Scrapie prions selectively modify the stress response in neuroblastoma cells

The fundamental event underlying scrapie infection seems to be a conformational change in the prion protein. To investigate proteins that might feature in the conversion of the cellular prion protein (PrPC) into the scrapie isoform (PrPSc), we examined mouse neuroblastoma N2a cells for the expression and cellular distribution of heat shock proteins (Hsps), some of which function as molecular chaperones. In scrapie-infected N2a (ScN2a) cells, Hsp72 and Hsp28 were not induced by heat shock, sodium arsenite, or an amino acid analog, in contrast to uninfected control N2a cells, while other inducible Hsps were increased by these treatments. Following heat shock of the N2a cells, constitutively expressed Hsp73 was translocated from the cytoplasm into the nucleus and nucleolus. In contrast, the distribution of Hsp73 in ScN2a cells was not altered by heat shock; the discrete cytoplasmic structures containing Hsp73 were largely resistant to detergent extraction. These alterations in the expression and subcellular translocation of specific Hsps in ScN2a cells may reflect the cellular response to the accumulation of PrPSc. Whether any of these Hsps feature in the conversion of PrPC into PrPSc or the pathogenesis of prion diseases remains to be established.     

Chaperone-supervised conversion of prion protein to its protease-resistant form

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet. The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.     

Prions

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.     

The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation

BACKGROUND: A conformational change seems to represent the major difference between the scrapie prion protein (PrPSc) and its normal cellular isoform (PrPC). We recently proposed a set of four helix bundle models for the three-dimensional structure of PrPC that are consistent with a variety of spectroscopic and genetic data. RESULTS: We report a plausible model for the three-dimensional structure of a biologically important fragment of PrPSc. The model of residues 108-218 was constructed by an approach that combines computational techniques and experimental data. The proposed structures of this fragment of PrPSc display a four-stranded beta-sheet covered on one face by two alpha-helices. Residues implicated in the prion species barrier are found to cluster on the solvent-accessible surface of the beta-sheet of one of the models. This interface could provide a structural template that would assist the conversion of PrPC to PrPSc and hence direct prion propagation. CONCLUSIONS: Molecular models of the PrP isoforms should prove very useful in developing structural hypotheses about the process by which PrPC is transformed into PrPSc, the mechanisms by which PrP gene mutations give rise to the inherited human prion diseases, and the species barrier that seems to protect humans from animal prions. It seems likely that PrPC represents a kinetically trapped intermediate in PrP folding.     

Genetics of prion diseases and prion diversity in mice

Linkage of the prion protein (PrP) and scrapie incubation time genes in mice provided strong evidence for the central role of PrP in determining susceptibility to prion disorders. Considerable evidence now argues that the prion protein and incubation time genes are identical. The mouse prion protein gene (Prn-p) may act both quantitatively and qualitatively in modulating prion incubation time. Differences at positions 108 and 189 between PrP-A and PrP-B allotypes can place constraints on interaction between the normal cellular and the scrapie-specific isoforms of PrP (PrPC and PrPSc), although the supply of PrPC available for post-translational conversion to PrPSc can also influence incubation time. Results using transgenic (Tg) mice in studies on scrapie 'strains' or isolates suggest that incubation time characteristics of scrapie isolates can be explained by these two properties of PrP. The final section of this report discusses the novel finding that uninoculated Tg mice overexpressing wild-type (wt) PrP transgenes spontaneously develop a late-onset degenerative neuromyopathy, broadening the spectrum of prion diseases and providing new information on PrP function in both normal and pathological states.     

The use of transgenic mice in the investigation of transmissible spongiform encephalopathies

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and to be identical to PrPSc (prion protein: scrapie form), a modified form of the normal host protein PrPC (prion protein: cellular form) which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. The authors generated homozygous Prnp(o/o) ('PrP knockout') mice and showed that, after inoculation with prions, these mice remained free from scrapie for at least two years while wild-type controls all died within six months. There was no propagation of prions in the Prnp(o/o) animals. Surprisingly, heterozygous Prnp(o/+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie despite high levels of infectious agent and PrPSc in the brain at an early stage. After introduction of murine PrP transgenes, Prnp(o/o) mice became highly susceptible to mouse--but not to hamster--prions, while the insertion of Syrian hamster PrP transgenes rendered the mice susceptible to hamster prions but much less susceptible to mouse prions. These complementation experiments enabled the application of reverse genetics. The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.     

Sulfated glycans stimulate endocytosis of the cellular isoform of the prion protein, PrPC, in cultured cells

There is currently no effective therapy for human prion diseases. However, several polyanionic glycans, including pentosan sulfate and dextran sulfate, prolong the incubation time of scrapie in rodents, and inhibit the production of the scrapie isoform of the prion protein (PrPSc), the major component of infectious prions, in cultured neuroblastoma cells. We report here that pentosan sulfate and related compounds rapidly and dramatically reduce the amount of PrPC, the non-infectious precursor of PrPSc, present on the cell surface. This effect results primarily from the ability of these agents to stimulate endocytosis of PrPC, thereby causing a redistribution of the protein from the plasma membrane to the cell interior. Pentosan sulfate also causes a change in the ultrastructural localization of PrPC, such that a portion of the protein molecules are shifted into late endosomes and/or lysosomes. In addition, we demonstrate, using PrP-containing bacterial fusion proteins, that cultured cells express saturable and specific surface binding sites for PrP, many of which are glycosaminoglycan molecules. Our results raise the possibility that sulfated glycans inhibit prion production by altering the cellular localization of PrPC precursor, and they indicate that endogenous proteoglycans are likely to play an important role in the cellular metabolism of both PrPC and PrPSc.     

A trend of molecular genetics on prion diseases and prion protein

Infectious amyloid filaments designated as prion rods or scrapie associated fibrils (SAF) present in brain tissues affected by transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker disease (GSS) and kuru of humans, and scrapie of sheep. A hydrophobic glycoprotein, PrPSc is a major component of SAF, and is known to be associated with the infectivity of these diseases. Both PrPSc and the normal isoform of this glycoprotein, PrPC are encoded by a single host gene, PrP gene, and the conversion of PrPC to PrPSc is a posttranslational event. Several mutations on the PrP gene are associated with variations of the phenotype and the occurrence in familial CJD and GSS.     

Oral administration of (-)catechin protects against ischemia-reperfusion-induced neuronal death in the gerbil

Prions diseases are fatal neurodegenerative disorders resulting from conformational changes in the prion protein from the normal cellular form, PrPC, to the infectious scrapie isoform, PrPSc. High resolution structures for PrPC are now available, and biochemical investigations are shedding light on the nature and determinants of the conformational transition. Together, these studies are beginning to provide a framework to describe structure-function relationships of the prion protein.     

The prion diseases

The human prion diseases are fatal neurodegenerative maladies that may present as sporadic, genetic, or infectious illnesses. The sporadic form is called Creutzfeldt-Jakob disease (CJD) while the inherited disorders are called familial (f) CJD, Gerstmann-Straussler-Scheinker (GSS) disease and fatal familial insomnia (FFI). Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high beta-sheet content. In fCJD, GSS, and FFI, mutations in the PrP gene located on the short arm of chromosome 20 are the cause of disease. Considerable evidence argues that the prion diseases are disorders of protein conformation.     

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of approximately 142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an alpha-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular beta-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.     

Identification of intermediate steps in the conversion of a mutant prion protein to a scrapie-like form in cultured cells

The central causative event in infectious, familial, and sporadic forms of prion disease is thought to be a conformational change that converts the cellular isoform of the prion protein (PrPC) into the scrapie isoform (PrPSc) that is the primary constituent of infectious prion particles. To provide a model system for analyzing the mechanistic details of this critical transformation, we have previously prepared cultured Chinese hamster ovary cells that stably express mouse PrP molecules carrying mutations homologous to those seen in familial prion diseases of humans. In the present work, we have analyzed the kinetics with which a PrP molecule containing an insertional mutation associated with Creutzfeldt-Jakob disease acquires several biochemical properties characteristic of PrPSc. Within 10 min of pulse labeling, the mutant protein undergoes a molecular alteration that is detectable by a change in Triton X-114 phase partitioning and phenyl-Sepharose binding. After 30 min of labeling, a detergent-insoluble and protease-sensitive form of the protein appears. After a chase period of several hours, the protein becomes protease-resistant. Incubation of cells at 18 degrees C or treatment with brefeldin A inhibits acquisition of detergent insolubility and protease resistance but does not affect Triton X-114 partitioning and phenyl-Sepharose binding. Our results support a model in which conversion of mutant PrPs to a PrPSc-like state proceeds in a stepwise fashion via a series of identifiable biochemical intermediates, with the earliest step occurring during or very soon after synthesis of the polypeptide in the endoplasmic reticulum.     

The prionoses and other conformational disorders

The basic pathogenesis of numerous neurodegenerative disorders is now thought to be related to abnormal protein conformation. The common theme in all these diseases is the conversion of a normal cellular and/or circulating protein into an insoluble, aggregated, beta-sheet rich form which is deposited in the brain, sometimes in the form of amyloid. These deposits are toxic and produce neuronal dysfunction and death. The most common of these illnesses is Alzheimer's disease (AD), in which a central event is the conversion of the normal soluble amyloid beta (sA beta) peptide to amyloid beta (A beta) within neuritic plaques and cerebral vessels. A unique category of the conformational conditions are prion related diseases (or prionoses), where the etiology is thought to be related to conversion of the normal prion protein, PrPC, into an infectious and pathogenic form, PrPSc. In the case of AD and the prionoses, the conformational change can be influenced by the presence of mutations in various gene products, as well as by chaperone proteins. Apolipoprotein E is thought to act as such a chaperone protein in AD; however, among the prionoses such a protein has been hypothesized to exist only by indirect evidence and is called "protein X". Our growing understanding of the mechanisms involved in this category of diseases, raises the possibility of therapeutic approaches based directly on the prevention and reversal of pathologic protein conformation.     

Mapping the prion protein using recombinant antibodies

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPC but absent from PrPSc.     

Recombinant scrapie-like prion protein of 106 amino acids is soluble

The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.     

Transgenetic investigations of prion diseases of humans and animals

Prions cause transmissible and genetic neurodegenerative diseases. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrPSc), which is encoded by a chromosomal gene. Although the PrP gene is single copy, transgenic mice with both alleles of the PrP gene ablated develop normally. A post-translational process, as yet unidentified, converts the cellular prion protein (PrPC) into PrPSc. Scrapie incubation times, neuropathology and prion synthesis in transgenic mice are controlled by the PrP gene. Mutations in the PrP gene are genetically linked to development of neurodegeneration. Transgenic mice expressing mutant PrP spontaneously develop neurological dysfunction and spongiform neuropathology. Investigations of prion diseases using transgenesis promise to yield much new information about these once enigmatic disorders.     

The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells

Prions are thought to consist of infectious proteins that cause transmissible spongiform encephalopathies. According to overwhelming evidence, the pathogenic prion protein PrPSc converts its host encoded isoform PrPC into insoluble aggregates of PrPSc, concomitant with pathological modifications (for review, see refs. 1-3). Although the physiological role of PrPC is poorly understood, studies with PrP knockout mice demonstrated that PrPC is required for the development of prion diseases. Using the yeast two-hybrid technology in Saccharomyces cerevisiae, we identified the 37-kDa laminin receptor precursor (LRP) as interacting with the cellular prion protein PrPC. Mapping analysis of the LRP-PrP interaction site in S. cerevisiae revealed that PrP and laminin share the same binding domain (amino acids 161 to 180) on LRP. The LRP-PrP interaction was confirmed in vivo in insect (Sf9) and mammalian cells (COS-7). The LRP level was increased in scrapie-infected murine N2a cells and in brain and spleen of scrapie-infected mice. In contrast, the LRP concentration was not significantly altered in these organs from mice infected with the bovine spongiform encephalopathic agent (BSE), which have a lower PrPSc accumulation. LRP levels, however, were dramatically increased in brain and pancreas, slightly increased in the spleen and not altered in the liver of crapie-infected hamsters. These data show that enhanced LRP concentrations are correlated with PrPSc accumulation in organs from mice and hamsters. The laminin receptor precursor, which is highly conserved among mammals and is located on the cell surface, may act as a receptor or co-receptor for the prion protein on mammalian cells.     

Heat-shock proteins and molecular chaperones: implications for pathogenesis, diagnostics, and therapeutics

Cells react to physical (e.g., heat) or chemical (e.g., anoxia, low pH) stressors, mounting a stress (heat-shock) response. Most genes are turned down or off, while a few are activated. The latter encode the stress or heat-shock proteins (Hsps), whose levels increase in stressed cells. Various Hsps are molecular chaperones. These, and other molecular chaperones that are not Hsps, help the other cellular proteins to achieve their native state (correct folding or functional conformation), reach their final destination (e.g., the endoplasmic reticulum or the mitochondria), resist denaturing by stressors, and regain the native state after partial denaturation. Thus the Hsps and molecular chaperones occupy the stage's center whenever and wherever there is cellular and tissue injury caused by local or systemic stressors via protein damage. This feature, their participation in protein folding and transport, and their evolutionary conservation within the three phylogenetic domains, strongly suggest a vital role for Hsps and molecular chaperones. Their importance in pathogenesis, and as diagnostic markers and prognostic indicators, is beginning to be appreciated. The role of Hsps and molecular chaperones in cell recovery from injury by a variety of noxae of clinical and surgical relevance is also being assessed. Consequently, the potential of these molecules (and corresponding genes) as targets for treatment or as therapeutic tools is emerging and is being explored. Stroke, myocardial infarction, inflammatory syndromes, infectious and parasitic diseases, autoimmune disorders, cancer, and aging are but some examples of conditions in which Hsps and molecular chaperones are being scrutinized. The era of Hsp and molecular chaperone pathology has dawned. It is likely that genetic and acquired defects of Hsp and molecular chaperone structure and function will be identified, and will play a primary, or auxiliary but determinant, role in disease.     

Evidence that GABA transmission mediates context-specific extinction of learned fear

Recent evidence has suggested that molecular chaperones participate in the conformational change between the normal cellular prion protein (PrPC) and its scrapie isoform (PrPSc). To study a role of PrPC in the regulation of expression of heat shock proteins (HSPs), a group of molecular chaperones, heat-induced expression of major HSPs (HSP105, HSP90alpha, HSP72, HSC70, HSP60, and HSP25) was investigated in cultured skin fibroblasts isolated from the mice homogeneous for a disrupted PrP gene (PrP-/- mice) by Western blot analysis and immunocytochemistry. Two lines of fibroblasts were established and designated SFK derived from the PrP-/- mice and SFH derived from the PrP+/+ mice, respectively. In both SFK and SFH cells, HSP105, HSP72, and HSP25 were expressed at low levels under unstressed conditions but they were induced markedly following exposure to heat stress (43 degreesC/20 min) at 3-72 h postrecovery. In both cell types, HSC70 and HSP60 were expressed at high levels under unstressed conditions and their levels remained unchanged after heat shock treatment. HSP90alpha was undetectable in both cell types under any conditions examined. The pattern of expression, induction, and subcellular location of HSP105, HSP72, HSC70, HSP60, and HSP25 was not significantly different between SFK and SFH cells under unstressed and heat-stressed conditions. Furthermore, the levels of constitutive expression of HSP105, HSC70, HSP60, and HSP25 were similar between the brain tissues isolated from the PrP-/- and PrP+/+ mice. These results indicate that HSP induction is not affected by either the existence or the absence of PrPC in the cells.