Hamster Prion Research Articles

Guanidine hydrochloride extraction and detection of prion proteins in mouse and hamster prion diseases by ELISA.

Current detection of transmissible spongiform encephalopathy (TSE) relies on the proteolytic generation of a protease-resistant core from the scrapie isoform of prion protein (PrP(Sc)) followed by immunoblotting. This process is non-quantitative, time-consuming, and technically demanding. Recently, an alternative in vitro test for TSE based on the differential extraction of brain homogenates using guanidine hydrochloride followed by DELFIA (Dissociation Enhanced Lanthanide FluoroImmunoAssay) has been developed. In the present study, this approach was adopted using a panel of anti-PrP monoclonal antibodies (MAbs) in conventional sandwich enzyme-linked immunosorbent assay (ELISA) to investigate hamster and two distinct strains of mouse prion diseases. Although PrP species were present in both soluble and insoluble fractions from normal as well as TSE samples, only the PrP species in the insoluble fractions from the latter samples were protease-resistant. In addition, certain anti-PrP MAb pairs could distinguish the PrP species in infected brains from those in the normal samples. The ability to differentiate disease-associated PrP isoforms without proteinase K digestion could serve as a panacea for developing a reliable and rapid diagnostic test for prion diseases.

Alternative prion structural changes revealed by high pressure.

At high temperature, recombinant hamster prion protein (SHaPrP(90-231)) undergoes aggregation and changes from a predominantly alpha-helical to beta-sheet conformation. We then applied high pressure (200 MPa) to the beta-sheet-rich conformation. The aggregation was reversed, and the original tertiary and secondary structures were recovered at ambient pressure, after pressure release. The application of a pressure of 200 MPa thus allowed studying the heat-induced equilibrium refolding in the absence of protein aggregation. Prion protein unfolding as a function of high pressure was also investigated. Simple two-state, reversible unfolding transitions were observed, as monitored by spectral changes in the UV and fluorescence of the hydrophobic probe 8-anilino-1-naphthalene sulfonate. However, these heat- and pressure-induced conformers differed in their unfolding free energy. At pressures over 400 MPa, strong thioflavin-T binding was observed, suggesting a further structural change to a metastable oligomeric structure.

Optimized overproduction, purification, characterization and high-pressure sensitivity of the prion protein in the native (PrP C-like) or amyloid (PrP Sc-like) conformation

Overproduction and purification of the prion protein is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. We have developed a method for the effective cloning, overexpression in Escherichia coli and purification to homogeneity of Syrian golden hamster prion protein (SHaPrP 90–231). A high level of overexpression, resulting in the formation of inclusion bodies, was obtained under the control of the T7-inducible promoter of the pET15b plasmid. The protein required denaturation, reduction and refolding steps to become soluble and attain its native conformation. Purification was carried out by differential centrifugation, gel filtration and reverse phase chromatography. An improved cysteine oxidation protocol using oxidized glutathione under denaturing conditions, resulted in the recovery of a higher yield of chromatographically pure protein. About 10 mg of PrP protein per liter of bacterial culture was obtained. The recombinant protein was identified by monoclonal antibodies and its integrity was confirmed by electrospray mass spectrometry (ES/MS), whereas correct folding was assessed by circular dichroism (CD) spectroscopy. This protein had the structural characteristics of PrP C and could be converted to an amyloid structure sharing biophysical and biochemical properties of the pathologic form (PrP Sc). The sensitivity of these two forms to high pressure was investigated. We demonstrate the potential of using pressure as a thermodynamic parameter to rescue trapped aggregated prion conformations into a soluble state, and to explore new conformational coordinates of the prion protein conformational landscape.

BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein.

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.

Motor dysfunction and gliosis with preserved dopaminergic markers in human α-synuclein A30P transgenic mice

α-Synuclein is a major component of Lewy bodies (LBs) in the substantia nigra and cortex in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), and in glial inclusions in multiple systems atrophy (MSA). Mutations in α-synuclein have been associated with autosomal dominant forms of PD. We investigated the clinical and neuropathological effects of overexpression of human α-synuclein, α-synuclein A30P, and α-synuclein A53T under the control of the hamster prion protein (PrP) promoter; 5–15× endogenous levels of protein expression were achieved with widespread neuronal, including nigral, transgene expression. High expression of α-synuclein A30P in the Tg5093 line was associated with a progressive motor disorder with rigidity, dystonia, gait impairment, and tremor. Histological analysis of this line showed aberrant expression of the protein in cell soma and progressive CNS gliosis, but no discrete Lewy body-like α-synuclein inclusions could be identified. Biochemical analysis demonstrated α-synuclein fragmentation. Despite strong expression of the transgene in the nigra, there was no specific deterioration of the nigrostriatal dopaminergic system as assessed by quantitation of nigral tyrosine hydroxylase (TH) containing neurons, striatal TH immunoreactivity, dopamine levels, or dopamine receptor number and function. Lower expressing lines had no specific behavioral or histopathological phenotype. Thus, high expression of mutant human α-synuclein resulted in a progressive motor and widespread CNS gliotic phenotype independent of dopaminergic dysfunction in the Tg5093 line.

Molecular dynamics simulations of alanine rich beta-sheet oligomers: Insight into amyloid formation.

The aggregation observed in protein conformational diseases is the outcome of significant new beta-sheet structure not present in the native state. Peptide model systems have been useful in studies of fibril aggregate formation. Experimentally, it was found that a short peptide AGAAAAGA is one of the most highly amyloidogenic peptides. This peptide corresponds to the Syrian hamster prion protein (ShPrP) residues 113-120. The peptide was observed to be conserved in all species for which the PrP sequence has been determined. We have simulated the stabilities of oligomeric AGAAAAGA and AAAAAAAA (A8) by molecular dynamic simulations. Oligomers of both AGAAAAGA and AAAAAAAA were found to be stable when the size is 6 to 8 (hexamer to octamer). Subsequent simulation of an additional alpha-helical AAAAAAAA placed on the A8-octamer surface has revealed molecular events related to conformational change and oligomer growth. Our study addresses both the minimal oligomeric size of an aggregate seed and the mechanism of seed growth. Our simulations of the prion-derived 8-residue amyloidogenic peptide and its variant have indicated that an octamer is stable enough to be a seed and that the driving force for stabilization is the hydrophobic effect.

Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc?

A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein.

Prion-Protein-Specific Aptamer Reduces PrPSc Formation

The critical initial event in the pathophysiology of transmissible spongiform encephalopathies (TSEs) appears to be the conversion of the cellular prion protein (PrP(C)) into the abnormal isoform PrP(Sc). This isoform forms high-molecular-weight protease K (PK) resistant aggregates that accumulate in the central nervous system of affected individuals. We have selected nuclease-resistant 2'-amino-2'-deoxypyrimidine-modified RNA aptamers which recognize a peptide comprising amino acid residues 90-129 of the human prion protein with high specificity. This domain of prion proteins is thought to be functionally important for the conversion of PrP(C) into its pathogenic isoform PrP(Sc) and is highly homologous among prion proteins of various species including mouse, hamster, and man. Consequently, aptamer DP7 binds to the full-length human, mouse, and hamster prion protein. At low concentrations in the growth medium of persistently prion-infected neuroblastoma cells, aptamer DP7 significantly reduced the relative proportion of de novo synthesized PK-resistant PrP(Sc) within only 16 h. These findings may open the door towards a rational development of a new class of drugs for the therapy or prophylaxis of prion diseases.

A Change in the Conformation of Prions Accompanies the Emergence of a New Prion Strain

To investigate the role of the pathogenic prion protein (PrP Sc) in controlling susceptibility to foreign prions, two Syrian hamster (SHa) prion strains, Sc237 and DY, were transmitted to transgenic mice expressing chimeric SHa/mouse PrP genes, Tg(MH2M). First passage of SHa(Sc237) prions exhibited prolonged incubation times, diagnostic of a species barrier. PrP Sc of the new MH2M(Sc237) strain possessed different structural properties from those of SHa(Sc237), as demonstrated by relative conformational stability measurements. This change was accompanied by a disease phenotype different from the SHa(Sc237) strain. Conversely, transmission of SHa(DY) prions to Tg(MH2M) mice showed no species barrier, and the MH2M(DY) strain retained the conformational and disease-specific properties of SHa(DY). These results suggest a causal relationship between species barriers, changes in PrP Sc conformation, and the emergence of new prion strains.

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90–231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90–231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in β-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of α-helical structure. This conformation of PrP with a high content of α-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.

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.

Thioredoxin converts the Syrian hamster (29-231) recombinant prion protein to an insoluble form

The prion protein (PrP) is an essential, and probably the only, component of the infectious agent responsible for the transmissible spongiform encephalopathies. In its cellular (PrP C) form, it is a soluble, α-helix–rich protein of yet unknown function attached to the outer membrane of neurons through a glycosylphosphatidyl inositol anchor. In its pathogenic, “scrapie” form (PrP Sc), it appears as an aggregate showing no detectable covalent modifications but displaying a profoundly altered conformation enriched in β-sheet structure. Reduction of the single disulfide bridge in the prion protein with millimolar concentrations of dithiothreitol results in transformation of the α-helix–rich to the β-sheet–rich conformation, with concomitant decrease in solubility. We report here that thioredoxin coupled with thioredoxin reductase and NADPH efficiently reduces recombinant Syrian hamster (29-231) prion protein under physiologically relevant conditions. The reduced prion protein immediately becomes insoluble and precipitates, although it does not gain significant resistance to proteinase K. The thioredoxin/thioredoxin reductase system is ∼7000 times more efficient than dithiothreitol.

PoPMuSiC, an algorithm for predicting protein mutant stability changes: application to prion proteins.

A novel tool for computer-aided design of single-site mutations in proteins and peptides is presented. It proceeds by performing in silico all possible point mutations in a given protein or protein region and estimating the stability changes with linear combinations of database-derived potentials, whose coefficients depend on the solvent accessibility of the mutated residues. Upon completion, it yields a list of the most stabilizing, destabilizing or neutral mutations. This tool is applied to mouse, hamster and human prion proteins to identify the point mutations that are the most likely to stabilize their cellular form. The selected mutations are essentially located in the second helix, which presents an intrinsic preference to form beta-structures, with the best mutations being T183-->F, T192-->A and Q186-->A. The T183 mutation is predicted to be by far the most stabilizing one, but should be considered with care as it blocks the glycosylation of N181 and this blockade is known to favor the cellular to scrapie conversion. Furthermore, following the hypothesis that the first helix might induce the formation of hydrophilic beta-aggregates, several mutations that are neutral with respect to the structure's stability but improve the helix hydrophobicity are selected, among which is E146-->L. These mutations are intended as good candidates to undergo experimental tests.

Species-barrier-independent prion replication in apparently resistant species

Transmission of prions between mammalian species is thought to be limited by a "species barrier," which depends on differences in the primary structure of prion proteins in the infecting inoculum and the host. Here we demonstrate that a strain of hamster prions thought to be nonpathogenic for conventional mice leads to prion replication to high levels in such mice but without causing clinical disease. Prions pathogenic in both mice and hamsters are produced. These results demonstrate the existence of subclinical forms of prion infection with important public health implications, both with respect to iatrogenic transmission from apparently healthy humans and dietary exposure to cattle and other species exposed to bovine spongiform encephalopathy prions. Current definitions of the species barrier, which have been based on clinical end-points, need to be fundamentally reassessed.

NMR structures of three single-residue variants of the human prion protein.

The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121-230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrP(C)) that is formed by the C-terminal part of helix 3, with residues 218-230, and a loop of residues 166-172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized "protein X," and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121-230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized "conformational markers" could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166-172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166-172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).

NMR structure of the bovine prion protein.

The NMR structures of the recombinant 217-residue polypeptide chain of the mature bovine prion protein, bPrP(23-230), and a C-terminal fragment, bPrP(121-230), include a globular domain extending from residue 125 to residue 227, a short flexible chain end of residues 228-230, and an N-terminal flexibly disordered "tail" comprising 108 residues for the intact protein and 4 residues for bPrP(121-230), respectively. The globular domain contains three alpha-helices comprising the residues 144-154, 173-194, and 200-226, and a short antiparallel beta-sheet comprising the residues 128-131 and 161-164. The best-defined parts of the globular domain are the central portions of the helices 2 and 3, which are linked by the only disulfide bond in bPrP. Significantly increased disorder and mobility is observed for helix 1, the loop 166-172 leading from the beta-strand 2 to helix 2, the end of helix 2 and the following loop, and the last turn of helix 3. Although there are characteristic local differences relative to the conformations of the murine and Syrian hamster prion proteins, the bPrP structure is essentially identical to that of the human prion protein. On the other hand, there are differences between bovine and human PrP in the surface distribution of electrostatic charges, which then appears to be the principal structural feature of the "healthy" PrP form that might affect the stringency of the species barrier for transmission of prion diseases between humans and cattle.

Molecular dynamics of mouse and Syrian hamster PrP: Implications for activity

Molecular dynamics computer simulations have been performed on Mouse (Mo) and Syrian Hamster (SHa) prion proteins. These proteins differ, primarily, in that the SHa form incorporates additional residues at the C-terminus and also includes a segment of the unstructured N-terminal region that is required for infectivity. The 1-ns simulations have been analyzed by using a combination of dynamical cross-correlation maps, residue-residue contact plots, digital filtering, and residue-based root-mean-square deviations. The results show that the extra residues present in the SHa form at the C- and N-termini produce changes in the stability of key regions of the protein. The loop region between strand S2 and helix B that contains part of the proposed discontinuous binding site for the chaperone, protein X, is found to be more stable in SHa than in the Mo protein; these results are consistent with the NMR data of James et al. (James et al. Proc Natl Acad Sci USA 1997;94:10086-10091). In addition, a degree of flexibility within the region between and including strand S1 and helix A is also shown in SHa, which is not present in the Mo form; the cross-correlation maps suggest that this is a consequence of the additional unstructured N-terminal region. Furthermore, the extra residues in the N-terminal region of SHa are found to form a beta-bridge with the beta-sheet, within which critical point mutations associated with prion diseases lie. The implications of these results for the conformational interconversion pathway of the prion protein are discussed.

Digitally filtered molecular dynamics: The frequency specific control of molecular dynamics simulations

A new method for modifying the course of a molecular dynamics computer simulation is presented. Digitally filtered molecular dynamics (DFMD) applies the well-established theory of digital filters to molecular dynamics simulations, enabling atomic motion to be enhanced or suppressed in a selective manner solely on the basis of frequency. The basic theory of digital filters and its application to molecular dynamics simulations is presented, together with the application of DFMD to the simple systems of single molecules of water and butane. The extension of the basic theory to the condensed phase is then described followed by its application to liquid phase butane and the Syrian hamster prion protein. The high degree of selectivity and control offered by DFMD, and its ability to enhance the rate of conformational change in butane and in the prion protein, is demonstrated.

The role of hydrophobicity patterns in prion folding as revealed by recurrence quantification analysis of primary structure.

It has been suggested that the number and strength of local contacts are the major factors governing conformation accessibility of model two ground-state polypeptide chains. This phenomenology has been posed as a possible factor influencing prion folding. To test this conjecture, recurrence quantification analysis was applied to two model 36mers, and the Syrian hamster prion protein. A unique divergence of the radius function for the recurrence quantification variable %DET of hydrophobicity patterns was observed for both 36mers, and in a critical region of the hamster prion protein. This divergence suggests a partition between strong short- and long-range hydrophobicity patterns, and may be an important factor in prion phenomenology, along with other global thermodynamic factors.

NMR solution structure of the human prion protein.

The NMR structures of the recombinant human prion protein, hPrP(23-230), and two C-terminal fragments, hPrP(90-230) and hPrP(121-230), include a globular domain extending from residues 125-228, for which a detailed structure was obtained, and an N-terminal flexibly disordered "tail." The globular domain contains three alpha-helices comprising the residues 144-154, 173-194, and 200-228 and a short anti-parallel beta-sheet comprising the residues 128-131 and 161-164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167-171, the residues 187-194 at the end of helix 2, and the residues 219-228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187-193 in helix 2 and 219-226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23-230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167-171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.