No abstract

Conversion of the cellular prion protein (PrP C ) into its altered conformation, PrPSc , is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher ␤-sheet content and characteristics similar to those of PrP Sc . RNA molecules can also interact with PrP and potentially modulate PrP C to PrP Sc conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP C 3 PrP Sc conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.Prion diseases can be infectious, sporadic, or inherited (1). Regardless of their origin, they are related to modifications of a ubiquitous membrane-anchored protein, the prion protein (PrP).3 Through a poorly understood process, the cellular PrP isoform (PrP C ), an ␣-helix-rich protein, undergoes a profound conformational change, acquiring higher ␤-sheet content; the latter isoform is known as PrP Sc (Sc from scrapie) and is the only known component of the infectious prion particle (1-4).The protein-only hypothesis postulates that PrP Sc "multiplies" by catalyzing the conversion of PrP C into a likeness of itself, thus becoming responsible for its own propagation (5). This hypothesis is based strongly on the fact that PrP knock-out mice are resistant to prion infection, suggesting that endogenous PrP is necessary for prion propagation and infection (6). It was also suggested, however, that an additional unknown factor could influence the PrP C to PrP Sc conversion (7-10). This molecule would act by lowering the free energy barrier between PrP C and PrP Sc and triggering conversion (11,12). In this field, a great number of biological macromolecules have emerged as candidates for conversion catalysts. Cellular adhesion molecules, nucleic acids (NAs), basal membrane molecules, and sulfated glycans, among other biological macromolecules, have been reported to interact with PrP C and to induce its conversion in...

Protein misfolding has been implicated in a large number of diseases termed protein- folding disorders (PFDs), which include Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington’s disease, and type II diabetes. In these diseases, large quantities of incorrectly folded proteins undergo aggregation, destroying brain cells and other tissues.The interplay between ligand binding and hydration is an important component of the formation of misfolded protein species. Hydration drives various biological processes, including protein folding, ligand binding, macromolecular assembly, enzyme kinetics, and signal transduction. The changes in hydration and packing, both when proteins fold correctly or when folding goes wrong, leading to PFDs, are examined through several biochemical, biophysical, and structural approaches. Although in many cases the binding of a ligand such as a nucleic acid helps to prevent misfolding and aggregation, there are several examples in which ligands induce misfolding and assembly into amyloids. This occurs simply because the formation of structured aggregates (such as protofibrillar and fibrillar amyloids) involves decreases in hydration, formation of a hydrogen-bond network in the secondary structure, and burying of nonpolar amino acid residues, processes that also occur in the normal folding landscape. In this Account, we describe the present knowledge of the folding and misfolding of different proteins, with a detailed emphasis on mammalian prion protein (PrP) and tumoral suppressor protein p53; we also explore how ligand binding and hydration together influence the fate of the proteins.Anfinsen’s paradigm that the structure of a protein is determined by its amino acid sequence is to some extent contradicted by the observation that there are two isoforms of the prion protein with the same sequence: the cellular and the misfolded isoform. The cellular isoform of PrP has a disordered N-terminal domain and a highly flexible, not-well-packed C-terminal domain, which might account for its significant hydration. When PrP binds to biological molecules, such as glycosaminoglycans and nucleic acids, the disordered segments appear to fold and become less hydrated. Formation of the PrP−nucleic acid complex seems to accelerate the conversion of the cellular form of the protein into the disease-causing isoform. For p53, binding to some ligands, including nucleic acids, would prevent misfolding of the protein. Recently, several groups have begun to analyze the folding−misfolding of the individual domains of p53, but several questions remain unanswered. We discuss the implications of these findings for understanding the productive and incorrect folding pathways of these proteins in normal physiological states and in human disease, such as prion disorders and cancer. These studies are shown to lay the groundwork for the development of new drugs.

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The conversion of cellular prion protein (PrP(C)) into the pathological conformer PrP(Sc) requires contact between both isoforms and probably also requires a cellular factor, such as a nucleic acid or a glycosaminoglycan (GAG). Little is known about the structural features implicit in the GAG-PrP interaction. In the present work, light scattering, fluorescence, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy were used to describe the chemical and physical properties of the murine recombinant PrP 23-231 interaction with low molecular weight heparin (LMWHep) at pH 7.4 and 5.5. LMWHep interacts with rPrP 23-231, thereby inducing transient aggregation. The interaction between murine rPrP and heparin at pH 5.5 had a stoichiometry of 2:1 (LMWHep:rPrP 23-231), in contrast to a 1:1 binding ratio at pH 7.4. At binding equilibrium, NMR spectra showed that rPrP complexed with LMWHep had the same general fold as that of the free protein, even though the binding can be indicated by significant changes in few residues of the C-terminal domain, especially at pH 5.5. Notably, the soluble LMWHep:rPrP complex prevented RNA-induced aggregation. We also investigated the interaction between LMWHep and the deletion mutants rPrP Δ51-90 and Δ32-121. Heparin did not bind these constructs at pH 7.4 but was able to interact at pH 5.5, indicating that this glycosaminoglycan binds the octapeptide repeat region at pH 7.4 but can also bind other regions of the protein at pH 5.5. The interaction at pH 5.5 was dependent on histidine residues of the murine rPrP 23-231. Depending on the cellular milieu, the PrP may expose different regions that can bind GAG. These results shed light on the role of GAGs in PrP conversion. The transient aggregation of PrP may explain why some GAGs have been reported to induce the conversion into the misfolded, scrapie conformation, whereas others are thought to protect against conversion. The acquired resistance of the complex against RNA-induced aggregation explains some of the unique properties of the PrP interaction with GAGs.

Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF).

Despite being referred to as the guardian of the genome, when impacted by mutations, p53 can lose its protective functions and become a renegade. The malignant transformation of p53 occurs on multiple levels, such as altered DNA binding properties, acquisition of novel cellular partners, or associating into different oligomeric states. The consequences of these transformations can be catastrophic. Ongoing studies have implicated different oligomeric p53 species as having a central role in cancer biology; however, the correlation between p53 oligomerization status and oncogenic activities in cancer progression remains an open conundrum. In this review, we summarize the roles of different p53 oligomeric states in cancer and discuss potential research directions for overcoming aberrant p53 function associated with them. We address how misfolding and prion-like amyloid aggregation of p53 seem to play a crucial role in cancer development. The misfolded and aggregated states of mutant p53 are prospective targets for the development of novel therapeutic strategies against tumoral diseases.

The conversion of the prion protein (PrP) into scrapie PrP (PrP(Sc)) is a central event in prion diseases. Several molecules work as cofactors in the conversion process, including glycosaminoglycans (GAGs). GAGs exhibit a paradoxical effect, as they convert PrP into protease-resistant PrP (PrP-res) but also exert protective activity. We compared the stability and aggregation propensity of PrP and the heparin-PrP complex through the application of different in vitro aggregation approaches, including real-time quaking-induced conversion (RT-QuIC). Transmissible spongiform encephalopathy-associated forms from mouse and hamster brain homogenates were used to seed RT-QuIC-induced fibrillization. In our study, interaction between heparin and cellular PrP (PrP(C)) increased thermal PrP stability, leading to an 8-fold decrease in temperature-induced aggregation. The interaction of low-molecular-weight heparin (LMWHep) with the PrP N- or C-terminal domain affected not only the extent of PrP fibrillization but also its kinetics, lowering the reaction rate constant from 1.04 to 0.29 s(-1) and increasing the lag phase from 12 to 19 h in RT-QuIC experiments. Our findings explain the protective effect of heparin in different models of prion and prion-like neurodegenerative diseases and establish the groundwork for the development of therapeutic strategies based on GAGs.