Binding of prion proteins to lipid membranes

A key molecular event in prion diseases is the conversion of the normal cellular form of the prion protein (PrPC) to an aberrant form known as the scrapie isoform, PrPSc. Under normal physiological conditions PrPC is attached to the outer leaflet of the plasma membrane via a GPI-anchor. It has been proposed that a direct interaction between PrP and lipid membranes could be involved in the conversion of PrPC to its disease-associated corrupted conformation, PrPSc. Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of recombinant PrP isoforms to model lipid membranes using surface plasmon resonance spectroscopy. The binding of alpha- and beta-PrP to negatively charged lipid membranes of POPG, zwitterionic membranes of DPPC, and model raft membranes composed of DPPC, cholesterol, and sphingomyelin is compared at pH 7 and 5, to simulate the environment at the plasma membrane and within endosomes, respectively. It is found that PrP binds strongly to lipid membranes. The strength of the association of PrP with lipid membranes depends on the protein conformation and pH, and involves both hydrophobic and electrostatic lipid-protein interactions. Competition binding measurements established that the binding of alpha-PrP to lipid membranes follows a decreasing order of affinity to POPG>DPPC>rafts.     

Prion protein helix1 promotes aggregation but is not converted into beta-sheet

Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).     

DNA converts cellular prion protein into the beta-sheet conformation and inhibits prion peptide aggregation

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.     

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.     

Self-assembly of recombinant prion protein of 106 residues

The central event in the pathogenesis of prion diseases is a profound conformational change of the prion protein (PrP) from an alpha-helical (PrP(C)) to a beta-sheet-rich isoform (PrP(Sc)). The elucidation of the mechanism of conformational transition has been complicated by the challenge of collecting high-resolution biophysical data on the relatively insoluble aggregation-prone PrP(Sc) isoform. In an attempt to facilitate the structural analysis of PrP(Sc), a redacted chimeric mouse-hamster PrP of 106 amino acids (MHM2 PrP106) with two deletions (Delta23-88 and Delta141-176) was expressed and purified from Escherichia coli. PrP106 retains the ability to support PrP(Sc) formation in transgenic mice, implying that it contains all regions of PrP that are necessary for the conformational transition into the pathogenic isoform [Supattapone, S., et al. (1999) Cell 96, 869-878]. Unstructured at low concentrations, recombinant unglycosylated PrP106 (rPrP106) undergoes a concentration-dependent conformational transition to a beta-sheet-rich form. Following the conformational transition, rPrP106 possesses properties similar to those of PrP(Sc)106, such as high beta-sheet content, defined tertiary structure, resistance to limited digestion by proteinase K, and high thermodynamic stability. In GdnHCl-induced denaturation studies, a single cooperative conformational transition between the unstructured monomer and the assembled beta-oligomer was observed. After proteinase K digestion, the oligomers retain an intact core with unusually high beta-sheet content (>80%). Using mass spectrometry, we discovered that the region of residues 134-215 of rPrP106 is protected from proteinase K digestion and possesses a solvent-independent propensity to adopt a beta-sheet-rich conformation. In contrast to the PrP(Sc)106 purified from the brains of neurologically impaired animals, multimeric beta-rPrP106 remains soluble, providing opportunities for detailed structural studies.     

Similarity between the C-terminal domain of the prion protein and chimpanzee cytomegalovirus glycoprotein UL9

Prion diseases are a group of fatal neurodegenerative disorders associated with structural conversion of a normal, mostly alpha-helical cellular prion protein, PrP(C), into a pathogenic beta-sheet-rich conformation, PrP(Sc). The structure of PrP(C) is well studied, whereas the insolubility of PrP(Sc) makes the characterization of its structure problematic. No proteins similar to PrP, except for its paralog with the same fold, PrP-Doppel, are known. However, PrP-Doppel does not undergo a structural transition into a beta-sheet-rich conformation. Structural information from proteins that share a weak but significant sequence similarity with PrP may be used to gain additional insights into the conformation of PrP(Sc). We construct a sequence profile corresponding to the structured domain of PrP and use this profile to search the SWISS-PROT and TrEMBL databases. We identify a significant sequence similarity between PrP and chimpanzee cytomegalovirus glycoprotein UL9. This glycoprotein scores higher than all PrP-Doppel sequences. Fold recognition methods assign a mainly-beta fold to UL9. Owing to the observed sequence similarity with PrP and a putative mainly-beta fold, the UL9 glycoprotein may represent a potential target for experimental structure determination aimed at obtaining a structural template for PrP(Sc) modeling.     

Characterization of recombinant, membrane-attached full-length prion protein

An abnormal isoform, PrP(Sc), of the normal cellular prion protein (PrP(C)) is the major component of the causative agent of prion diseases. Both isoforms were found to possess the same covalent structures, including a C-terminal glycosylphosphatidylinositol anchor, but different secondary and tertiary structures. In this study, a variant of full-length PrP with an unpaired cysteine at the C terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as a model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment. Covalent coupling of PrP to liposomes did not result in significant structural changes observable by far-UV circular dichroism. Moreover, limited proteolysis experiments failed to detect changes in the stability of liposome-bound PrP relative to soluble PrP. These data suggest that the requirement of raft localization for the PrP(C) to PrP(Sc) conversion, observed previously in cell culture models, is not because of a direct influence of raft lipids on the structure and stability of membranebound PrP(C) but caused by other factors, e.g. increased local PrP concentrations or high effective concentrations of membrane-associated conversion factors. The availability of recombinant PrP covalently attached to liposomes provides the basis for systematic in vitro conversion assays with recombinant PrP on the surface of membranes. In addition, our results indicate that the three-dimensional structure of mammalian PrP(C) in membranes is identical to that of recombinant PrP in solution.     

pH-dependent prion protein conformation in classical Creutzfeldt-Jakob disease.

In transmissible spongiform encephalopathies, the cellular prion protein (PrP(C)) undergoes a conformational change from a prevailing alpha-helical structure to a beta-sheet-rich, protease-resistant isoform, termed PrP(Sc). PrP(C) has two characteristics: a high affinity for Cu(2+) and a strong pH-dependent conformation. Lines of evidence indicate that PrP(Sc) conformation is dependent on copper and that acidic conditions facilitate the conversion of PrP(C) --> PrP(Sc). In each species, PrP(Sc) exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrP(Sc) have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrP(Sc) under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrP(Sc), associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrP(Sc) types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrP(Sc) in sCJD.     

Chaperonin-mediated de novo generation of prion protein aggregates.

The infectious prion protein, PrP(Sc), a predominantly beta-sheet aggregate, is derived from PrP(C), the largely alpha-helical cellular isoform of PrP. Conformational conversion of PrP(C) into PrP(Sc) has been suggested to involve a chaperone-like factor. Here we report that the bacterial chaperonin GroEL, a close homolog of eukaryotic Hsp60, can catalyze the aggregation of chemically denatured and of folded, recombinant PrP in a model reaction in vitro. Aggregates form upon ATP-dependent release of PrP from chaperonin and have certain properties of PrP(Sc), including a high beta-sheet content, the ability to bind the dye Congo red, detergent-insolubility and increased protease-resistance. A conserved sequence segment of PrP (residues 90-121), critical for PrP(Sc) generation in vivo, is also required for chaperonin-mediated aggregate formation in vitro. Initial binding of refolded, alpha-helical PrP to chaperonin is mediated by the unstructured N-terminal segment of PrP (residues 23-121) and is followed by a rearrangement of the globular PrP core-domain. These results show that chaperonins of the Hsp60 class can, in principle, mediate PrP aggregation de novo, i.e. independently of a pre-existent PrP(Sc) template.     

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.     

Elongated oligomers assemble into mammalian PrP amyloid fibrils

In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly alpha-helical state into beta-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a beta form of recombinant mouse PrP (residues 91-231). The beta-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrP Sc. Using electron microscopy and image processing, we obtained three-dimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of approximately 60 A. The repeating unit along each protofilament can be accounted for by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-beta amyloid fold.     

Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrP(C) and PrP(Sc) have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrP(C) to PrP(Sc), but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.     

Interaction of the cellular prion protein with raft-like lipid membranes

Conversion of the cellular isoform of the prion protein (PrP(C)) into the disease-associated isoform (PrP(Sc)) plays a key role in the development of prion diseases. Within its cellular pathway, PrP(C) undergoes several posttranslational modifications, i.e., the attachment of two N-linked glycans and a glycosyl phosphatidyl inositol (GPI) anchor, by which it is linked to the plasma membrane on the exterior cell surface. To study the interaction of PrP(C) with model membranes, we purified posttranslationally modified PrP(C) from transgenic Chinese hamster ovary (CHO) cells. The mono-, di- and oligomeric states of PrP(C) free in solution were analyzed by analytical ultracentrifugation. The interaction of PrP(C) with model membranes was studied using both lipid vesicles in solution and lipid bilayers bound to a chip surface. The equilibrium and mechanism of PrP(C) association with the model membranes were analyzed by surface plasmon resonance. Depending on the degree of saturation of binding sites, the concentration of PrP(C) released from the membrane into aqueous solution was estimated at between 10(-9) and 10(-7) M. This corresponds to a free energy of the insertion reaction of -48 kJ/mol. Consequences for the conversion of PrP(C) to PrP(Sc) are discussed.     

Beta-PrP form of human prion protein stimulates production of monoclonal antibodies to epitope 91-110 that recognise native PrPSc

Prion diseases are associated with accumulation of strain-dependent biochemically distinct, disease-related isoforms (PrP(Sc)) of host-encoded prion protein (PrP(C)). PrP(Sc) is characterised by increased beta-sheet content, detergent insolubility and protease resistance. Recombinant alpha-PrP adopts a PrP(C)-like conformation, while beta-PrP conformationally resembles PrP(Sc), to these we raised 81 monoclonal antibodies in Prnp(0/0) mice. The N-terminal residues 91-110 are highly immunogenic in beta-PrP-immunised mice and of (17/41) anti-beta-PrP antibodies that could be epitope-mapped, approximately 70%, recognised this segment. In contrast, only 3/40 anti-alpha-PrP antibodies could be mapped and none interacted with this region, instead recognising residues 131-150, 141-160 and 171-190. Native PrP(C) was recognised by both antibody groups, but only anti-beta-PrP antibodies directed to 91-110 residues recognised native PrP(Sc) with high affinity, where in addition, species heterogeneity was also evident. Within the six anti-beta-PrP antibodies studied, they all recognised PK-treated native human and mouse PrP(Sc), four failed to recognise PK-treated native bovine PrP(Sc), one of which also did not recognise native PK-treated ovine PrP(Sc), showing the epitope becomes exposed on unfolding and disaggregation. These results demonstrate strain-dependent variations in chain conformation and packing within the 91-110 region of PrP(Sc).     

Monoacylated cellular prion protein modifies cell membranes, inhibits cell signaling, and reduces prion formation

Prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into a disease related, protease-resistant isoform (PrP(Sc)). In these studies, a cell painting technique was used to introduce PrP(C) to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrP(C) resulted in increased PrP(Sc) formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A(2) (cPLA(2)). In contrast, although PrP(C) lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrP(C)-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA(2). It remained within cells for longer than PrP(C) with a conventional GPI anchor and was not converted to PrP(Sc). Moreover, the addition of high amounts of PrP(C)-G-lyso-PI displaced cPLA(2) from PrP(Sc)-containing lipid rafts, reduced the activation of cPLA(2), and reduced PrP(Sc) formation in all three cell lines. In addition, ScGT1 cells treated with PrP(C)-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrP(C) modified the local membrane microenvironments that control cell signaling, the fate of PrP(C), and hence PrP(Sc) formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.     

Hydration and packing effects on prion folding and beta-sheet conversion. High pressure spectroscopy and pressure perturbation calorimetry studies

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we compare the stability against pressure and the thermomechanical properties of the alpha-helical and beta-sheet conformations of recombinant murine prion protein, designated as alpha-rPrP and beta-rPrP, respectively. High temperature induces aggregates and a large gain in intermolecular antiparallel beta-sheet (beta-rPrP), a conformation that shares structural similarity with PrP(Sc). alpha-rPrP is highly stable, and only pressures above 5 kilobars (1 kilobar = 100 MegaPascals) cause reversible denaturation, a process that leads to a random and turnrich conformation with concomitant loss of alpha-helix, as measured by Fourier transform infrared spectroscopy. In contrast, aggregates of beta-rPrP are very sensitive to pressure, undergoing transition into a dissociated species that differs from the denatured form derived from alpha-rPrP. The higher susceptibility to pressure of beta-rPrP can be explained by its less hydrated structure. Pressure perturbation calorimetry supports the view that the accessible surface area of alpha-rPrP is much higher than that of beta-rPrP, which explains the lower degree of hydration of beta-rPrP. Our findings shed new light on the mechanism of prion conversion and show how water plays a prominent role. Our results allow us to propose a volume and free energy diagram of the different species involved in the conversion and aggregation. The existence of different folded conformations as well as different denatured states of PrP may explain the elusive character of its conversion into a pathogenic form.     

An unusual soluble beta-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.     

PrPSc binding antibodies are potent inhibitors of prion replication in cell lines

Conversion of the cellular alpha-helical prion protein (PrP(C)) into a disease-associated isoform (PrP(Sc)) is central to the pathogenesis of prion diseases. Molecules targeting either normal or disease-associated isoforms may be of therapeutic interest, and the antibodies binding PrP(C) have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrP(Sc) inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrP(C) were relatively inefficient inhibitors of ScRov cell PrP(Sc) accumulation compared with antibodies that additionally targeted disease-associated PrP isoforms. Although the mechanism by which these monoclonal antibodies inhibit prion replication is unclear, some of the data suggest that antibodies might actively increase PrP(Sc) turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents.     

Clustered negative charges on the lipid membrane surface induce beta-sheet formation of prion protein fragment 106-126

The conformational conversion of prion protein (PrP) from an alpha-helix-rich normal cellular isoform (PrPC) to a beta-sheet-rich pathogenic isoform (PrPSc) is a key event in the development of prion diseases, and it takes place in caveolae, cavelike invaginations of the plasma membrane. A peptide homologous to residues 106-126 of human PrP (PrP106-126) is known to share several properties with PrPSc, e.g., the capability to form a beta-sheet and toxicity against PrPC-expressing cells. PrP106-126 is thus expected to represent a segment of PrP that is involved in the formation of PrPSc. We have examined the effect of lipid membranes containing negatively charged ganglioside, an important component of caveolae, on the secondary structure of PrP106-126 by circular dichroism. The peptide forms an alpha-helical or a beta-sheet structure on the ganglioside-containing membranes. The beta-sheet content increases with an increase of the peptide:lipid ratio, indicating that the beta-sheet formation is linked with self-association of the positively charged peptide on the negatively charged membrane surface. Analogous beta-sheet formation is also induced by membranes composed of negatively charged and neutral glycerophospholipids with high and low melting temperatures, respectively, in which lateral phase separation and clustering of negatively charged lipids occur as shown by Raman spectroscopy. Since ganglioside-containing membranes also exhibit lateral phase separation, clustered negative charges are concluded to be responsible for the beta-sheet formation of PrP106-126. In caveolae, clustered ganglioside molecules are likely to interact with the residue 106-126 region of PrPC to promote the PrPC-to-PrPSc conversion.     

Aggregation and fibrillization of the recombinant human prion protein huPrP90-231

According to the "protein-only" hypothesis, the critical step in the pathogenesis of prion diseases is the conformational transition between the normal (PrP(C)) and pathological (PrP(Sc)) isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90-231 of the human prion protein (huPrP90-231). Incubation of the protein under acidic conditions (pH 3.6-5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an alpha-helical conformation to a beta-sheet structure and oligomerization of huPrP90-231 into large molecular weight aggregates. No stable monomeric beta-sheet-rich folding intermediate of the protein could be detected in the present experiments. Kinetic analysis of the data indicates that the formation of beta-sheet structure and protein oligomerization likely occur concomitantly. The beta-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrP(Sc)). Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrP(Sc)-like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90-231 to an oligomeric beta-sheet structure is the presence of salt.