Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein

Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrP^Sc of the host-encoded prion protein (PrP^c). A profound conformational change of PrP^c underlies formation of PrP^Sc and prion propagation involves conversion of PrP^c substrate by direct interaction with PrP^Sc template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrP^C into PrP^Sc. To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrP^Sc recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated.     

PrPST,a Soluble,Protease Resistant and Truncated PrP Form Features in the Pathogenesis of a Genetic Prion Disease

While the conversion of PrP^C into PrP^Sc in the transmissible form of prion disease requires a preexisting PrP^Sc seed, in genetic prion disease accumulation of disease related PrP could be associated with biochemical and metabolic modifications resulting from the designated PrP mutation. To investigate this possibility, we looked into the time related changes of PrP proteins in the brains of TgMHu2ME199K/wt mice, a line modeling for heterozygous genetic prion disease linked to the E200K PrP mutation. We found that while oligomeric entities of mutant E199KPrP exist at all ages, aggregates of wt PrP in the same brains presented only in advanced disease, indicating a late onset conversion process. We also show that most PK resistant PrP in TgMHu2ME199K mice is soluble and truncated (PrP^ST), a pathogenic form never before associated with prion disease. We next looked into brain samples from E200K patients and found that both PK resistant PrPs, PrP^ST as in TgMHu2ME199K mice, and “classical” PrP^Sc as in infectious prion diseases, coincide in the patient''s post mortem brains. We hypothesize that aberrant metabolism of mutant PrPs may result in the formation of previously unknown forms of the prion protein and that these may be central for the fatal outcome of the genetic prion condition.     

Polo-like kinase 3 (PLK3) mediates the clearance of the accumulated PrP mutants transiently expressed in cultured cells and pathogenic PrPSc in prion infected cell line via protein interaction

Polo-like kinases (PLKs) family has long been known to be critical for cell cycle and recent studies have pointed to new dimensions of PLKs function in the nervous system. Our previous study has verified that the levels of PLK3 in the brain are severely downregulated in prion-related diseases. However, the associations of PLKs with prion protein remain unclear. In the present study, we confirmed that PrP protein constitutively interacts with PLK3 as determined by both in vitro and in vivo assays. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. Overexpression of PLK3 did not affect the level of wild-type PrP, but significantly decreased the levels of the mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed to be independent of its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrPs. Moreover, PLK3 overexpression in a scrapie infected cell line caused notable reduce of PrP^Sc level in a dose-dependent manner, but had minimal effect on the expression of PrP^C in its normal partner cell line. Our findings here confirmed the molecular interaction between PLK3 and PrP and outlined the regulatory activity of PLK3 on the degradation of abnormal PrPs, even its pathogenic isoform PrP^Sc. We, therefore, assume that the recovery of PLK3 in the early stage of prion infection may be helpful to prevent the toxic accumulation of PrP^Sc in the brain tissues.     

NMR Structure of the Human Prion Protein with the Pathological Q212P Mutation Reveals Unique Structural Features

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrP^C) conformer, denoted as infectious scrapie isoform or PrP^Sc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrP^Sc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β₂–α₂ loop region. This structure might provide new insights into the early events of conformational transition of PrP^C into PrP^Sc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β₂–α₂ loop and α₃ helix.     

Mammalian prion amyloid formation in bacteria

Mammalian prion proteins (PrPs) that cause transmissible spongiform encephalopathies are misfolded conformations of the host cellular PrP. The misfolded form, the scrapie PrP (PrP^Sc), can aggregate into amyloid fibrils that progressively accumulate in the brain, evolving to a pathological phenotype. A particular characteristic of PrP^Sc is to be found as different strains, related to the diversity of conformational states it can adopt. Prion strains are responsible for the multiple phenotypes observed in prion diseases, presenting different incubation times and diverse deposition profiles in the brain. PrP biochemical properties are also strain-dependent, such as different digestion pattern after proteolysis and different stability. Although they have long been studied, strain formation is still a major unsolved issue in prion biology. The recreation of strain-specific conformational features is of fundamental importance to study this unique pathogenic phenomenon. In our recent paper, we described that murine PrP, when expressed in bacteria, forms amyloid inclusion bodies that possess different strain-like characteristics, depending on the PrP construct. Here, we present an extra-view of these data and propose that bacteria might become a successful model to generate preparative amounts of prion strain-specific assemblies for high-resolution structural analysis as well as for addressing the determinants of infectivity and transmissibility.     

The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

PrP^Sc, a misfolded and aggregated form of the cellular prion protein PrP^C, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP^C in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP^C and PrP^Sc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP^C. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP^C. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP^Sc. Other antibodies immunoprecipitate PrP^C, but not PrP^Sc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP^C and PrP^Sc. Amino-proximal antibodies were found to react with repetitive PrP^C epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.     

Early Appearance but Lagged Accumulation of Detergent-Insoluble Prion Protein in the Brains of Mice Inoculated with a Mouse-Adapted Creutzfeldt–Jakob Disease Agent

SUMMARY 1. To elucidate mechanisms for the generation of the detergent-insoluble, proteinase K-resistant prion protein (PrP^Sc) from the detergent-soluble, proteinase K-sensitive PrP (PrP^C) and the replication of the infectious agent in prion diseases, we followed the kinetics of detergent-insoluble PrP and PrP^Sc levels, infectious titers, and associated pathological changes in the brains of mice inoculated with a mouse-adapted Creutzfeldt–Jakob disease agent.2. PrP^Sc in brain homogenate and detergent-insoluble PrP enriched by two-cycle ultracentrifugation were detected by immunoblotting and their relative amounts were estimated according to a standard curve plotted between the amount of PrP and signal intensity on immunoblotting. The titer of infectivity was determined by the incubation periods of mice inoculated with the unfractionated homogenate on the basis of a standard curve plotted between the titer and incubation period.3. Detergent-insoluble PrP became detectable 4 weeks postinoculation (p.i.) well before the detection of PrP^Sc. The low level of detergent-insoluble PrP continued until dramatic accumulation occurred at 14 weeks p.i., correlating well with the accumulation of PrP^Sc and development of pathological changes. The infectious titer was undetectable at 4 weeks p.i. and its logarithmic increase occurred 10 weeks p.i. preceding the logarithmic accumulation of PrPs.4. The lag time of detergent-insoluble PrP accumulation and the discrepancy between infectious titers and PrPs observed during the early period after inoculation suggest a slow and rate-limiting step for the detergent-insoluble PrP to become the infectious agent-associated PrP^Sc.     

Identifying critical sites of PrPc-PrPSc interaction in prion-infected cells by dominant-negative inhibition

A direct physical interaction of the prion protein isoforms is a key element in prion conversion. Which sites interact first and which parts of PrP^c are converted subsequently is presently not known in detail. We hypothesized that structural changes induced by PrP^Sc interaction occur in more than one interface and subsequently propagate within the PrP^C substrate, like epicenters of structural changes. To identify potential interfaces we created a series of systematically-designed mutant PrPs and tested them in prion-infected cells for dominant-negative inhibition (DNI) effects. This showed that mutant PrPs with deletions in the region between first and second α-helix are involved in PrP-PrP interaction and conversion of PrP^C into PrP^Sc. Although some PrPs did not reach the plasma membrane, they had access to the locales of prion conversion and PrP^Sc recycling using autophagy pathways. Using other series of mutant PrPs we already have identified additional sites which constitute potential interaction interfaces. Our approach has the potential to characterize PrP-PrP interaction sites in the context of prion-infected cells. Besides providing further insights into the molecular mechanisms of prion conversion, this data may help to further elucidate how prion strain diversity is maintained.     

Conformational diversity in prion protein variants influences intermolecular β‐sheet formation

A conformational transition of normal cellular prion protein (PrP^C) to its pathogenic form (PrP^Sc) is believed to be a central event in the transmission of the devastating neurological diseases known as spongiform encephalopathies. The common methionine/valine polymorphism at residue 129 in the PrP influences disease susceptibility and phenotype. We report here seven crystal structures of human PrP variants: three of wild‐type (WT) PrP containing V129, and four of the familial variants D178N and F198S, containing either M129 or V129. Comparison of these structures with each other and with previously published WT PrP structures containing M129 revealed that only WT PrPs were found to crystallize as domain‐swapped dimers or closed monomers; the four mutant PrPs crystallized as non‐swapped dimers. Three of the four mutant PrPs aligned to form intermolecular β‐sheets. Several regions of structural variability were identified, and analysis of their conformations provides an explanation for the structural features, which can influence the formation and conformation of intermolecular β‐sheets involving the M/V129 polymorphic residue.     

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.     

Immunopurification of Pathological Prion Protein Aggregates

Background

Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP^C) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP^Sc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP^C molecules. A great deal of effort has been devoted to developing protocols for purifying PrP^Sc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP^Sc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.

Principal Findings

We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP^Sc and mutant PrP aggregates at electrophoretic homogeneity. PrP^Sc purified from prion-infected mice was able to seed misfolding of PrP^C in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons.

Significance

The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.     

Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion

Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrP^C into the infectious misfolded isoform PrP^Sc. Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrP^Sc destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121–231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders.     

In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP^C) converts into a misfolded isoform (PrP^Sc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP^C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP^Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP^Sc-specific conformational epitopes. In contrast to PrP^Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP^Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP^Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP^Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.     

Cellular uptake of the prion protein fragment PrP106-126 in vitro

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP^c). This pathological isoform (PrP^Sc) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP^Sc in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP^Sc-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP^c expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP^Sc in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.     

Implications of prion adaptation and evolution paradigm for human neurodegenerative diseases

There is a growing body of evidence indicating that number of human neurodegenerative diseases, including Alzheimer disease, Parkinson disease, fronto-temporal dementias, and amyotrophic lateral sclerosis, propagate in the brain via prion-like intercellular induction of protein misfolding. Prions cause lethal neurodegenerative diseases in humans, the most prevalent being sporadic Creutzfeldt-Jakob disease (sCJD); they self-replicate and spread by converting the cellular form of prion protein (PrP^C) to a misfolded pathogenic conformer (PrP^Sc). The extensive phenotypic heterogeneity of human prion diseases is determined by polymorphisms in the prion protein gene, and by prion strain-specific conformation of PrP^Sc. Remarkably, even though informative nucleic acid is absent, prions may undergo rapid adaptation and evolution in cloned cells and upon crossing the species barrier. In the course of our investigation of this process, we isolated distinct populations of PrP^Sc particles that frequently co-exist in sCJD. The human prion particles replicate independently and undergo competitive selection of those with lower initial conformational stability. Exposed to mutant substrate, the winning PrP^Sc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to the lowest stability. Thus, the evolution and adaptation of human prions is enabled by a dynamic collection of distinct populations of particles, whose evolution is governed by the selection of progressively less stable, faster replicating PrP^Sc conformers. This fundamental biological mechanism may explain the drug resistance that some prions gained after exposure to compounds targeting PrP^Sc. Whether the phenotypic heterogeneity of other neurodegenerative diseases caused by protein misfolding is determined by the spectrum of misfolded conformers (strains) remains to be established. However, the prospect that these conformers may evolve and adapt by a prion-like mechanism calls for the reevaluation of therapeutic strategies that target aggregates of misfolded proteins, and argues for new therapeutic approaches that will focus on prior pathogenetic steps.     

Anti-prion activity of an RNA aptamer and its structural basis

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP^C) of PrP into an abnormal form (PrP^Sc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP^C is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP^Sc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP^C, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.     

Retrotranslocation of prion proteins from the endoplasmic reticulum by preventing GPI signal transamidation

Neurodegeneration in diseases caused by altered metabolism of mammalian prion protein (PrP) can be averted by reducing PrP expression. To identify novel pathways for PrP down-regulation, we analyzed cells that had adapted to the negative selection pressure of stable overexpression of a disease-causing PrP mutant. A mutant cell line was isolated that selectively and quantitatively routes wild-type and various mutant PrPs for ER retrotranslocation and proteasomal degradation. Biochemical analyses of the mutant cells revealed that a defect in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an unprocessed GPI-anchoring signal sequence that directs both ER retention and efficient retrotranslocation of PrP. An unprocessed GPI signal was sufficient to impart ER retention, but not retrotranslocation, to a heterologous protein, revealing an unexpected role for the mature domain in the metabolism of misprocessed GPI-anchored proteins. Our results provide new insights into the quality control pathways for unprocessed GPI-anchored proteins and identify transamidation of the GPI signal sequence as a step in PrP biosynthesis that is absolutely required for its surface expression. As each GPI signal sequence is unique, these results also identify signal recognition by the GPI-transamidase as a potential step for selective small molecule perturbation of PrP expression.     

Live Cell Fluorescence Resonance Energy Transfer Predicts an Altered Molecular Association of Heterologous PrPSc with PrPC

Prion diseases result from the accumulation of a misfolded isoform (PrP^Sc) of the normal host prion protein (PrP^C). PrP^Sc propagates by templating its conformation onto resident PrP^C to generate new PrP^Sc. Although the nature of the PrP^Sc-PrP^C complex is unresolved, certain segments or specific residues are thought to feature critically in its formation. The polymorphic residue 129 is one such site under considerable study. We combined transmission studies with a novel live cell yeast-based fluorescence resonance energy transfer (FRET) system that models the molecular association of PrP in a PrP^Sc-like state, as a way to explore the role of residue 129 in this process. We show that a reduction in efficiency of prion transmission between donor PrP^Sc and recipient PrP^C that are mismatched at residue 129 correlates with a reduction in FRET between PrP-129M and PrP-129V in our yeast model. We further show that this effect depends on the different secondary structure propensities of Met and Val, rather than the specific amino acids. Finally, introduction of the disease-associated P101L mutation (mouse- equivalent) abolished FRET with wild-type mouse PrP, whereas mutant PrP-P101L displayed high FRET with homologous PrP-P101L, as long as residue 129 matched. These studies provide the first evidence for a physical alteration in the molecular association of PrP molecules differing in one or more residues, and they further predict that the different secondary structure propensities of Met and Val define the impaired association observed between PrP^Sc and PrP^C mismatched at residue 129.     

PrP106-126 amide causes the semi-penetrated poration in the supported lipid bilayers

A major hallmark of prion diseases is the cerebral amyloid accumulation of the pathogenic PrP^Sc, an abnormally misfolded, protease-resistant, and β-sheet rich protein. PrP106-126 is the key domain responsible for the conformational conversion and aggregation of PrP. It shares important physicochemical characteristics with PrP^Sc and presents similar neurotoxicity as PrP^Sc. By combination of fluorescence polarization, dye release assay and in situ time-lapse atomic force microscopy (AFM), we investigated the PrP106-126 amide interacting with the large unilamellar vesicles (LUVs) and the supported lipid bilayers (SLBs). The results suggest that the interactions involve a poration-mediated process: firstly, the peptide binding results in the formation of pores in the membranes, which penetrate only half of the membranes; subsequently, PrP106-126 amide undergoes the poration-mediated diffusion in the SLBs, represented by the formation and expansion of the flat high-rise domains (FHDs). The possible mechanisms of the interactions between PrP106-126 amide and lipid membranes are proposed based on our observations.