”

Rabbit PrP Is Partially Resistant to in vitro Aggregation Induced by Different Biological Cofactors

“…Recombinant rabbit PrP c exhibits different electrostatic potential distribution than MuPrP, which has a larger positively charged surface (Wen et al, 2010a;Silva et al, 2011;Matos et al, 2020). This may affect interactions with negatively charged cofactors and, therefore, likely vary with pH.…”

“…DNA can bind with high affinity to the prion protein in vitro and modulate its aggregation (Cordeiro et al, 2001;Macedo et al, 2012). It has also been shown that the DNA structure leads to distinct interactions with MuPrP 90−231 (Matos et al, 2020). In this study, we analyzed two DNA sequences previously described as high-affinity cofactors of MuPrP and evaluated whether they have different binding profiles in their interactions with MuPrP and RaPrP.…”

“…Nucleic acids have been proposed to act as catalysts in the conversion of PrP C to a PrP Sc -like form (Cordeiro et al, 2001;Cordeiro and Silva, 2005). More recently, DNA-induced prionlike conversion in a physiological process was also observed, with specific DNA aptamers modulating PrP liquid-liquid phase separation (Matos et al, 2020). The binding of nucleic acids can thus influence the unfolding of proteins in both disease-related and physiological situations.…”

“…Recently, PrP-DNA interactions were investigated in a more targeted manner, selecting short single-stranded NA sequences that bind with high affinity and specificity to the extended C-terminal domain of the prion protein (PrP 90−231 ). A selected 25-mer apatmer (A1) was able to modulate the phase transition of PrP -from LLPS to aggregation (Matos et al, 2020).…”