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.     

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.     

The octapeptide repeats in mammalian prion protein constitute a pH-dependent folding and aggregation site

Structural studies of mammalian prion protein at pH values between 4.5 and 5.5 established that the N-terminal 100 residue domain is flexibly disordered. Here, we show that at pH values between 6.5 and 7.8, i.e. the pH at the cell membrane, the octapeptide repeats in recombinant human prion protein hPrP(23-230) encompassing the highly conserved amino acid sequence PHGGGWGQ are structured. The nuclear magnetic resonance solution structure of the octapeptide repeats at pH 6.2 reveals a new structural motif that causes a reversible pH-dependent PrP oligomerization. Within the aggregation motif the segments HGGGW and GWGQ adopt a loop conformation and a beta-turn-like structure, respectively. Comparison with the crystal structure of HGGGW-Cu(2+) indicates that the binding of copper ions induces a conformational transition that presumably modulates PrP aggregation. The knowledge that the cellular prion protein is immobilized on the cell surface along with our results suggests a functional role of aggregation in endocytosis or homophilic cell adhesion.     

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.     

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.     

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.     

The binding of the molecular chaperone Hsc70 to the prion protein PrP is modulated by pH and copper

Conformational transitions in the prion protein (PrP) are thought to be central to the pathogenesis of the transmissible spongiform encephalopathies (TSE), such as Creutzfeldt-Jacob disease and bovine spongiform encephalopathy. Studies of prion phenomena in yeast have shown that molecular chaperones play an important role in prion related conformational transitions. Here, we investigated the interaction of the molecular chaperone Hsc70 (HSPA8) with recombinant PrP in vitro using an ELISA based assay. Hsc70 bound to PrP in a saturable manner over a range of temperatures and binding was greatest at low pH. Surprisingly, Hsc70 bound more avidly to native recombinant PrP than to denatured PrP or other potential clients, such as denatured luciferase or rhodanese. Hsc70 binding to native PrP was enhanced by incubation with Cu²⁺ at low pH. The Hsc70 binding sites in PrP were analysed using a synthetic PrP-derived peptide array. The binding of Hsc70 to PrP was reminiscent of the published ovine PrP to bovine PrP binding data and included two potential regions of binding that correspond to the proposed ‘protein X’ binding sites in PrP. Synthetic peptides corresponding to these sites specifically inhibited the Hsc70 interaction with native PrP, further demonstrating that Hsc70 might interact with PrP via this epitope. The data suggest that molecular chaperones could modulate important PrP conformational transitions or protein–protein interactions in TSE pathogenesis.     

Identification of a common sphingolipid-binding domain in Alzheimer, prion, and HIV-1 proteins.

The V3 loop of the human immunodeficiency virus (HIV)-1 surface envelope glycoprotein gp120 is a sphingolipid-binding domain mediating the attachment of HIV-1 to plasma membrane microdomains (rafts). Sphingolipid-induced conformational changes in gp120 are required for HIV-1 fusion. Galactosylceramide and sphingomyelin have been detected in highly purified preparations of prion rods, suggesting that the prion protein (PrP) may interact with selected sphingolipids. Moreover, a major conformational transition of the Alzheimer beta-amyloid peptide has been observed upon interaction with sphingolipid-containing membranes. Structure similarity searches with the combinatorial extension method revealed the presence of a V3-like domain in the human prion protein PrP and in the Alzheimer beta-amyloid peptide. In each case, synthetic peptides derived from the predicted V3-like domain were found to interact with monomolecular films of galactosylceramide and sphingomyelin at the air-water interface. The V3-like domain of PrP is a disulfide-linked loop (Cys(179)-Cys(214)) that includes the E200K mutation site associated with familial Creutzfeldt-Jakob disease. This mutation abrogated sphingomyelin recognition. The identification of a common sphingolipid-binding motif in gp120, PrP, and beta-amyloid peptide underscores the role of lipid rafts in the pathogenesis of HIV-1, Alzheimer, and prion diseases and may provide new therapeutic strategies.     

Insight into early events in the aggregation of the prion protein on lipid membranes

The key molecular event underlying prion diseases is the conversion of the monomeric and α-helical cellular form of the prion protein (PrP^C) to the disease-associated state, which is aggregated and rich in β-sheet (PrP^Sc). The molecular details associated with the conversion of PrP^C into PrP^Sc are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces β-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the α-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.     

Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.     

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.     

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.     

Disulfide bond as a structural determinant of prion protein membrane insertion

Conversion of the normal soluble form of prion protein, PrP (PrP^C), to proteinase K-resistant form (PrP^Sc) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from α-helix to β-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111–135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.     

The role of rafts in the fibrillization and aggregation of prions

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrP(C)) to the disease-specific form (PrP(Sc)). The transition from PrP(C) to PrP(Sc) involves a major conformational change, resulting in amorphous aggregates and/or fibrillar amyloid deposits. Here several lines of evidence implicating membranes in the conversion of PrP are reviewed with a particular emphasis on the role of lipid rafts in the conformational transition of prion proteins. New correlations between in vitro biophysical studies and findings from cell biology work on the role of rafts in prion conversion are highlighted and a mechanism for the role of rafts in prion conversion is proposed.     

Immobilized prion protein undergoes spontaneous rearrangement to a conformation having features in common with the infectious form

It is hypothesized that infectious prions are generated as the cellular form of the prion protein (PrP(C)) undergoes pronounced conformational change under the direction of an infectious PrP(Sc) template. Conversion to the infectious conformer is particularly associated with major structural rearrangement in the central portion of the protein (residues 90-120), which has an extended flexible structure in the PrP(C) isoform. Using a panel of recombinant antibodies reactive with different parts of PrP, we show that equivalent major structural rearrangements occur spontaneously in this region of PrP immobilized on a surface. In contrast, regions more towards the termini of the protein remain relatively unaltered. The rearrangements occur even under conditions where individual PrP molecules should not contact one another. The propensity of specific unstructured regions of PrP to spontaneously undergo large and potentially deleterious conformational changes may have important implications for prion biology.     

A role for His155 in binding of human prion peptide144-167 to immobilised prion protein

The interactions and conformational changes that lead to the conversion of the normal prion protein (PrP(c)) to its pathogenic form, PrP(sc), are still being elucidated. Using Surface Plasma Resonance (SPR), we provide evidence that a synthetic peptide (PrP(144-167)) corresponding to residues comprising the alpha helix 1-beta strand 2 domain of PrP(c) is able to interact and bind to immobilised recombinant human PrP (rHuPrP) in a dose-dependent manner. The interaction is pH dependent with an increase in binding observed as the pH is lowered, particularly between pH 6.5 and pH 5.5 suggesting a specific role for His(155) in the interaction, confirmed by covalent modification of this residue in the peptide with diethylpyrocarbonate (DEPC). Circular dichroism analysis of PrP(144-167) revealed no secondary structure motifs across the pH range investigated. Possible pH related structural changes of immobilised rHuPrP are also discussed with regard to the increased affinity for PrP(144-167).     

Spontaneous conformational change within the prion protein—implications for disease pathogenesis?

A recent paper by Leclerc et al(1) describes how recombinant hamster prion protein can undergo a spontaneous change in conformation to a structure that has features in common with PrP(Sc). Structural change in the host prion protein, PrP(C) to an insoluble and aggregated form with increased beta-sheet content (PrP(Sc)) is central to the pathology of prion diseases.(2) A detailed understanding of the nature of these conformational changes will increase our knowledge of the molecular basis of prion pathology. These findings may have implications for how the disease is initiated and provide a format for further investigation.     

Solution structure of the E200K variant of human prion protein. Implications for the mechanism of pathogenesis in familial prion diseases

Prion propagation in transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrP(C), into a pathogenic conformer, PrP(Sc). Hereditary forms of the disease are linked to specific mutations in the gene coding for the prion protein. To gain insight into the molecular basis of these disorders, the solution structure of the familial Creutzfeldt-Jakob disease-related E200K variant of human prion protein was determined by multi-dimensional nuclear magnetic resonance spectroscopy. Remarkably, apart from minor differences in flexible regions, the backbone tertiary structure of the E200K variant is nearly identical to that reported for the wild-type human prion protein. The only major consequence of the mutation is the perturbation of surface electrostatic potential. The present structural data strongly suggest that protein surface defects leading to abnormalities in the interaction of prion protein with auxiliary proteins/chaperones or cellular membranes should be considered key determinants of a spontaneous PrP(C) --> PrP(Sc) conversion in the E200K form of hereditary prion disease.     

Interaction of the 106-126 prion peptide with lipid membranes and potential implication for neurotoxicity

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the cellular prion protein (PrP(C)). In the present work, we were interested to study the mode of prion protein interaction with the membrane using the 106-126 peptide and small unilamellar lipid vesicles as model. As previously demonstrated, we showed by MTS assay that PrP 106-126 induces alterations in the human neuroblastoma SH-SY5Y cell line. We demonstrated for the first time by lipid-mixing assay and by the liposome vesicle leakage test that PrP 106-126, a non-tilted peptide, induces liposome fusion thus a potential cell membrane destabilization, as supported by membrane integrity assay (LDH). By circular dichroism (CD) analysis we showed that the fusogenic property of PrP 106-126 in the presence of liposome is associated with a predominantly beta-sheet structure. These data suggest that the fusogenic property associated with a predominant beta-sheet structure exhibited by the prion peptides contributes to the neurotoxicity of these peptides by destabilizing cellular membranes. The latter might be attached at the membrane surface in a parallel orientation as shown by molecular modeling.     

Aggregation and fibrillization of prions in lipid membranes

A key molecular event in prion diseases is the conversion of PrP (prion protein) from its normal cellular form (PrP(c)) into the disease-specific form (PrP(Sc)). The transition from PrP(c) to PrP(Sc) involves a major conformational change, resulting in amorphous aggregates and/or fibrillar amyloid deposits. Here, we review several lines of evidence implicating membranes in the conversion of PrP, and summarize recent results from our own work on the role of lipid membranes in conformational transitions of prion proteins. By establishing new correlations between in vivo biological findings with in vitro biophysical results, we propose a role for lipid rafts in prion conversion, which takes into account the structural heterogeneity of PrP in different lipid environments.