α-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

PrP overdrive

Knockout of the cellular prion protein (PrP^C) in mice is tolerated, as is complete elimination of the protein’s N-terminal domain. However, deletion of select short segments between the N- and C-terminal domains is lethal. How can one reconcile this apparent paradox? Research over the last few years demonstrates that PrP^C undergoes α-cleavage in the vicinity of residue 109 (mouse sequence) to release the bioactive N1 and C1 fragments. In biophysical studies, we recently characterized the action of relevant members of the ADAM (A Disintegrin And Metalloproteinase) enzyme family (ADAM8, 10, and 17) and found that they all produce α-cleavage, but at 3 distinct cleavage sites, with proteolytic efficiency modulated by the physiologic metals copper and zinc. Remarkably, the shortest lethal deletion segment in PrP^C fully encompasses the three α-cleavage sites. Analysis of all reported PrP^C deletion mutants suggests that elimination of α-cleavage, coupled with retention of the protein’s N-terminal residues, segments 23–31 and longer, confers the lethal phenotype. Interestingly, these N-terminal residues are implicated in the activation of several membrane proteins, including synaptic glutamate receptors. We propose that α-cleavage is a general mechanism essential for downregulating PrP^C’s intrinsic activity, and that blockage of proteolysis leads to constitutively active PrP^C and consequent dyshomeostasis.     

Separate mechanisms act concurrently to shed and release the prion protein from the cell

The cellular prion protein (PrP^C) is attached to the cell membrane via its glycosylphosphatidylinositol (GPI)-anchor and is constitutively shed into the extracellular space. Here, three different mechanisms are presented that concurrently shed PrP^C from the cell. The fast α-cleavage released a N-terminal fragment (N1) into the medium and the extreme C-terminal cleavage shed soluble full-length (FL-S) PrP and C-terminally cleaved (C1-S) fragments outside the cell. Also, a slow exosomal release of full-length (FL) and C1-fragment (C1) was demonstrated. The three separate mechanisms acting simultaneously, but with different kinetics, have to be taken into consideration when elucidating functional roles of PrP^C and also when processing of PrP^C is considered as a target for intervention in prion diseases. Further, in this study it was shown that metalloprotease inhibitors affected the extreme C-terminal cleavage and shedding of PrP^C. The metalloprotease inhibitors did not influence the α-cleavage or the exosomal release. Taken together, these results are important for understanding the different mechanisms acting in parallel in the shedding and cleavage of PrP^C.     

The PrPC C1 fragment derived from the ovine A136R154R171 PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrPC protein in vitro

Expression of the cellular prion protein (PrP^C) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrP^C comprised of 25% more C1 fragment than PrP^C from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrP^C variants. We propose that the increased α-cleavage of ovine ARR PrP^C contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrP^C β-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.     

��-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

Microdeletions within the hydrophobic core region of cellular prion protein alter its topology and metabolism

The cellular prion protein (PrP^C) is a GPI-anchored cell-surface protein. A small subset of PrP^C molecules, however, can be integrated into the ER-membrane via a transmembrane domain (TM), which also harbors the most highly conserved regions of PrP^C, termed the hydrophobic core (HC). A mutation in HC is associated with prion disease resulting in an enhanced formation of a transmembrane form (^CtmPrP), which has thus been postulated to be a neurotoxic molecule besides PrP^Sc. To elucidate a possible physiological function of the transmembrane domain, we created a set of mutants carrying microdeletions of 2-8 aminoacids within HC between position 114 and 121. Here, we show that these mutations display reduced propensity for transmembrane topology. In addition, the mutants exhibited alterations in the formation of the C1 proteolytic fragment, which is generated by α-cleavage during normal PrP^C metabolism, indicating that HC might function as recognition site for the protease(s) responsible for PrP^C α-cleavage. Interestingly, the mutant G113V, corresponding to a hereditary form of prion disease in humans, displayed increased ^CtmPrP topology and decreased α-cleavage in our in vitro assay. In conclusion, HC represents an essential determinant for transmembrane PrP topology, whereas the high evolutionary conservation of this region is rather based upon preservation of PrP^C α-cleavage, thus highlighting the biological importance of this cleavage.     

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes α-Cleavage of PrP<Superscript>C</Superscript>

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrP^Sc of the cellular prion protein PrP^C. Expression of PrP^C is a prerequisite for prion infection, and conformational conversion of PrP^C is induced upon its direct interaction with PrP^Sc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrP^C as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrP^Sc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrP^C α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrP^C at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aβ oligomers characteristic for Alzheimer’s disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrP^C and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.     

Taking advantage of physiological proteolytic processing of the prion protein for a therapeutic perspective in prion and Alzheimer diseases

Prion and Alzheimer diseases are fatal neurodegenerative diseases caused by misfolding and aggregation of the cellular prion protein (PrP^C) and the β-amyloid peptide, respectively. Soluble oligomeric species rather than large aggregates are now believed to be neurotoxic. PrP^C undergoes three proteolytic cleavages as part of its natural life cycle, α-cleavage, β-cleavage, and ectodomain shedding. Recent evidences demonstrate that the resulting secreted PrP^C molecules might represent natural inhibitors against soluble toxic species. In this mini-review, we summarize recent observations suggesting the potential benefit of using PrP^C-derived molecules as therapeutic agents in prion and Alzheimer diseases.     

Unexpected Tolerance of α-Cleavage of the Prion Protein to Sequence Variations

The cellular form of the prion protein, PrP^C, undergoes extensive proteolysis at the α site (109K↓H110). Expression of non-cleavable PrP^C mutants in transgenic mice correlates with neurotoxicity, suggesting that α-cleavage is important for PrP^C physiology. To gain insights into the mechanisms of α-cleavage, we generated a library of PrP^C mutants with mutations in the region neighbouring the α-cleavage site. The prevalence of C1, the carboxy adduct of α-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the α-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, α-cleavage was size-dependently impaired by deletions within the domain 106–119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that α-cleavage is executed by an α-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP^C.     

The P’s and Q’s of cellular PrP-Aβ interactions

Prion disease research has opened up the “black-box” of neurodegeneration, defining a key role for protein misfolding wherein a predominantly alpha-helical precursor protein, PrP^C, is converted to a disease-associated, β-sheet enriched isoform called PrP^Sc. In Alzheimer disease (AD) the Aβ peptide derived from the β-amyloid precuror protein APP folds in β-sheet amyloid. Early thoughts along the lines of overlap may have been on target,¹ but were eclipsed by a simultaneous (but now anachronistic) controversy over the role of PrP^Sc in prion diseases.^2,3 Nonetheless, as prion diseases such as Creutzfeldt-Jakob Disease (CJD) are themselves rare and can include an overt infectious mode of transmission, and as familial prion diseases and familial AD involve different genes, an observer might reasonably have concluded that prion research could occasionally catalyze ideas in AD, but could never provide concrete overlaps at the mechanistic level. Surprisingly, albeit a decade or three down the road, several prion/AD commonalities can be found within the contemporary literature. One important prion/AD overlap concerns seeded spread of Aβ aggregates by intracerebral inoculation much like prions,⁴ and, with a neuron-to-neuron ‘spreading’ also reported for pathologic forms of other misfolded proteins, Tau^5,6 and α-synuclein in the case of Parkinson Disease.^7,8 The concept of seeded spread has been discussed extensively elsewhere, sometimes under the rubric of “prionoids”⁹, and lies outside the scope of this particular review where we will focus upon PrP^C. From this point the story can now be subdivided into four strands of investigation: (1) pathologic effects of Aβ can be mediated by binding to PrP^C,¹⁰ (2) the positioning of endoproteolytic processing events of APP by pathologic (β-cleavage + γ-cleavage) and non-pathologic (α-cleavage + γ-cleavage) secretase pathways is paralleled by seemingly analogous α- and β-like cleavage of PrP^C (Fig. 1) (3) similar lipid raft environments for PrP^C and APP processing machinery,^11-13 and perhaps in consequence, overlaps in repertoire of the PrP^C and APP protein interactors (“interactomes”),^14,15 and (4) rare kindreds with mixed AD and prion pathologies.¹⁶ Here we discuss confounds, consensus and conflict associated with parameters that apply to these experimental settings.     

Separate mechanisms act concurrently to shed and release the prion protein from the cell

The cellular prion protein (PrP^C) is attached to the cell membrane via its glycosylphosphatidylinositol (GPI)-anchor and is constitutively shed into the extracellular space. Here, three different mechanisms are presented that concurrently shed PrP^C from the cell. The fast α-cleavage released a N-terminal fragment (N1) into the medium and the extreme C-terminal cleavage shed soluble full-length (FL-S) PrP and C-terminally cleaved (C1-S) fragments outside the cell. Also, a slow exosomal release of full-length (FL) and C1-fragment (C1) was demonstrated. The three separate mechanisms acting simultaneously, but with different kinetics, have to be taken into consideration when elucidating functional roles of PrP^C and also when processing of PrP^C is considered as a target for intervention in prion diseases. Further, in this study it was shown that metalloprotease inhibitors affected the extreme C-terminal cleavage and shedding of PrP^C. The metalloprotease inhibitors did not influence the α-cleavage or the exosomal release. Taken together, these results are important for understanding the different mechanisms acting in parallel in the shedding and cleavage of PrP^C.     

The cellular prion protein (PrPC) as neuronal receptor for α-synuclein

The term ‘prion-like’ is used to define some misfolded protein species that propagate intercellularly, triggering protein aggregation in recipient cells. For cell binding, both direct plasma membrane interaction and membrane receptors have been described for particular amyloids. In this respect, emerging evidence demonstrates that several β-sheet enriched proteins can bind to the cellular prion protein (PrP^C). Among other interactions, the physiological relevance of the binding between β-amyloid and PrP^C has been a relevant focus of numerous studies. At the molecular level, published data point to the second charged cluster domain of the PrP^C molecule as the relevant binding domain of the β-amyloid/PrP^C interaction. In addition to β-amyloid, participation of PrP^C in binding α-synuclein, responsible for neurodegenerative synucleopathies, has been reported. Although results indicate relevant participation of PrP^C in the spreading of α-synuclein in living mice, the physiological relevance of the interaction remains elusive. In this comment, we focus our attention on summarizing current knowledge of PrP^C as a receptor for amyloid proteins and its physiological significance, with particular focus on α-synuclein.     

The role of the prion protein in the internalization of α-synuclein amyloids

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein amyloids in several regions of the brain. α-Synuclein fibrils are able to spread via cell-to-cell transfer, and once inside the cells, they can template the misfolding and aggregation of the endogenous α-synuclein. Multiple mechanisms have been shown to participate in the process of propagation: endocytosis, tunneling nanotubes and macropinocytosis. Recently, we published a research showing that the cellular form of the prion protein (PrP^C) acts as a receptor for α-synuclein amyloid fibrils, facilitating their internalization through and endocytic pathway. This interaction occurs by a direct interaction between the fibrils and the N-terminal domain of PrP^C. In cell lines expressing the pathological form of PrP (PrP^Sc), the binding between PrP^C and α-synuclein fibrils prevents the formation and accumulation of PrP^Sc, since PrP^C is no longer available as a substrate for the pathological conversion templated by PrP^Sc. On the contrary, PrP^Sc deposits are cleared over passages, probably due to the increased processing of PrP^C into the neuroprotective fragments N1 and C1. Starting from these data, in this work we present new insights into the role of PrP^C in the internalization of protein amyloids and the possible therapeutic applications of these findings.     

Selective Processing and Metabolism of Disease-Causing Mutant Prion Proteins

Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP^C). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP^Sc form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP^C to PrP^Sc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP^Sc in familial prion disease.     

Does the tail wag the dog? How the structure of a glycosylphosphatidylinositol anchor affects prion formation

There is increasing interest in the role of the glycosylphosphatidylinositol (GPI) anchor attached to the cellular prion protein (PrP^C). Since GPI anchors can alter protein targeting, trafficking and cell signaling, our recent study examined how the structure of the GPI anchor affected prion formation. PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc in prion-infected neuronal cell lines and in scrapie-infected primary cortical neurons. In uninfected neurons desialylated PrP^C was associated with greater concentrations of gangliosides and cholesterol than PrP^C. In addition, the targeting of desialylated PrP^C to lipid rafts showed greater resistance to cholesterol depletion than PrP^C. The presence of desialylated PrP^C caused the dissociation of cytoplasmic phospholipase A₂ (cPLA₂) from PrP-containing lipid rafts, reduced the activation of cPLA₂ and inhibited PrP^Sc production. We conclude that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation.     

Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds,Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate,Chlorpromazine, and U18666A,in Prion-Infected Mouse Neuroblastoma Cells

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP^Sc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP^Sc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP^C) and PrP^Sc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP^Sc levels without altering intracellular distribution of PrP^Sc. PPS did not change the distribution and levels of PrP^C, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP^C to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP^Sc formation. In contrast, CPZ and U18666A initiated the redistribution of PrP^Sc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP^C. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP^Sc redistribution by CPZ or U18666A partly antagonized PrP^Sc degradation, suggesting that the transfer of PrP^Sc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP^Sc degradation. This study revealed that precise analysis of the intracellular dynamics of PrP^C and PrP^Sc provides important information for understanding the mechanism of anti-prion agents.     

Laminin‐γ1 chain and stress inducible protein 1 synergistically mediate PrPC‐dependent axonal growth via Ca2+ mobilization in dorsal root ganglia neurons

Prion protein (PrP^C) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP^C interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP^C co‐opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross‐talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP^C‐mediated axonogenesis in peripheral neurons in response to STI1 and laminin‐γ1 chain‐derived peptide (Ln‐γ1). STI1 and Ln‐γ1 promoted robust axonogenesis in wild‐type neurons, whereas no effect was observed in neurons from PrP^C‐null mice. PrP^C binding to Ln‐γ1 or STI1 led to an increase in intracellular Ca²⁺ levels via distinct mechanisms: STI1 promoted extracellular Ca²⁺ influx, and Ln‐γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln‐γ1, but depends on, C‐type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP^C‐mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP^C. These results suggest a role for PrP^C as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.     

The P’s and Q’s of cellular PrP-Aβ interactions

Prion disease research has opened up the “black-box” of neurodegeneration, defining a key role for protein misfolding wherein a predominantly alpha-helical precursor protein, PrP^C, is converted to a disease-associated, β-sheet enriched isoform called PrP^Sc. In Alzheimer disease (AD) the Aβ peptide derived from the β-amyloid precuror protein APP folds in β-sheet amyloid. Early thoughts along the lines of overlap may have been on target,¹ but were eclipsed by a simultaneous (but now anachronistic) controversy over the role of PrP^Sc in prion diseases.^2,3 Nonetheless, as prion diseases such as Creutzfeldt-Jakob Disease (CJD) are themselves rare and can include an overt infectious mode of transmission, and as familial prion diseases and familial AD involve different genes, an observer might reasonably have concluded that prion research could occasionally catalyze ideas in AD, but could never provide concrete overlaps at the mechanistic level. Surprisingly, albeit a decade or three down the road, several prion/AD commonalities can be found within the contemporary literature. One important prion/AD overlap concerns seeded spread of Aβ aggregates by intracerebral inoculation much like prions,⁴ and, with a neuron-to-neuron ‘spreading’ also reported for pathologic forms of other misfolded proteins, Tau^5,6 and α-synuclein in the case of Parkinson Disease.^7,8 The concept of seeded spread has been discussed extensively elsewhere, sometimes under the rubric of “prionoids”⁹, and lies outside the scope of this particular review where we will focus upon PrP^C. From this point the story can now be subdivided into four strands of investigation: (1) pathologic effects of Aβ can be mediated by binding to PrP^C,¹⁰ (2) the positioning of endoproteolytic processing events of APP by pathologic (β-cleavage + γ-cleavage) and non-pathologic (α-cleavage + γ-cleavage) secretase pathways is paralleled by seemingly analogous α- and β-like cleavage of PrP^C (Fig. 1) (3) similar lipid raft environments for PrP^C and APP processing machinery,^11-13 and perhaps in consequence, overlaps in repertoire of the PrP^C and APP protein interactors (“interactomes”),^14,15 and (4) rare kindreds with mixed AD and prion pathologies.¹⁶ Here we discuss confounds, consensus and conflict associated with parameters that apply to these experimental settings.     

Differential stability of the bovine prion protein upon urea unfolding

Prion diseases, or transmissible spongiform encephalopathies, are a group of infectious neurological diseases associated with the structural conversion of an endogenous protein (PrP) in the central nervous system. There are two major forms of this protein: the native and noninfectious cellular form, PrP^C; and the misfolded, infectious, and proteinase K‐resistant form, PrP^Sc. The C‐terminal domain of PrP^C is mainly α‐helical in structure, whereas PrP^Sc in known to aggregate into an assembly of β‐sheets, forming amyloid fibrils. To identify the regions of PrP^C potentially involved in the initial steps of the conversion to the infectious conformation, we have used high‐resolution NMR spectroscopy to characterize the stability and structure of bovine recombinant PrP^C (residues 121 to 230) during unfolding with the denaturant urea. Analysis of the 800 MHz ¹H NMR spectra reveals region‐specific information about the structural changes occurring upon unfolding. Our data suggest that the dissociation of the native β‐sheet of PrP^C is a primary step in the urea‐induced unfolding process, while strong hydrophobic interactions between helices α1 and α3, and between α2 and α3, stabilize these regions even at very high concentrations of urea.     

Role of Lipid Rafts and GM1 in the Segregation and Processing of Prion Protein

The prion protein (PrP^C) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrP^C is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrP^C raft association, together with raft lipid composition, appears essential for the conversion of PrP^C into the scrapie isoform PrP^Sc, and the development of prion disease. Controversial findings were reported on the nature of PrP^C-containing rafts, as well as on the distribution of PrP^C between rafts and non-raft membranes. We investigated PrP^C/ganglioside relationships and their influence on PrP^C localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrP^C conformations coexist: in lipid rafts PrP^C is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrP^Sc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrP^C conformation and interaction with lipid bilayers, without modifying PrP^C distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.