Nonspecific Prion Protein–Nucleic Acid Interactions Lead to Different Aggregates and Cytotoxic Species

Abstract

A misfolded form of the prion protein (PrP) is the primary culprit in mammalian prion diseases. It has been shown that nucleic acids catalyze the misfolding of cellular PrP into a scrapie-like conformer. It has also been observed that the interaction of PrP with nucleic acids is nonspecific and that the complex can be toxic to cultured cells. No direct correlation has yet been drawn between changes in PrP structure and toxicity due to nucleic acid binding. Here we asked whether different aggregation, stability, and toxicity effects are detected when nonrelated DNA sequences interact with recombinant PrP. Using spectroscopic techniques to analyze PrP tertiary and secondary structure and cellular assays to assess toxicity, we found that rPrP–DNA interactions lead to different aggregated species, depending on the sequence and size of the oligonucleotide tested. A 21-mer DNA sequence (D67) induced higher levels of aggregation and also dissimilar structural changes in rPrP, compared to binding to oligonucleotides with the same length and different nucleotide sequences or different GC contents. The rPrP–D67 complex induced significant cell dysfunction, which appears to be correlated with the biophysical properties of the complex. Although sequence specificity is not apparent for PrP–nucleic acid interactions, we believe that particular nucleic acid patterns, possibly related to GC content, oligonucleotide length, and structure, govern PrP recognition. Understanding the structural and cellular effects observed for PrP–nucleic acid complexes may shed light on the still mysterious pathology of the prion protein.

The “Jekyll and Hyde” Actions of Nucleic Acids on the Prion-like Aggregation of Proteins

“…Although a specific nucleic acid consensus sequence recognized by PrP has not been defined, there are some sequences to which PrP prefers to bind (43,44). The minimal size of the nucleic acid molecule that can convert PrP into PrP Sc -like species is also arguable (18,36,45).…”

“…It was shown that PrP can bind its own mRNA, which contains G4 motifs, causing it to form a quadruplex structure under particular conditions (47). It is becoming more and more obvious that PrP binds to structured nucleic acids, and both the nucleic acid and the protein undergo structural changes when this interaction occurs (29,43,44,48,49). Indeed, different prion strains display different interactions with RNA molecules when added as conversion cofactors under cell-free conditions (50).…”

The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition

“…Independent studies have shown that nucleic acid interactions facilitate prion fibril formation (27)(28)(29), although the mechanism of such processes is not known. Detailed knowledge about the in vivo substrates of PFAR and direct kinetic analysis will be required to corroborate our hypothesis about the involvement of PFAR in prion mechanisms.…”

Mammalian prion amyloid formation in bacteria

“…13,14 In fact, PrP is considered a 'promiscuous' protein because of its ability to bind to different classes of macromolecules, such as nucleic acids (NAs), lipids, glycosaminoglycans, or even metallic ions; all these cofactors have been shown to trigger changes in PrP conformation leading to aggregation and toxicity. [15][16][17][18][19] It appears that the minimal components necessary to facilitate PrP in vitro conversion and promote de novo prion formation with higher degree of infectivity are polyanions, mainly nucleic acid (NAs) molecules or lipids. [20][21][22] The challenging task of creating recombinant prions that hold strain-like conformational characteristics and infectivity responds to the difficulty in reproducing an in vitro environment with proper facilitating factors, such as molecular crowding, the presence of chaperones and proteases, continuous synthesis of the protein in the ribosome or the presence of interacting NAs or lipids.…”