Scrapie infectivity is independent of amyloid staining properties of the N-terminally truncated prion protein

The prion protein undergoes a profound conformational change when the cellular isoform (PrP(C)) is converted into the disease-causing form (PrP(Sc)). Limited proteolysis of PrP(Sc) produces PrP 27-30, which readily polymerizes into amyloid. To study the relationship between PrP amyloid and infectivity, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes alpha-helix formation, modified the ultrastructure of PrP amyloid and decreased the beta-sheet content as well as prion infectivity. HFIP reversibly decreased the binding of Congo red dye to the PrP amyloid rods while inactivation of prion infectivity was irreversible. In contrast, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolished Congo red binding. Solubilization using various solvents and detergents produced monomeric and dimeric PrP that lacked infectivity. Proteinase K resistance of detergent-treated PrP 27-30 showed no correlation with scrapie infectivity. Our results separate prion infectivity from the amyloid properties of PrP 27-30 and underscore the dependence of prion infectivity on PrP(Sc) conformation. These findings also demonstrate that the specific beta-sheet-rich structures required for prion infectivity can be differentiated from those required for amyloid formation.     

Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy.

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.     

Reconstitution of prion infectivity from solubilized protease-resistant PrP and nonprotein components of prion rods

The scrapie isoform of the prion protein, PrP(Sc), is the only identified component of the infectious prion, an agent causing neurodegenerative diseases such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Following proteolysis, PrP(Sc) is trimmed to a fragment designated PrP 27-30. Both PrP(Sc) and PrP 27-30 molecules tend to aggregate and precipitate as amyloid rods when membranes from prion-infected brain are extracted with detergents. Although prion rods were also shown to contain lipids and sugar polymers, no physiological role has yet been attributed to these molecules. In this work, we show that prion infectivity can be reconstituted by combining Me(2)SO-solubilized PrP 27-30, which at best contained low prion infectivity, with nonprotein components of prion rods (heavy fraction after deproteination, originating from a scrapie-infected hamster brain), which did not present any infectivity. Whereas heparanase digestion of the heavy fraction after deproteination (originating from a scrapie-infected hamster brain), before its combination with solubilized PrP 27-30, considerably reduced the reconstitution of infectivity, preliminary results suggest that infectivity can be greatly increased by combining nonaggregated protease-resistant PrP with heparan sulfate, a known component of amyloid plaques in the brain. We submit that whereas PrP 27-30 is probably the obligatory template for the conversion of PrP(C) to PrP(Sc), sulfated sugar polymers may play an important role in the pathogenesis of prion diseases.     

The role of prion peptide structure and aggregation in toxicity and membrane binding

Prion diseases are neurodegenerative disorders associated with a conformational change in the normal cellular isoform of the prion protein, PrP(C), to an abnormal scrapie isoform, PrP(SC). Unlike the alpha-helical PrP(C), the protease-resistant core of PrP(SC) is predominantly beta-sheet and possesses a tendency to polymerize into amyloid fibrils. We performed experiments with two synthetic human prion peptides, PrP(106-126) and PrP(127-147), to determine how peptide structure affects neurotoxicity and protein-membrane interactions. Peptide solutions possessing beta-sheet and amyloid structures were neurotoxic to PC12 cells in vitro and bound with measurable affinities to cholesterol-rich phospholipid membranes at ambient conditions, but peptide solutions lacking stable beta-sheet structures and amyloid content were nontoxic and possessed less than one tenth of the binding affinities of the amyloid-containing peptides. Regardless of structure, the peptide binding affinities to cholesterol-depleted membranes were greatly reduced. These results suggest that the beta-sheet and amyloid structures of the prion peptides give rise to their toxicity and membrane binding affinities and that membrane binding affinity, especially in cholesterol-rich environments, may be related to toxicity. Our results may have significance in understanding the role of the fibrillogenic cerebral deposits associated with some of the prion diseases in neurodegeneration and may have implications for other amyloidoses.     

Properties of scrapie prion protein liposomes

Purified scrapie prions contain one identifiable macromolecule, PrP 27-30, which polymerizes into rod-shaped amyloids. The rods can be dissociated with retention of scrapie infectivity upon incorporation of PrP 27-30 into detergent-lipid-protein complexes (DLPC) as well as liposomes. As measured by end-point titration, scrapie infectivity was increased greater than 100-fold upon dissociating the rods into liposomes. The incorporation of PrP 27-30 into liposomes was demonstrated by immunoelectron microscopy using colloidal gold. Detergent extraction of prion liposomes followed by chloroform/methanol extraction resulted in the reappearance of rods, indicating that this process is reversible. Scrapie prion infectivity in rods and liposomes was equally resistant to inactivation by irradiation at 254 nm and was unaltered by exposure to nucleases. A variety of lipids used for producing DLPC and liposomes did not alter infectivity. Fluorescently labeled PrP 27-30 in liposomes was used to study its entry into cultured cells. Unlike the rods which remained as large fluorescent extracellular masses, the PrP 27-30 in liposomes rapidly entered the cells and was seen widely distributed within the interior of the cell. PrP 27-30 is derived by limited proteolysis from a larger protein designated PrP(Sc) which is membrane bound. PrP(Sc) in membrane fractions was solubilized by incorporation in DLPC, thus preventing its aggregation into amyloid rods. The functional solubilization of scrapie prion proteins in DLPC and liposomes offers new approaches to the study of prion structure and the mechanism by which they cause brain degeneration.     

Amyloid-beta-sheet formation at the air-water interface

An amyloid(1-40) solution rich in coil, turn, and alpha-helix, but poor in beta-sheet, develops monolayers with a high beta-sheet content when spread at the air-water interface. These monolayers are resistant to repeated compression-dilatation cycles and interaction with trifluoroethanol. The secondary structure motifs were detected by circular dichroism (CD) in solution and with infrared reflection-absorption spectroscopy (IRRAS) at the interface. Hydrophobic influences are discussed for the structure conversion in an effort to understand the completely unknown reason for the natural change of the normal prion protein cellular (PrP(C)) into the abnormal prion protein scrapie (PrP(Sc)).     

Synthetic miniprion PrP106

Elucidation of structure and biological properties of the prion protein scrapie (PrP(Sc)) is fundamental to an understanding of the mechanism of conformational transition of cellular (PrP(C)) into disease-specific isoforms and the pathogenesis of prion diseases. Unfortunately, the insolubility and heterogeneity of PrP(Sc) have limited these studies. The observation that a construct of 106 amino acids (termed PrP106 or miniprion), derived from mouse PrP and containing two deletions (Delta 23-88, Delta 141-176), becomes protease-resistant when expressed in scrapie-infected neuroblastoma cells and sustains prion replication when expressed in PrP(0/0) mice prompted us to generate a corresponding synthetic peptide (sPrP106) to be used for biochemical and cell culture studies. sPrP106 was obtained successfully with a straightforward procedure, which combines classical stepwise solid phase synthesis with a purification strategy based on transient labeling with a lipophilic chromatographic probe. sPrP106 readily adopted a beta-sheet structure, aggregated into branched filamentous structures without ultrastructural and tinctorial properties of amyloid, exhibited a proteinase K-resistant domain spanning residues 134-217, was highly toxic to primary neuronal cultures, and induced a remarkable increase in membrane microviscosity. These features are central properties of PrP(Sc) and make sPrP106 an excellent tool for investigating the molecular basis of the conformational conversion of PrP(C) into PrP(Sc) and prion disease pathogenesis.     

Local structural plasticity of the prion protein. Analysis of NMR relaxation dynamics

A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.     

Molecular characteristics of the major scrapie prion protein

A major protein was identified that purifies with the scrapie agent extracted from infected hamster brains. The protein, designated PrP 27-30, was differentiated from other proteins in purified fractions containing the scrapie agent by its microheterogeneity (Mr 27000-30000) and its unusual resistance to protease digestion. PrP 27-30 was found in all fractions enriched for scrapie prions by discontinuous sucrose gradient sedimentation or sodium dodecyl sarcosinate-agarose gel electrophoresis. It is unlikely that PrP 27-30 is a pathologic product because it was found in fractions isolated from the brains of hamsters sacrificed prior to the appearance of histopathology. If PrP 27-30 is present in normal brain, its concentration must be 100-fold lower than that found in equivalent fractions from scrapie-infected hamsters. Three protease-resistant proteins similar to PrP 27-30 were found in fractions obtained by discontinuous sucrose gradient sedimentation of scrapie-infected mouse brain. These proteins were not evident in corresponding fractions prepared from normal mouse brain. One-dimensional peptide maps comparing PrP 27-30 and normal hamster brain proteins of similar molecular weight demonstrated that PrP 27-30 has a primary structure which is distinct from these normal proteins. Heating substantially purified scrapie fractions to 100 degrees C in sodium dodecyl sulfate inactivated the prion and rendered PrP 27-30 susceptible to protease digestion. Though the scrapie agent appears to be hydrophobic, PrP 27-30 remained in the aqueous phase after extraction with organic solvents, indicating that it is probably not a proteolipid. PrP 27-30 is the first structural component of the scrapie prion to be identified.     

Discriminating scrapie and bovine spongiform encephalopathy isolates by infrared spectroscopy of pathological prion protein

For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27-30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27-30. However, FT-IR spectroscopy revealed that PrP27-30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in beta-sheet and other secondary structure elements of PrP27-30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27-30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species.     

Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein

Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127).     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.     

Temperature-induced conformational changes in amyloid beta(1-40) peptide investigated by simultaneous FT-IR microspectroscopy with thermal system

Temperature-dependent secondary structures of the amyloid beta(1-40) peptide in the solid state were studied by simultaneous Fourier transform infrared/differential scanning calorimetry (FT-IR/DSC) microspectroscopic system with the heating-cooling-reheating cycle. The result indicates that a thermal transition temperature at 45 degrees C was easily obtained from the three-dimensional plot of the transmission FT-IR spectra as a function of temperature. Furthermore, the thermal-dependent conformational transformations, due to denaturation and aggregation, of solid amyloid beta(1-40) were mainly evidenced by reducing the compositions from 37 to 20-24% for alpha-helical and random coil structures but increasing the components from 27 to 45% for intermolecular beta-sheet structures. Thermal-irreversible behavior and a poor thermal stability of solid amyloid beta(1-40) were also observed from the poor restoration of the secondary conformational changes in the heated sample.     

Disruption of prion rods generates 10-nm spherical particles having high alpha-helical content and lacking scrapie infectivity.

An abnormal isoform of the prion protein (PrP) designated PrPSc is the major, or possibly the only, component of infectious prions. Structural studies of PrPSc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrPSc. After ultracentrifugation at 100,000 x g for 1 h, approximately 30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of approximately 6S, approximately 10 mm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% alpha-helical content; addition of 25% acetonitrile induced aggregates high in beta sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity.     

Assembly of natural and recombinant prion protein into fibrils

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , PrP 27-30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrP Sc from PrP C and the assembly of rods from PrP 27-30, we solubilized Syrian hamster (sol SHa) PrP 27-30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27-30 adopted a beta-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27-30 in a soluble, beta-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27-30, but also with native SHaPrP C . Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90-231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27-30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27-30 or PrP C were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89-230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.     

Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis.

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.     

Ultrastructural studies on scrapie prion protein crystals obtained from reverse micellar solutions.

The structural transition from the cellular prion protein (PrPC) that is rich in alpha-helices to the pathological form (PrPSc) that has a high beta-sheet content seems to be the fundamental event underlying the prion diseases. Determination of the structure of PrPSc and the N-terminally truncated PrP 27-30 has been complicated by their insolubility. Here we report the solubilization of PrP 27-30 through a system of reverse micelles that yields monomeric and dimeric PrP. Although solubilization of PrP 27-30 was not accompanied by any recognizable change in secondary structure as measured by FTIR spectroscopy, it did result in a loss of prion infectivity. The formation of small two- and three-dimensional crystals upon exposure to uranyl salts argues that soluble PrP 27-30 possesses considerable tertiary structure. The crystals of PrP 27-30 grown from reverse micellar solutions suggest a novel crystallization mechanism that might be applicable for other membrane proteins. A variety of different crystal lattices diffracted up to 1.85 nm by electron microscopy. Despite the lack of measurable biological activity, the structure of PrP 27-30 in these crystals may provide insight into the structural transition that occurs during PrPSc formation.     

Amyloid protein of Gerstmann-Sträussler-Scheinker disease (Indiana kindred) is an 11 kd fragment of prion protein with an N-terminal glycine at codon 58.

Gerstmann-Sträussler-Scheinker (GSS) disease is a familial neurological disorder pathologically characterized by amyloid deposition in the cerebrum and cerebellum. The GSS amyloid is immunoreactive to antisera raised against the hamster prion protein (PrP) 27-30. This is a proteinase K-resistant glycoprotein of 27-30 kd that is derived from an abnormal isoform of a neuronal glycoprotein of 33-35 kd designated PrPSc and is a molecular marker of amyloid fibrils isolated from animals with scrapie and humans with related disorders. We have purified and characterized proteins extracted from amyloid plaque cores isolated from two patients of the Indiana kindred of GSS disease. We found that the major component of GSS amyloid is an 11 kd degradation product of PrP, whose N-terminus corresponds to the glycine residue at position 58 of the amino acid sequence deduced from the human PrP cDNA. In addition, amyloid fractions contained larger PrP fragments with apparently intact N-termini and amyloid P component. These findings suggest that the disease process leads to proteolytic cleavage of PrP, generating an amyloidogenic peptide that polymerizes into insoluble fibrils. The N-terminal cleavage of PrP in GSS disease occurs at a tryptophan-glycine peptide bond identical to that cleaved by proteinase K in vitro to generate PrP 27-30 from hamster PrPSc at codon 90. Since no mutations of the structural PrP gene have been found in the Indiana family of GSS disease, it is conceivable that factors other than the primary structure of PrP play a crucial role in the process of amyloid formation and the development of clinical neurologic dysfunction.     

Structural dependence of the cellular isoform of prion protein on solvent: spectroscopic characterization of an intermediate conformation.

Using circular dichroism, fluorescence, and infrared spectroscopies, we studied the secondary structure of purified hamster PrP(C) in the presence of the mild, nonionic detergent octylglucoside. Under these native conditions, PrP(C) displayed an unexpectedly high beta-sheet component, intermediate between the values previously reported for PrP(Sc) and an isoform of PrP(C) isolated in a zwitterionic detergent. The structure of PrP(C) appeared to depend strongly on the detergent and/or phase. Switching from octylglucoside to zwitterion 3-14 drastically modified PrP secondary structure by increasing the alpha-helix while abolishing the beta-sheet component. In contrast, the conformation of PrP(C) in zwitterion was highly stable, since reverting to octylglucoside did not restore the original native structure. These and other results show that native PrP(C) in octylglucoside has some of the conformational characteristics that make the protein susceptible to conversion into PrP(Sc). Most importantly, this is the first study to demonstrate the intrinsic plasticity of the full-length native PrP(C) isolated from animal brains.     

The role of the 132-160 region in prion protein conformational transitions

The native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.