Rare Large Scale Subdomain Motions in Prion Protein can Initiate Aggregation

Abstract

The prion protein is thought to induce prion diseases by changing its conformation from the cellular form, PrP^c, into the infectious Scrapie-form, PrP^Sc. Little is known about the structural and dynamical features of this conformational change. We here introduce a novel concept that involves rare large scale motions between the subdomains βl-αl-β2 and α2-α3 in the carboxy-terminal, globular part of PrP. The interface between these two subdomains carries most pathogenic mutations known to be associated with prion diseases. Based on computational simulations as well as experimental results we propose that such a large scale motion subsequently destabilizes large parts of the cellular conformer PrP^c, thus, rendering it prone to structural rearrangements, including aggregation of now partially unfolded parts of the PrP sequence. We hypothesize that such large scale motions occur as a rare event even under equilibrium conditions and that the interaction of such partially destabilized PrP^c-conformers, which we named PrP^c*, contributes to the formation of pathogenic oligomeric species of the prion protein.     

Altered prion protein glycosylation in the aging mouse brain

The normal cellular prion protein (PrP(C)) is a glycoprotein with two highly conserved potential N-linked glycosylation sites. All prion diseases, whether inherited, infectious or sporadic, are believed to share the same pathogenic mechanism that is based on the conversion of the normal cellular prion protein (PrP(C)) to the pathogenic scrapie prion protein (PrP(Sc)). However, the clinical and histopathological presentations of prion diseases are heterogeneous, depending not only on the strains of PrP(Sc) but also on the mechanism of diseases, such as age-related sporadic vs. infectious prion diseases. Accumulated evidence suggests that N-linked glycans on PrP(C) are important in disease phenotype. A better understanding of the nature of the N-linked glycans on PrP(C) during the normal aging process may provide new insights into the roles that N-linked glycans play in the pathogenesis of prion diseases. By using a panel of 19 lectins in an antibody-lectin enzyme-linked immunosorbent assay (ELISA), we found that the lectin binding profiles of PrP(C) alter significantly during aging. There is an increasing prevalence of complex oligosaccharides on the aging PrP(C), which are features of PrP(Sc). Taken together, this study suggests a link between the glycosylation patterns on PrP(C) during aging and PrP(Sc).     

The effect of disease-associated mutations on the folding pathway of human prion protein

Propagation of transmissible spongiform encephalopathies is believed to involve the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). An important step toward understanding the mechanism of this conversion is to elucidate the folding pathway(s) of the prion protein. We reported recently (Apetri, A. C., and Surewicz, W. K. (2002) J. Biol. Chem. 277, 44589-44592) that the folding of wild-type prion protein can best be described by a three-state sequential model involving a partially folded intermediate. Here we have performed kinetic stopped-flow studies for a number of recombinant prion protein variants carrying mutations associated with familial forms of prion disease. Analysis of kinetic data clearly demonstrates the presence of partially structured intermediates on the refolding pathway of each PrP variant studied. In each case, the partially folded state is at least one order of magnitude more populated than the fully unfolded state. The present study also reveals that, for the majority of PrP variants tested, mutations linked to familial prion diseases result in a pronounced increase in the thermodynamic stability, and thus the population, of the folding intermediate. These data strongly suggest that partially structured intermediates of PrP may play a crucial role in prion protein conversion, serving as direct precursors of the pathogenic PrP(Sc) isoform.     

Lactoferrin induces cell surface retention of prion protein and inhibits prion accumulation

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrP(C)) into its pathogenic, amyloidogenic isoform (PrP(Sc)) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin (TF) family, which accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer's disease and Pick's disease. In the present study, we have examined the effects of LF on PrP(Sc) formation by using cell culture models. Bovine LF inhibited PrP(Sc) accumulation in scrapie-infected cells in a time- and dose-dependent manner, whereas TF was not inhibitory. Bioassays of LF-treated cells demonstrated prolonged incubation periods compared with non-treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrP(C) by diminishing its internalization and was capable of interacting with PrP(C) in addition to PrP(Sc). Furthermore, LF partially inhibited the formation of protease-resistant PrP as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities.     

Rdj2, a J protein family member, interacts with cellular prion PrP(C)

PrP(C) is a glycosylphosphatidylinositol (GPI) anchored glycoprotein of unknown function. Misfolding of normal cellular PrP(C) to the pathogenic PrP(Sc) is the hallmark of prion diseases (transmissible spongiform encephalopathies). Prion diseases are characterized by extensive neurodegeneration and early death. Understanding how PrP(C) maintains its correct conformation is a major endeavor of current inquiry. Here we demonstrate a novel interaction between PrP(C) and the J protein family member, Rdj2 (DjA2; Dj3, Dnj3, Cpr3, and Hirip4). The importance of the J protein family in the cellular folding machinery has been recognized for many years. The PrP(C)/Rdj2 association was direct and concentration-dependent. Other J proteins such as CSPalpha and auxilin did not associate with PrP(C) in the absence of ATP, demonstrating the specificity of the PrP(C)/J protein interaction. These findings suggest that the J protein family serves as a 'folding catalyst' for PrP(C) and implicates Rdj2 as a factor in the protection against prion diseases.     

Cellular phenotyping of secretory and nuclear prion proteins associated with inherited prion diseases.

The pathogenic mechanisms leading from mutations in the prion protein (PrP) gene to infectious disease are not understood. To investigate the possibility that cellular processing of mutant prion protein may contribute to the formation of infectious particles, a mouse PrP model system has been established using the green fluorescent protein. Three novel PrP mutants were examined employing this model system and compared with wild type as well as known mutant PrPs. Two Creutzfeldt-Jakob disease-associated PrP mutants, PrP T188K and PrP T188R, revealed a secretory pathway to the cell membrane and PrP(Sc)-like properties, i.e. enhanced proteinase K resistance and detergent insolubility similar to other mutant PrPs associated with familial prion diseases. Moreover, a recently described disease-related truncated PrP mutant, PrP Q160(Stop), showed an almost exclusive localization in the nucleus and a catabolism along the proteasomal pathway. Therefore, various distinct pathological mechanisms may cause prion diseases, and aberrant cellular processing may be included in the pathogenesis of prion diseases.     

Evolving views in prion glycosylation: functional and pathological implications

Prion diseases form a group of neurodegenerative disorders with the unique feature of being transmissible. These diseases involve a pathogenic protein, called PrP(Sc) for the scrapie isoform of the cellular prion protein (PrP(C)) which is an abnormally-folded counterpart of PrP(C). Many questions remain unresolved concerning the function of PrP(C) and the mechanisms underlying prion replication, transmission and neurodegeneration. PrP(C) is a glycosyl-phosphatidylinositol-anchored glycoprotein expressed at the cell surface of neurons and other cell types. PrP(C) may be present as distinct isoforms depending on proteolytic processing (full length and truncated), topology(GPI-anchored, transmembrane or soluble) and glycosylation (non- mono- and di-glycosylated). The present review focuses on the implications of PrP(C) glycosylation as to the function of the normal protein, the cellular pathways of conversion into PrP(Sc), the diversity of prion strains and the related selective neuronal targeting.     

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.     

Comparative computational analysis of prion proteins reveals two fragments with unusual structural properties and a pattern of increase in hydrophobicity associated with disease-promoting mutations

Prion diseases are a group of neurodegenerative disorders associated with conversion of a normal prion protein, PrPC, into a pathogenic conformation, PrPSc. The PrPSc is thought to promote the conversion of PrPC. The structure and stability of PrPC are well characterized, whereas little is known about the structure of PrPSc, what parts of PrPC undergo conformational transition, or how mutations facilitate this transition. We use a computational knowledge-based approach to analyze the intrinsic structural propensities of the C-terminal domain of PrP and gain insights into possible mechanisms of structural conversion. We compare the properties of PrP sequences to those of a PrP paralog, Doppel, and to the distributions of structural propensities observed in known protein structures from the Protein Data Bank. We show that the prion protein contains at least two sequence fragments with highly unusual intrinsic propensities, PrP(114-125) and helix B. No segments with unusual properties were found in Doppel protein, which is topologically identical to PrP but does not undergo structural rearrangements. Known disease-promoting PrP mutations form a statistically significant cluster in the region comprising helices B and C. Due to their unusual properties, PrP(114-125) and the C terminus of helix B may be considered as primary candidates for sites involved in conformational transition from PrPC to PrPSc. The results of our study also show that most PrP mutations associated with neurodegenerative disorders increase local hydrophobicity. We suggest that the observed increase in hydrophobicity may facilitate PrP-to-PrP or/and PrP-to-cofactor interactions, and thus promote structural conversion.     

Conformational polymorphism of wild-type and mutant prion proteins: Energy landscape analysis

Conformational transitions are thought to be the prime mechanism of prion diseases. In this study, the energy landscapes of a wild-type prion protein (PrP) and the D178N and E200K mutant proteins were mapped, enabling the characterization of the normal isoforms (PrP(C)) and partially unfolded isoforms (PrP(PU)) of the three prion protein analogs. It was found that the three energy landscapes differ in three respects: (i) the relative stability of the PrP(C) and the PrP(PU) states, (ii) the transition pathways from PrP(C) to PrP(PU), and (iii) the relative stability of the three helices in the PrP(C) state. In particular, it was found that although helix 1 (residues 144-156) is the most stable helix in wild-type PrP, its stability is dramatically reduced by both mutations. This destabilization is due to changes in the charge distribution that affects the internal salt bridges responsible for the greater stability of this helix in wild-type PrP. Although both mutations result in similar destabilization of helix 1, they a have different effect on the overall stability of PrP(C) and of PrP(PU) isoforms and on structural properties. The destabilization of helix 1 by mutations provides additional evidences to the role of this helix in the pathogenic transition from the PrP(C) to the pathogenic isoform PrP(SC).     

Ablation of cellular prion protein expression affects mitochondrial numbers and morphology

The cellular prion protein (PrP(C)), predominantly expressed in the central nervous system, is required for pathogenesis of prion neurodegenerative diseases and its conversion into a pathogenic isoform (PrP(Sc)) is a common feature of disease. While the physiological function of PrP(C) remains unclear, accumulating evidence indicates a role for PrP(C) in oxidative homeostasis in vivo and suggests that PrP(C) may be involved in the cellular response to oxidative stress. Mice in which PrP(C) expression has been ablated are viable and develop normally. Here we show that in an inbred line of mice, in tissues that normally express PrP at moderate to high levels, ablation of PrP(C) results in reduced mitochondrial numbers, unusual mitochondrial morphology, and elevated levels of mitochondrial manganese-dependent superoxide dismutase antioxidant enzyme. These observations may have relevance to the pathogenic mechanism for this group of fatal neurodegenerative conditions.     

Chemical chaperones interfere with the formation of scrapie prion protein.

The fundamental event in prion diseases involves a conformational change in one or more of the alpha-helices of the cellular prion protein (PrP(C)) as they are converted into beta-sheets during the formation of the pathogenic isoform (PrP(Sc)). Here, we show that exposure of scrapie-infected mouse neuroblastoma (ScN2a) cells to reagents known to stabilize proteins in their native conformation reduced the rate and extent of PrP(Sc) formation. Such reagents include the cellular osmolytes glycerol and trimethylamine N-oxide (TMAO) and the organic solvent dimethylsulfoxide (DMSO), which we refer to as 'chemical chaperones' because of their influence on protein folding. Although the chemical chaperones did not appear to affect the existing population of PrP(Sc) molecules in ScN2a cells, they did interfere with the formation of PrP(Sc) from newly synthesized PrP(C). We suggest that the chemical chaperones act to stabilize the alpha-helical conformation of PrP(C) and thereby prevent the protein from undergoing a conformational change to produce PrP(Sc). These observations provide further support for the idea that prions arise due to a change in protein conformation and reveal potential strategies for preventing PrP(Sc) formation.     

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.     

Molecular interactions between prions as seeds and recombinant prion proteins as substrates resemble the biological interspecies barrier in vitro

Prion diseases like Creutzfeldt-Jakob disease in humans, Scrapie in sheep or bovine spongiform encephalopathy are fatal neurodegenerative diseases, which can be of sporadic, genetic, or infectious origin. Prion diseases are transmissible between different species, however, with a variable species barrier. The key event of prion amplification is the conversion of the cellular isoform of the prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)). We developed a sodiumdodecylsulfate-based PrP conversion system that induces amyloid fibril formation from soluble α-helical structured recombinant PrP (recPrP). This approach was extended applying pre-purified PrP(Sc) as seeds which accelerate fibrillization of recPrP. In the present study we investigated the interspecies coherence of prion disease. Therefore we used PrP(Sc) from different species like Syrian hamster, cattle, mouse and sheep and seeded fibrillization of recPrP from the same or other species to mimic in vitro the natural species barrier. We could show that the in vitro system of seeded fibrillization is in accordance with what is known from the naturally occurring species barriers.     

Trapping prion protein in the endoplasmic reticulum impairs PrPC maturation and prevents PrPSc accumulation

The conversion of the normal cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrP(C) and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrP(C) we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrP(C) and on PrP(Sc) formation. We found that ER-targeted anti-prion scFvs retain PrP(C) in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrP(C), with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrP(C) acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrP(Sc) accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.     

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.     

Local structural plasticity of the prion protein. Analysis of NMR relaxation dynamics

A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.     

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.     

Zinc, copper, and carnosine attenuate neurotoxicity of prion fragment PrP106-126

Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of β-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited β-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (β-alanyl histidine) significantly inhibited both the β-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases.     

Production of cattle lacking prion protein

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.