Untitled

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP C ) to a disease-specific isomer (PrP Sc ), shifting from a predominantly ␣-helical conformation to one dominated by ␤-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP Sc , is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant ␤-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a ␤-rich conformation. This precursor state is almost as compact as the folded PrP C structure and, as it assembles, only residues 126 -227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

Section: Discussionsupporting

confidence: 50%

Section: Resultsmentioning

confidence: 99%

Conformational Properties of β-PrP

Hosszu

Trevitt

Jones

et al. 2009

“…It has been suggested that oligomerization of PrP can occur through the formation of intermolecular disulfide bonds (50,51). However, oligomers of mPrP-(121-231) were not disassembled even by boiling in the presence of 140 mM dithiothreitol and SDS (Fig.…”

Section: Discussionmentioning

confidence: 94%

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

Martins

Frosoni

Martínez

et al. 2006

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP C , into a misfolded, protease-resistant form, PrP Sc . Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106 -120 from the so-called amyloidogenic core of PrP (residues 106 -126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65°C. Under partially denaturing conditions (65°C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65°C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65°C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65°C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP Sc can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106 -120 of PrP and does not require other cellular factors or a PrP Sc template.The conformational conversion of the normal cellular isoform of the prion protein, PrP C , 4 into an abnormal pathological isoform, PrP Sc , underlies a group of fatal neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases, which includes Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle (1). Neuropathologically, prion diseases are characterized by neuronal loss and astrogliosis and often by spongiform degeneration of the brain and deposition of amyloid plaques (1). The unique feature of these diseases is that, in addition to sporadic and inherited forms, they may be acquired by transmission of an infectious agent. The "proteinonly" hypothesis of prion propagation postulates that the abnormal isoform, PrP Sc , acts as a transmissible agent of the disease and self-propagates its pathological conformation using PrP C as a substrate (1).PrP Sc is defined as an aggregated form of PrP that is largely resistant to proteinase K (PK) digestion under conditions in which PrP C and most other proteins are readily degraded (2). In addition to their different stabilities to proteolytic degradation, the secondary, tertiary, and quaternary structures of PrP C and PrP Sc also differ (3-7...

“…A simple observation that emerges from the examination of the structures of all mammalian recombinant PrPs 1 that have been solved so far in their ␣-helix-rich forms is that they have a highly positively charged N-terminal tail that is flexibly disordered and a stable globular C-terminal domain (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). It is becoming clear that these structural features must have some functional significance.…”

mentioning

confidence: 99%

Characterization of 2′-Fluoro-RNA Aptamers That Bind Preferentially to Disease-associated Conformations of Prion Protein and Inhibit Conversion

Rhie

Kirby

Sayer

et al. 2003

173

148

We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2 -fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrP Sc than for recombinant PrP C and inhibited the accumulation of PrP res in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from nonspecific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, ␣-helix-rich and ␤-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the ␤-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the ␣-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the ␣-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is ␤-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.