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Prion diseases are rare fatal neurodegenerative conditions that affect humans and animals. The human diseases include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, and fatal familial insomnia. Most cases of human prion [...]

Extracellular plaques of amyloid-β and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and [...]

The self-templating conformations of yeast prion proteins act as epigenetic elements of inheritance. Yeast prions might provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits. However, this hypothesis is highly controversial. Prions that create new traits have not been found in wild strains, leading to the perception that they are rare 'diseases' of laboratory cultivation. Here we biochemically test approximately 700 wild strains of Saccharomyces for [Ψ.sup.+]] or [[MOT3.sup.+]], and find these prions in many. They conferred diverse phenotypes that were frequently beneficial under selective conditions. Simple meiotic re-assortment of the variation harboured within a strain readily fixed one such trait, making it robust and prion-independent. Finally, we genetically screened for unknown prion elements. Fully one-third of wild strains harboured them. These, too, created diverse, often beneficial phenotypes. Thus, prions broadly govern heritable traits in nature, in a manner that could profoundly expand adaptive opportunities., The heritable variation that drives new forms and functions is generally ascribed to mutations in the genetic code. We previously proposed an entirely different pathway for creating heritable phenotypic diversity [...]

Prions are infectious, self-propagating protein aggregates that have been identified in evolutionarily divergent members of the eukaryotic domain of life. Nevertheless, it is not yet known whether prokaryotes can support the formation of prion aggregates. Here we demonstrate that the yeast prion protein Sup35 can access an infectious conformation in Escherichia coli cells and that formation of this material is greatly stimulated by the presence of a transplanted [[PSI.sup.+]] inducibility factor, a distinct prion that is required for Sup35 to undergo spontaneous conversion to the prion form in yeast. Our results establish that the bacterial cytoplasm can support the formation of infectious prion aggregates, providing a heterologous system in which to study prion biology. Sup35 | [[PSI.sup.*]] inducibility factor I amyloid doi/ 10.1073/pnas.0913280107

Prions are infectious proteins composed of the abnormal disease-causing isoform [PrP.sup.Sc], which induces conformational conversion of the host-encoded normal cellular prion protein [PrP.sup.C] to additional [PrP.sup.Sc]. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of [PrP.sup.Sc] with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk [PrP.sup.C], strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer [PrP.sup.C]. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with [PrP.sup.Sc] conforrnational compatibility, determines prion strain mutation in these cervids. doi: 10.1126/science.1187107

When the translation termination factor Sup35 adopts the prion state, [[PSI.sup.+]], the read-through of stop codons increases, uncovering hidden genetic variation and giving rise to new, often beneficial, phenotypes. Evidence suggests that prion induction involves a process of maturation, but this has never been studied in detail. To do so, we used a visually tractable prion model consisting of the Sup35 prion domain fused to GFP (PrD-GFP) and overexpressed it to achieve induction in many cells simultaneously. PrD-GFP first assembled into Rings as previously described. Rings propagated for many generations before the protein transitioned into a Dot structure. Dots transmitted the [[PSI.sup.+]] phenotype through mating and meiosis, but Rings did not. Surprisingly, the underlying amyloid conformation of PrD-GFP was identical in Rings and Dots. However, by electron microscopy, Rings consisted of very long uninterrupted bundles of fibers, whereas Dot fibers were highly fragmented. Both forms were deposited at the IPOD, a biologically ancient compartment for the deposition of irreversibly aggregated proteins that we propose is the site of de novo prion induction. We find that oxidatively damaged proteins are also localized there, helping to explain how proteotoxic stresses increase the rate of prion induction. Curing PrD-GFP prions, by inhibiting Hsp104's fragmentation activity, reversed the induction process: Dot cells produced Rings before PrD-GFP reverted to the soluble state. Thus, formation of the genetically transmissible prion state is a two-step process that involves an ancient system for the asymmetric inheritance of damaged proteins and heritable changes in the extent of prion fragmentation. yeast prion | fiber fragmentation | IPOD (Insoluble Protein Deposit) | prion inheritance | asymmetric damage distribution doi/10.1073/pnas.1003895107

Peroxiredoxins (Prxs) are ubiquitous antioxidants that protect cells against oxidative stress. We show that the yeast Tsa1/Tsa2 Prxs colocalize to ribosomes and function to protect the Sup35 translation termination factor against oxidative stress--induced formation of its heritable [[PSI.sup.+]] prion conformation. In a tsa1 tsa2 [[psi.sup.-]] [[PIN.sup.+]] strain, the frequency of [[PSI.sup.+]] de novo formation is significantly elevated. The Tsa1/Tsa2 Prxs, like other 2-Cys Prxs, have dual activities as peroxidases and chaperones, and we show that the peroxidase activity is required to suppress spontaneous de novo [[PSI.sup.+]] prion formation. Molecular oxygen is required for [[PSI.sup.+]] prion formation as growth under anaerobic conditions prevents prion formation in the tsa1 tsa2 mutant. Conversely, oxidative stress conditions induced by exposure to hydrogen peroxide elevates the rate of de novo [[PSI.sup.+]] prion formation leading to increased suppression of all three termination codons in the tsa1 tsa2 mutant. Altered translational fidelity in [[PSI.sup.+]] strains may provide a mechanism that promotes genetic variation and phenotypic diversity (True HL, Lindquist SL (2000) Nature 407:477-483). In agreement, we find that prion formation provides yeast cells with an adaptive advantage under oxidative stress conditions, as elimination of the [[PSI.sup.+]] prion from tsa1 tsa2 mutants renders the resulting [[psi.sup.-]] [[pin.sup.-]] cells hypersensitive to hydrogen peroxide. These data support a model in which Prxs function to protect the ribosomal machinery against oxidative damage, but when these systems become overwhelmed, [[PSI.sup.+]] prion formation provides a mechanism for uncovering genetic traits that aid survival during oxidative stress conditions. doi/10.1073/pnas.1000347107

Inability to forro new memories is an early clinical sign of Alzheimer's disease (AD). There is ample evidence that the amyloid-[beta] (A[beta]) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of A[beta] are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of A[beta]-mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic A[[beta].sub.1-42] oligomers impaired consolidation of the Iong-term recognition memory, whereas mature A[[beta].sub.1-42] fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-A[beta] antibody. It has been suggested that the cellular prion protein (Pr[P.sup.c]) mediates the impairment of synaptic plasticity induced by A[beta]. We confirmed that A[[beta].sub.1-42] oligomers interact with Pr[P.sup.c], with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that A[[beta].sub.1-42] oligomers are responsible for cognitive impairment in AD and that Pr[P.sup.c] is not required. Alzheimer | neurotoxicity | object recognition test | surface plasmon resonance | protein aggregation doi/10.1073/pnas.0911829107

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurodegenerative diseases that affect many mammalian species. Some examples include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, Creutzfeldt-Jakob [...]

Prion infection is characterized by the conversion of host cellular prion protein (Pr[P.sup.C) into disease-related conformers (Pr[P.sup.Sc]) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for Pr[P.sup.C] rather than Pr[P.sup.Sc]. We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for Pr[P.sup.C] and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native Pr[P.sup.C] conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular [beta]-sheet. The importance of this residue in mediating protein-protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease. Creutzfeldt-Jakob disease | PrP-Fab complex | monoclonal antibody | prion therapeutics

[URE3] is a prion of the yeast Ure2 protein. Hsp40 is a cochaperone that regulates Hsp70 chaperone activity. When overexpressed, the Hsp40 Ydj1p cures yeast of [URE3], but the Hsp40 Sis1p does not. On the basis of biochemical data Ydj1p has been proposed to cure [URE3] by binding soluble Ure2p and preventing it from joining prion aggregates. Here, we mutagenized Ydj1p and find that disrupting substrate binding, dimerization, membrane association, or ability to transfer substrate to Hsp70 had little or no effect on curing. J-domain point mutations that disrupt functional interactions of Ydj1p with Hsp70 abolished curing, and the J domain alone cured [URE3]. Consistent with heterologous J domains possessing similar Hsp70 regulatory activity, the Sis1p J domain also cured [URE3]. We further show that Ydj1p is not essential for [URE3] propagation and that depletion of Ure2p is lethal in cells lacking Ydj1p. Our data imply that curing of [URE3] by overproduced Ydj1p does not involve direct interaction of Ydj1p with Ure2p but rather works through regulation of Hsp70 through a specific J-protein/Hsp70 interaction.

Abstract: There have been many studies of the morphology, behavioral audiograms, and population structure of the false killer whale (Pseudorca crassidens), but sequencing, mapping, and functional and comparative genomics studies [...]

Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form ([PrP.sup.C]) to the scrapie form ([PrP.sup.Sc]) is still unknown. Here we discovered a chemical chaperone to stabilize the [PrP.sup.C] conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a 'pocket' created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states ([PrP.sup.*]) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N- [4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced [PrP.sup.Sc]. Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E 196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of [PrP.sup.C], will open the way to the development of novel anti-prion drugs. anti-prion compound | binding sites | chemical chaperone | dynamics-based drug discovery | transmissible spongiform encephalopathy

Guanidine hydrochloride (Gdn-HCl) blocks the propagation of yeast prions by inhibiting Hsp104, a molecular chaperone that is absolutely required for yeast prion propagation. We had previously proposed that ongoing cell division is required for Gdn.HCl-induced loss of the [[PSI.sup.+]] prion. Subsequently, Wu et al. [Wu Y, Greene LE, Masison DC, Eisenberg E (2005)Proc Natl Acad Sci USA 102:12789--12794] claimed to show that Gdn x HCl can eliminate the [[PSI.sup.+]] prion from u-factor-arrested cells leading them to propose that in Gdn x HCl-treated cells the prion aggregates are degraded by an Hsp104-independent mechanism. Here we demonstrate that the results of Wu et al. can be explained by an unusually high rate of a-factor-induced cell death in the [[PSI.sup.+]] strain (780-1D) used in their studies. What appeared to be no growth in their experiments was actually no increase in total cell number in a dividing culture through a counterbalancing level of cell death. Using media-exchange experiments, we provide further support for our original proposal that elimination of the [[PSI.sup.+]] prion by Gdn x HCl requires ongoing cell division and that prions are not destroyed during or after the evident curing phase. propagons | Hsp104 | [alpha]-factor

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein [Prp.sup.Sc]), we have been interested in how these peptides interact with [PrP.sup.Sc]. After screening peptides from the entire human PrP sequence, we found two peptides ([PrP.sub.19-30] and [PrP.sub.100-111]) capable of binding full-length [PrP.sup.Sc] in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein ([Prp.sup.C]). The limit of detection for captured [PrP.sup.Sc] was calculated to be 8 amol from a [approximately equal to] 105-fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to [PrP.sup.Sc] over [PrP.sup.C]. Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and [PrP.sup.Sc]. Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-[PrP.sup.Sc] interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in [PrP.sup.Sc], is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of [PrP.sup.C] may play a role in the recruitment of [PrP.sup.C] to [PrP.sup.Ssc]. plasma | Creutzfeldt--Jakob disease | detection | cationic interaction | diagnostic

Proteolytic processing of the amyloid precursor protein (APP) by [beta]-secretase, [beta]-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid [beta] (A[beta]) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (Pr[P.sup.C]), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, remains enigmatic. Because both APP and Pr[P.sup.C] are subject to proteolytic processing by the same zinc metal-loproteases, we tested the involvement of Pr[P.sup.C] in the proteolytic processing of APP. Cellular overexpression of Pr[P.sup.C] inhibited the [beta]-secretase cleavage of APP and reduced A[beta] formation. Conversely, depletion of Pr[P.sup.C] in mouse N2a cells by siRNA led to an increase in A[beta] peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, A[beta] levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the [beta]-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of Pr[P.sup.C] on the [beta]-secretase cleavage of APP required the localization of Pr[P.sup.C] to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of Pr[P.sup.C] via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic A[beta] is regulated by Pr[P.sup.C] and may have implications for both Alzheimer's and prion diseases. lipid raft | proteolysis | scrapie | glycosaminoglycan

The abnormal protein that causes Creutzfeldt-Jakob disease (CJD) can occur in other parts of the body besides the brain, according to a study of 36 patients who died of CJD. The researchers tested autopsy samples for the presence of the prion protein and found that one-third of the patients had prion protein in other parts of the body, including the spleen and muscles. CJD belongs to the same group of diseases as mad cow disease.

A major prion protein (PrP) mutant that forms amyloid fibrils in the diseased brain of patients with Gerstmann-Straussler-Scheinker syndrome (GSS) is a fragment of 7 kDa spanning from residues 81-82 to 144-153 of PrP. Analysis of ionic membrane currents, recorded with a libid bilayer technique, revealed that the wild-type fragment PrP(82-146) WT and the partially scrambled PrP(82-146) (127-146) SC are capable of forming heterogenous ion channels that are similar to those channels formed with PrP(106-126). In contrast, PrP(82-146) peptides in which the region from residue 106 to 126 had been scrambled (SC) showed a reduction in interaction with lipid membranes and did not form channels. The PrP(82-146) WT- and PrP(82-146) (127-146) SC-formed cation channels with fast kinetics are [Cu.sup.2+] sensitive and rifampicin (RIF) insensitive, whereas the time-dependent inactivating channels formed by these same peptides are both [Cu.sup.2+] and RIF insensitive. The presence of RIF in the solution before the addition of PrP(82-146) WT or PrP(82-146) (127-146) SC affected their incorporation into the lipid bilayers. PrP(82-146) WT and PrP(82-146) (127-146) SC fast cation channels formed in the presence of RIF appeared in an electrically semisilent state or an inactivated state. Increasing [[[Cd.sub.2+]].sub.cis] enhanced the incorporation of PrP(82-146) WT and PrP(82-146) (127-146) SC channels formed in the presence of RIF. We conclude that the major PrP mutant fragment in the diseased brain of GSS patients is prone to form channels in neuronal membranes, causing their dysfunction. We propose that [Cd.sup.2+] may accentuate the neurotoxicity of this channel-forming PrP fragment by enhancing its incorporation into the membrane. prion diseases; prion channels; amyloids; neurodegenerative diseases; membrane-linked pathologies; vacuolation; cytotoxic proteins

Although the prion protein (PrP) is known to be the causative agent of the neurodegenerative transmissible spongiform encephalopathies, its normal cellular function remains elusive. Octapeptide repeats in the N terminus of PrP bind metal ions and are required for the endocytosis of PrP upon exposure of cells to copper or zinc. As the concentration of zinc in the extracellular spaces of the brain is higher than that for copper, we put forward the hypothesis that PrP is involved in neuronal zinc homeostasis; PrP might be involved in transport of zinc into the cell or might act as a zinc sensor. In prion disease, when the protein undergoes a conformational change to the infectious form, this function of PrP in zinc homeostasis might be compromised.

In the filamentous fungus Podospora anserina, two phenomena are associated with polymorphism at the het-s locus, vegetative incompatibility and ascospore abortion. Two het-s alleles occur naturally, het-s and het-S. The het-s encoded protein is a prion propagating as a self-perpetuating amyloid aggregate. When prion-infected [Het-s] hyphae fuse with [Het-S] hyphae, the resulting heterokaryotic cells necrotize. [Het-s] and [Het-S] strains are sexually compatible. When, however, a female [Het-s] crosses with [Het-S], a significant percentage of het-S spores abort, in a way similar to spore killing in Neurospora and Podospora. We report here that sexual transmission of the [Het-s] prion after nonisogamous mating in the reproductive cycle of Podospora is responsible for the killing of het-S spores. Progeny of crosses between isogenic strains with distinct wild-type or introduced, ectopic het-s/S alleles were cytologically and genetically analyzed. The effect of het-s/S overexpression, ectopic het-s/S expression, absence of het-s expression, loss of [Het-s] prion infection, and the distribution patterns of HET-s/S-GFP proteins were categorized during meiosis and ascospore formation. This study unveiled a het-S spore-killing system that is governed by dosage of and interaction between the [Het-s] prion and the HET-S protein. Due to this property of the [Het-s] prion, the het-s allele acts as a meiotic drive element favoring maintenance of the prion-forming allele in natural populations.

Disease-related amyloidogenic propensity has been unexpectedly found in proteins driven to adopt a monomeric uncomplexed state at high concentrations under near-physiological conditions. This situation occasionally arises in new health treatments, such as kidney dialysis. Assuming that under such conditions a partial retention of native structure takes place, this work identifies a structural characteristic indicating amyloidogenic propensity: a high density of backbone hydrogen bonds exposed to water attack in monomeric structure. On this basis, we propose a diagnostic tool based on the identification of hydrogen bonds with a paucity of intramolecular dehydration or 'wrapping.' We use this predictor to identify potentially pathogenic mutations that foster amyloidogenic propensity in human prions. Such mutations either enhance the intramolecular dehydration of [beta]-sheet hydrogen bonds, thus stabilizing the nucleus for rearrangement into the scrapie fold, or contribute to the destabilization of the cellular form by introducing additional underwrapped hydrogen bonds. Our predictions are consistent with known disease-related mutations and lead to a cogent explanation of the pathogenic nature of specific mutations affecting the cellular prion protein structural wrapping. On the other hand, a different wrapping of a very similar fold, mouse doppel, induces a dramatically different level of amyloidogenic propensity, suggesting that the packing within the fold, and not the fold itself, contains the signal for aggregation. amyloidosis | prions | protein structure | structural wrapping I hydrogen bonds

We noticed that disease-related amyloidogenic proteins and especially cellular prion proteins have the highest proportion of incompletely desolvated backbone H bonds among soluble proteins. Such bonds are vulnerable to water attack and thus represent structural weaknesses. We have measured the adsorption of proteins onto phospholipid bilayers and found a strong correlation between the extent of underwrapping of backbone H bonds in the native structure of a protein and its extent of deposition on the bilayer: the less the H bond wrapping, the higher the propensity for protein-bilayer binding. These observations support the proposition that soluble proteins with amyloidogenic propensity and membrane proteins share a pervasive building motif: the underwrapped H bonds. Whereas in membrane proteins, this motif does not signal a structural vulnerability, in soluble proteins, it is responsible for their reactivity.

Abnormal prion proteins may be present in the nasal mucosa of patients with Creutzfeldt-Jakob disease, according to a study of nine patients. This disease is the human version of mad cow disease, which is technically called bovine spongiform encephalopathy, or BSE. The presence of the abnormal prion protein responsible for these two diseases in the nose means the protein may be transmitted by contaminated surgical instruments. A nasal biopsy could also be a means of diagnosing the disease in its early stages.

Although the transport of model proteins across the mammalian ER can be reconstituted with purified Sec61p complex, TRAM, and signal recognition particle receptor, some substrates, such as the prion protein (PrP), are inefficiently or improperly translocated using only these components. Here, we purify a factor needed for proper translocation of PrP and identify it as the translocon-associated protein (TRAP) complex. Surprisingly, TRAP also stimulates vectorial transport of many, but not all, other substrates in a manner influenced by their signal sequences. Comparative analyses of several natural signal sequences suggest that a dependence on TRAP for translocation is not due to any single physical parameter, such as hydrophobicity of the signal sequence. Instead, a functional property of the signal, efficiency of its post-targeting role in initiating substrate translocation, correlates inversely with TRAP dependence. Thus, maximal translocation independent of TRAP can only be achieved with a signal sequence, such as the one from prolactin, whose strong interaction with the translocon mediates translocon gating shortly after targeting. These results identify the TRAP complex as a functional component of the translocon and demonstrate that it acts in a substrate-specific manner to facilitate the initiation of protein translocation.

Prions are 'infectious' proteins. When Sup35, a yeast translation termination factor, is aggregated in its [PS[I.sup.+]] prion form its function is compromised. When Rnq1 is aggregated in its [PI[N.sup.+]] prion form, it promotes the de novo appearance of [PS[I.sup.+]]. Heritable variants (strains) of [PS[I.sup.+]] with distinct phenotypes have been isolated and are analogous to mammalian prion strains with different pathologies. Here, we describe heritable variants of the [PI[N.sup.+]] prion that are distinguished by the efficiency with which they enhance the de novo appearance of [PS[I.sup.+]]. Unlike [PS[I.sup.+]] variants, where the strength of translation termination corresponds to the level of soluble Sup35, the phenotypes of these [PI[N.sup.+]] variants do not correspond to levels of soluble Rnq1. However, diploids and meiotic progeny from crosses between either different [PS[I.sup.+]], or different [PI[N.sup.+]] variants, always have the phenotype of the parental variant with the least soluble Sup35 or Rnq1, respectively. Apparently faster growing prion variants cure cells of slower growing or less stable variants of the same prion. We also find that YDJ1 overexpression eliminates some but not other [PI[N.sup.+]] variants and that prions are destabilized by meiosis. Finally, we show that, like its affect on [PS[I.sup.+]] appearance, [PI[N.sup.+]] enhances the de novo appearance of [URE3]. Surprisingly, [PS[I.sup.+]] inhibited [URE3] appearance. These results reinforce earlier reports that heterologous prions interact, but suggest that such interactions can not only positively, but also negatively, influence the de novo generation of prions.

The yeast prion [PS[I.sup.+]] provides an epigenetic mechanism for the inheritance of new phenotypes through self-perpetuating changes in protein conformation. [PS[I.sup.+]] is a nonfunctional, ordered aggregate of the translation termination factor Sup35p that influences new Sup35 proteins to adopt the same state. The N-terminal region of Sup35p plays a central role in prion induction and propagation. The C-terminal region provides translation termination activity. The function of the highly charged, conformationally flexible middle region (M) is unknown. An M deletion mutant was capable of existing in either the prion or the nonprion state, but in either case it was mostly insoluble. Substituting a charged synthetic polypeptide for M restored solubility, but the prions formed by this variant were mitotically very unstable. Substituting charged flexible regions from two other proteins for M created variants that acquired prion states (defined as self-perpetuating changes in function transferred to them from wild-type [PS[I.sup.+]] elements), but had profoundly different properties. One was soluble in both the prion and the nonprion form, mitotically stable but meiotically unstable, and cured by guanidine HCl but not by alterations in heat shock protein 104 (Hspl04p). The other could only maintain the prion state in the presence of wild-type protein, producing Mendelian segregation patterns. The unique character of these M variants, all carrying the same N-terminal prion-determining region, demonstrate the importance of M for [PS[I.sup.+]] and suggest that a much wider range of epigenetic phenomena might be based on self-perpetuating, prion-like changes in protein conformation than suggested by our current methods for defining prion states.

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating inactive amyloid form of the Ure2 protein. Ure2p residues 1-65 constitute the prion domain, and the remaining C-terminal portion regulates nitrogen catabolism. We have examined the URE2 genes of wild-type isolates of S. cerevisiae and those of several pathogenic yeasts and a filamentous fungus. We find that the normal function of the S. cerevisiae Ure2p in nitrogen regulation is fully complemented by the Ure2p of Candida albicans, Candida glabrata, Candida kefyr, Candida maltosa, Saccharomyces bayanus, and Saccharomyces paradoxus, all of which have high homology in the C-terminal nitrogen regulation domain. However, there is considerable divergence of their N-terminal domains from that of Ure2p of S. cerevisiae. [URE[3.sup.sc]] showed efficient transmission into S. cerevisiae ure2[DELTA] cells if expressing a Ure2p of species within Saccharomyces. However, [URE[3.sup.sc]] did not seed self-propagating inactivation of the Ure2p's from the other yeasts. When overexpressed as a fusion with green fluorescent protein, residues 5-47 of the S. cerevisiae prion domain are necessary for curing the [URE3] prion. Residues 11-39 are necessary for an inactivating interaction with the full-length Ure2p. A nearly identical region is highly conserved among many of the yeasts examined in this study, despite the wide divergence of sequences found in other parts of the N-terminal domains.

We reported that expression of the cellular prion protein (Pr[P.sup.c]) rescues doppel (Dpl)-induced cerebellar degeneration in mice. To search for protein(s) that mediate this process, we fused the C-termini of mouse (Mo) PrP and Dpl to the Fc portion of an IgG. Although both MoPrP-Fc and MoDpl-Fc bound to many regions of the brain, we observed restricted binding to granule cells in the cerebellum, suggesting a scenario in which granule cells express a protein that mediates Dpl-induced neurodegeneration. Because granule cells do not express Pr[P.sup.c], it seems unlikely that MoPrP-Fc binding reflects a ligand that is involved in the conversion of Pr[P.sup.c] into Pr[P.sup.sc], the disease-causing isoform. In contrast, the dominant-negative MoPrP(Q218K)-Fc not only binds to granule cells but also binds to neurons of the molecular layer where Pr[P.sup.c] is expressed. These findings raise the possibility that the cells of the molecular layer express an auxiliary protein, provisionally designated protein X, which is involved in prion formation and is likely to be distinct from the protein that mediates Dpl-induced degeneration. Although the binding of the dominant-negative MoPrP(Q218K)-Fc to cells in the molecular layer expressing Pr[P.sup.c] is consistent with a scenario for the binding of MoPrP(Q218K)-Fc to protein X, the absence of Pr[P.sup.sc] deposition in the molecular layer requires that Pr[P.sup.sc], once formed there, be readily transported to the cerebellar white matter where Pr[P.sup.sc] is found. Identifying the ligands to which PrP-Fc, Dpl-Fc, and dominant-negative PrP bind may provide new insights into the functions of Pr[P.sup.c] and Dpl as well as the mechanism of Pr[P.sup.sc] formation.

Research has been conducted on prion protein. The transformation of the prion protein normal cellular isoform to the infectious prion protein has entailed a metastable locally disordered prion protein conformer, the stability and structural features of this conformer have been characterized via NMR spectroscopy and the results indicate that the conformer coexists in solution.

Our discovery of dominant-negative inhibition of prion formation in cultured cells provided an explanation for the resistance of some sheep to scrapie and humans to Creutzfeldt-Jakob disease. To determine whether dominant-negative inhibition occurs in vivo, we produced transgenic (Tg) mice expressing prion protein (PrP) with either the Q167R or Q218K mutation alone or in combination with wild-type (wt) PrP. Tg(MoPrP, Q167R)[Prnp.sup.0/0] mice expressing mutant PrP at levels equal to non-Tg mice remained healthy for >550 days, indicating that inoculation with prions did not cause disease. Immunoblots of brain homogenates and histologic analysis did not reveal abnormalities. Tg(MoPrP, Q167R)[Prnp.sup.+/+] mice expressing both mutant and wt PrP did not exhibit neurologic dysfunction, but their brains revealed low levels of the PrP pathogenic isoform (Pr[P.sup.Sc]), and sections showed numerous vacuoles and severe astrocytic gliosis at 300 days after inoculation. Both Tg(MoPrP, Q218K)[Prnp.sup.0/0] and Tg(MoPrP,Q218K)[Prnp.sup.+/+] mice expressing high levels of the transgene product remained healthy for >300 days after inoculation. Neither Pr[P.sup.Sc] nor neuropathologic changes were found. Our studies demonstrate that although dominant-negative inhibition of wt Pr[P.sup.Sc] formation occurs, expression of the dominant-negative PrP at the same level as wt PrP does not prevent prion formation completely. However, expression of dominant-negative PrP alone had no deleterious effects on the mice and did not support prion propagation.

It has been known that the structural transition from Pr[P.sup.C] to Pr[P.sup.Sc] leads to the prion formation. This putative conformational change challenges the central dogma of the protein folding theory--'one sequence, one structure.' Generally, scientists believe that there must be either a posttranslational modification or environmental factors involved in this event. However, all of the efforts to solve the mystery of the Pr[P.sup.C] to Pr[P.sup.Sc] transition have ended in vain so far. Here we provide evidence linking O-linked glycosylation to the structural transition based on prion peptide studies. We find that the O-linked [alpha]-GalNAc at Ser-135 suppresses the formation of amyloid fibril formation of the prion peptide at physiological salt concentrations, whereas the peptide with the same sugar at Ser-132 shows the opposite effect. Moreover, this effect is sugar specific. Replacing [alpha]-GalNAc with [beta]-GlcNAc does not yield the same effect.

Results indicate that the secondary structures and thermal-stabilities against denaturation of mouse and sheep prion proteins are influenced by the anions, which preferentially interact with the glycine residues in the N-terminal segment. The increase of the anion concentration on the prion surface destabilizes the protein structure.

Diverse human disorders, including the majority of neurodegenerative diseases, are thought to arise from the misfolding and aggregation of protein. We have recently described a novel technology to amplify cyclically misfolded proteins in vitro. This procedure, named protein misfolding cyclic amplification (PMCA), is conceptually analogous to DNA amplification by PCR and has tremendous implications for research and diagnosis. The PMCA concept has been proved on the amplification of prions implicated in the pathogenesis of transmissible spongiform encephalopathies. In this article we describe the rational behind PMCA and some of the many potential applications of this novel technology.

Inactivation of Hsp104 by guanidine is contended to be the mechanism by which guanidine cures yeast prions. We now find an Hsp104 mutation (D184N) that confers resistance to guanidine-curing of the yeast [[PSI.sup.+]] prion. In an independent screen we isolated an HSP104 allele altered in the same residue (D184Y) that dramatically impairs [[PSI.sup.+]] propagation in a temperature-dependent manner. Directed mutagenesis of HSP104 produced additional alleles that conferred varying degrees of resistance to guanidine-curing or impaired [[PSI.sup.+]] propagation. The mutations similarly affected propagation of the [URE3] prion. Basal and induced abundance of all mutant proteins was normal. Thermotolerance of cells expressing mutant proteins was variably resistant to guanidine, and the degree of thermotolerance did not correlate with [[PSI.sup.+]] stability. We thus show that guanidine cures yeast prions by inactivating Hsp104 and identify a highly conserved Hsp104 residue that is critical for yeast prion propagation. Our data suggest that Hsp104 activity can be reduced substantially without affecting [[PSI.sup.+]] stability, and that Hsp104 interacts differently with prion aggregates than with aggregates of thermally denatured protein.

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.

The [Het-s] infectious element of the filamentous fungus Podospora anserina is a prion. We have recently reported that recombinant HET-s protein aggregates in vitro into amyloid fibers. In vivo, the protein aggregates specifically in the [Het-s] prion strains. Here, we show that biolistic introduction of aggregated recombinant HET-s protein into fungal cells induces emergence of the [Het-s] prion with a high frequency. Thus, we demonstrate that prion infectivity can be created de novo, in vitro from recombinant protein in this system. Although the amyloid filaments formed from HET-s could transmit [Het-s] efficiently, neither the soluble form of the protein nor amorphous aggregates would do so. In addition, we have found that (i) [Het-s] infectivity correlates with the ability to convert HET-s to amyloids in vitro, (ii) [Het-s] infectivity is resistant to proteinase K digestion, and (iii) HET-s aggregates formed in vivo in [Het-s] strains have the ability to convert the recombinant protein to aggregates. Together, our data designate the HET-s amyloids as the molecular basis of [Het-s] prion propagation.

Research has been conducted on Sup35p protein from Saccharomuces cerevisiae. The function and prion-like properties of this protein in the fungal species other than S. cerevisiae have been investigated via the isolation and characterization of the gene from Candida albicans and the results are presented.

Research has been conducted on the copper binding protein, prion protein containing conserved octarepeat sequence spanning residues. The study of the copper binding site's atomic resolution view within the octarepeat is presented.

A conformational variant of the normal prion protein Pr[P.sup.c] is believed to be identical to Pr[P.sup.Sc], the agent that causes prion diseases. Recently, a novel protein, named Doppel (Dpl), was identified that shares significant biochemical and structural homology with Pr[P.sup.c]. In specific strains of Pr[P.sup.c]-deficient mouse lines, Dpl is overexpressed and causes a neurological disease. Dpl neurotoxicity is counteracted and prevented by Pr[P.sup.c], but the mechanism of antagonistic Pr[P.sup.c]-Dpl interaction remains elusive. In contrast to its homologue Pr[P.sup.c], initial studies suggest that Dpl is dispensable for prion disease progression and for the generation of Pr[P.sup.Sc]. Although we are only beginning to understand its function, the discovery of Dpl has already provided some answers to longstanding questions and is transforming our understanding of prion biology.

Research has been conducted on the mammalian central nervous system's physical disruption which can release fragments of tissue into the blood system. The dispersion of the central nervous system tissues within the internal organism of cattle has been investigated via tracking the spread of a marker organism inoculated during commercial stunning and the results are reported.

Variant Creutzfeldt-Jakob disease and scrapie are typically initiated by extracerebral exposure to prions, and exhibit early prion accumulation in germinal centers. Follicular dendritic cells (FDCs), whose development and maintenance in germinal centers depends on tumor necrosis factor (TNF) and lymphotoxin (LT) signaling, are thought to be indispensable for extraneural prion pathogenesis. Here, we administered prions intraperitoneally to mice deficient for TNF and LT signaling components. LT[[alpha].sup.-/-], LT[[beta].sup.-/-], LT[beta][R.sup.-/-], and [LT[[alpha].sup.-/-] x TNF[[alpha].sup.-/-] mice resisted infection and contained no infectivity in spleens and lymph nodes (when present). However, [TNFR1.sup.-/-], [TNFR2.sup.-/-], and some [TNF[[alpha].sup.-/-] mice developed scrapie similarly to wild-type mice. High prion titers were detected in lymph nodes, but not spleens, of [TNFR1.sup.-/-] and [TNF[[alpha].sup.-/-] mice despite absence of FDCs and germinal centers. Transfer of [TNFR1.sup.-1-] fetal liver cells into lethally irradiated [Prnp.sup.0/0] mice restored infectivity mainly in lymph nodes. Prion protein (PrP) colocalized with a minority of macrophages in tumor necrosis factor receptor (TNFR) [1.sup.-/-] lymph nodes. Therefore, prion pathogenesis can be restricted to lymphoreticular subcompartments, and mature follicular dendritic cells are dispensable for this process. Macrophage subsets are plausible candidates for lymphoreticular prion pathogenesis and neuroinvasion in the absence of FDCs, and may represent a novel target for postexposure prophylaxis.