Byron Caughey

The most infectious prion protein particles

Neurodegenerative diseases such as Alzheimers, Parkinsons and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrPres, a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrPsen) into PrPres (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrPres (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrPres-containing aggregates, PrPres was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17–27-nm (300–600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of ≤5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14–28 PrP molecules, are the most efficient initiators of TSE disease.

Prions and their partners in crime

Prions, the infectious agents of transmissible spongiform encephalopathies (TSEs), have defied full characterization for decades. The dogma has been that prions lack nucleic acids and are composed of a pathological, self-inducing form of the hosts prion protein (PrP). Recent progress in propagating TSE infectivity in cell-free systems has effectively ruled out the involvement of foreign nucleic acids. However, host-derived nucleic acids or other non-PrP molecules seem to be crucial. Interactions between TSE-associated PrP and its normal counterpart are also pathalogically important, so the physiological functions of normal PrP and how they might be corrupted by TSE infections have been the subject of recent research.

STRAIN-DEPENDENT DIFFERENCES IN BETA -SHEET CONFORMATIONS OF ABNORMAL PRION PROTEIN

Strain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm−1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629–1630 cm−1 and stronger band intensity at both 1616 and 1694–1695 cm−1. Because absorbances from 1616 to 1636 cm−1 of protein infrared spectra are attributed primarily to β-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with β-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.

Lysosomotropic Agents and Cysteine Protease Inhibitors Inhibit Scrapie-Associated Prion Protein Accumulation

We report that lysosomotropic agents and cysteine protease inhibitors inhibited protease-resistant prion protein accumulation in scrapie-infected neuroblastoma cells. The inhibition occurred without either apparent effects on normal prion protein biosynthesis or turnover or direct interactions with prion protein molecules. The findings introduce two new classes of inhibitors of the formation of protease-resistant prion protein.

Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of deer and elk, and little is known about its transmissibility to other species. An important factor controlling interspecies TSE susceptibility is prion protein (PrP) homology between the source and recipient species/genotypes. Furthermore, the efficiency with which the protease‐resistant PrP (PrP‐res) of one species induces the in vitro conversion of the normal PrP (PrP‐sen) of another species to the protease‐resistant state correlates with the cross‐species transmissibility of TSE agents. Here we show that the CWD‐associated PrP‐res (PrPCWD) of cervids readily induces the conversion of recombinant cervid PrP‐sen molecules to the protease‐resistant state in accordance with the known transmissibility of CWD between cervids. In contrast, PrPCWD‐induced conversions of human and bovine PrP‐sen were much less efficient, and conversion of ovine PrP‐sen was intermediate. These results demonstrate a barrier at the molecular level that should limit the susceptibility of these non‐cervid species to CWD.

Getting a Grip on Prions: Oligomers, Amyloids, and Pathological Membrane Interactions*

The prion (infectious protein) concept has evolved with the discovery of new self-propagating protein states in organisms as diverse as mammals and fungi. The infectious agent of the mammalian transmissible spongiform encephalopathies (TSE) has long been considered the prototypical prion, and recent cell-free propagation and biophysical analyses of TSE infectivity have now firmly established its prion credentials. Other disease-associated protein aggregates, such as some amyloids, can also have prion-like characteristics under certain experimental conditions. However, most amyloids appear to lack the natural transmissibility of TSE prions. One feature that distinguishes the latter from the former is the glycophosphatidylinositol membrane anchor on prion protein, the molecule that is corrupted in TSE diseases. The presence of this anchor profoundly affects TSE pathogenesis, which involves major membrane distortions in the brain, and may be a key reason for the greater neurovirulence of TSE prions relative to many other autocatalytic protein aggregates.

Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein.

The scrapie prion protein isoform, PrPSc, is a prion-associated marker that seeds the conformational conversion and polymerization of normal protease-sensitive prion protein (PrP-sen). This seeding activity allows ultrasensitive detection of PrPSc using cyclical sonicated amplification (PMCA) reactions and brain homogenate as a source of PrP-sen. Here we describe a much faster seeded polymerization method (rPrP-PMCA) which detects ≥50 ag of hamster PrPSc (≈0.003 lethal dose) within 2–3 d. This technique uses recombinant hamster PrP-sen, which, unlike brain-derived PrP-sen, can be easily concentrated, mutated and synthetically tagged. We generated protease-resistant recombinant PrP fibrils that differed from spontaneously initiated fibrils in their proteolytic susceptibility and by their infrared spectra. This assay could discriminate between scrapie-infected and uninfected hamsters using 2-μl aliquots of cerebral spinal fluid. This method should facilitate the development of rapid, ultrasensitive prion assays and diagnostic tests, in addition to aiding fundamental studies of structure and mechanism of PrPSc formation.

Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrPSc) into contiguous membranes

Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol‐anchor that associates with detergent‐resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease‐sensitive PrP isoform (PrP‐sen) to the protease‐resistant isoform (PrP‐res). A cell‐free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP‐sen and brain microsomes from scrapie‐infected mice as a source of PrP‐res. Interestingly, DRM‐associated PrP‐sen was not converted to PrP‐res until the PrP‐sen was either released from DRMs by treatment with phosphatidylinositol‐specific phospholipase C (PI‐PLC), or the combined membrane fractions were treated with the membrane‐fusing agent polyethylene glycol (PEG). PEG‐assisted conversion was optimal at pH 6–7, and acid pre‐treating the DRMs was not sufficient to permit conversion without PI‐PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP‐res during TSE infection requires (i) removal of PrP‐sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP‐res into the raft domains of recipient cells.

Specific binding of normal prion protein to the scrapie form via a localized domain initiates its conversion to the protease-resistant state.

In the transmissible spongiform encephalopathies, normal prion protein (PrP‐sen) is converted to a protease‐resistant isoform, PrP‐res, by an apparent self‐propagating activity of the latter. Here we describe new, more physiological cell‐free systems for analyzing the initial binding and subsequent conversion reactions between PrP‐sen and PrP‐res. These systems allowed the use of antibodies to map the sites of interaction between PrP‐sen and PrP‐res. Binding of antibodies (α219–232) to hamster PrP‐sen residues 219–232 inhibited the binding of PrP‐sen to PrP‐res and the subsequent generation of PK‐resistant PrP. However, antibodies to several other parts of PrP‐sen did not inhibit. The α219–232 epitope itself was not required for PrP‐res binding; thus, inhibition by α219–232 was likely due to steric blocking of a binding site that is close to, but does not include the epitope in the folded PrP‐sen structure. The selectivity of the binding reaction was tested by incubating PrP‐res with cell lysates or culture supernatants. Only PrP‐sen was observed to bind PrP‐res. This highly selective binding to PrP‐res and the localized nature of the binding site on PrP‐sen support the idea that PrP‐sen serves as a critical ligand and/or receptor for PrP‐res in the course of PrP‐res propagation and pathogenesis in vivo.

Sulfated glycans and elevated temperature stimulate PrPSc-dependent cell-free formation of protease-resistant prion protein

A conformational conversion of the normal, protease‐ sensitive prion protein (PrP‐sen or PrPC) to a protease‐resistant form (PrP‐res or PrPSc) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP‐res deposits in vivo, suggesting that they may facilitate PrP‐res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP‐res accumulation and serve as prophylactic anti‐TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP‐res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell‐free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP‐res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP‐sen that may potentiate its PrP‐res‐induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.