The conformational change of a host protein, PrP C , into a disease-associated isoform, PrP Sc , appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt–Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrP C and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrP Sc molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrP Sc molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrP Sc molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrP C molecules, copurified lipid molecules, and a synthetic polyanion.

Much evidence supports the hypothesis that the infectious agents of prion diseases are devoid of nucleic acid, and instead are composed of a specific infectious protein. This protein, PrP(Sc), seems to be generated by template-induced conformational change of a normally expressed glycoprotein, PrP(C) (ref. 2). Although numerous studies have established the conversion of PrP(C) to PrP(Sc) as the central pathogenic event of prion disease, it is unknown whether cellular factors other than PrP(C) might be required to stimulate efficient PrP(Sc) production. We investigated the biochemical amplification of protease-resistant PrP(Sc)-like protein (PrPres) using a modified version of the protein-misfolding cyclic amplification method. Here we report that stoichiometric transformation of PrP(C) to PrPres in vitro requires specific RNA molecules. Notably, whereas mammalian RNA preparations stimulate in vitro amplification of PrPres, RNA preparations from invertebrate species do not. Our findings suggest that host-encoded stimulatory RNA molecules may have a role in the pathogenesis of prion disease. They also provide a practical approach to improve the sensitivity of diagnostic techniques based on PrPres amplification.

Because the insolubility of the scrapie prion protein (PrP Sc ) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP Sc (PrP 27-30) and a miniprion (PrP Sc 106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 Å resolution. By comparing projection maps of PrP 27-30 and PrP Sc 106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP Sc . Only models featuring parallel ␤-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.electron microscopy ͉ image processing ͉ Nanogold labeling ͉ parallel ␤-helix ͉ amyloid structure C reutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, and other spongiform encephalopathies are caused by an aberrantly folded isoform (PrP Sc ) of the prion protein (PrP) (1). Replication of prions includes a profound change in the conformation of the cellular isoform of PrP (PrP C ) to form the highly insoluble PrP Sc . The insolubility of PrP Sc has thwarted attempts to investigate its structure by either x-ray crystallography or NMR spectroscopy. Our knowledge about the structure of PrP Sc is therefore rather limited (2).After treatment with proteinase K (PK), PrP Sc loses the N-terminal residues 23 to Ϸ89 (forming PrP 27-30), but retains infectivity. During purification, PrP 27-30 polymerizes into rod-shaped filaments with the tinctorial properties of amyloid (3, 4). X-ray fibril diffraction illustrated the amyloid nature of PrP 27-30; characteristic 4.7 Å reflections indicative of cross-␤ structure were observed (5). Optical spectroscopy revealed that PrP Sc and PrP 27-30 are substantially enriched in ␤-sheet structure (6-9). This finding is in sharp contrast to the predominantly ␣-helical fold of the three-helix-bundle structure of PrP C as determined by NMR spectroscopy and x-ray crystallography on refolded recombinant PrP (10 -18). Owing to the lack of high-resolution structural information for PrP Sc , predictive methods have been used to develop molecular models to codify the existing spectroscopic, immunological, and biochemical data (19).In attempts to simplify the structural analysis of PrP Sc , we systematically deleted parts of the prion protein. One of these constructs containing only 106 residues, PrP106 (⌬23-88, ⌬141-176), supported the propagation of prions (20,21). Transgenic mice expressing only PrP106 develop a histologically accurate neurodegenerative prion disease after inoculation with prions, and the resulting prions can...

We report that branched polyamines, including polyamidoamide dendimers, polypropyleneimine, and polyethyleneimine, are able to purge PrP Sc , the protease-resistant isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture. The removal of PrP Sc by these compounds depends on both the concentration of branched polymer and the duration of exposure. Chronic exposure of ScN2a cells to low noncytotoxic concentrations of branched polyamines for 1 wk reduced PrP Sc to an undetectable level, a condition that persisted at least 3 wk after removal of the compound. Structure-activity analysis revealed that a high surface density of primary amino groups is required for polyamines to eliminate PrP Sc effectively from cells. The removal of PrP Sc by branched polyamines is attenuated by chloroquine in living cells, and exposure of scrapie-infected brain extracts with branched polyamines at acidic pH rendered the PrP Sc susceptible to protease in vitro, suggesting that endosomes or lysozomes may be the site of action. Our studies suggest that branched polyamines might be useful therapeutic agents for treatment of prion diseases and perhaps a variety of other degenerative disorders.neurodegeneration ͉ protein conformation P rion diseases are a group of fatal neurodegenerative disorders that can occur in hereditary, sporadic, and infectious forms (1). These illnesses occur in humans and a variety of other animals (2). Prions are infectious proteins. The normal cellular form of the prion protein (PrP), designated PrP C , contains three ␣-helices and has little ␤-sheet; in contrast, the protein of the prions, denoted the scrapie form of PrP (PrP Sc ), is rich in ␤-sheet structure. The accumulation of PrP Sc in the central nervous system precedes neurologic dysfunction accompanied by neuronal vacuolation and astrocytic gliosis.The spectrum of human prion diseases includes kuru (3), Creutzfeldt-Jakob disease (CJD) (4), Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia (5, 6), and a new form of human prion disease, new variant CJD (nvCJD), which has emerged in Great Britain and France (7-9). Several lines of evidence have suggested a link between the nvCJD outbreak and a preceding epidemic of bovine spongiform encephalopathy (7,(10)(11)(12). Although it is too early to predict the number of nvCJD cases that might eventually arise in Great Britain and elsewhere (8), it is clear that effective therapeutics for prion diseases are urgently needed. Unfortunately, although a number of compounds including amphotericins, sulfated polyanions, Congo red dye, and anthracycline antibiotics, have been reported as prospective therapeutic agents (13-16), all have demonstrated only modest potential to impede prion propagation, and none have been shown to effect the removal of preexisting prions from an infected host.Here we report that noncytotoxic concentrations of branched polyamines can rapidly eliminate PrP Sc from chronically infected ScN2a cells. These compounds appear to act by stimulating normal ...

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.

Prion diseases are caused by an infectious protein (20,25). These invariably fatal illnesses cannot be cured using routine antimicrobial agents, and materials contaminated with prions cannot be disinfected by conventional methods. Therefore, it is important to identify compounds that can be used either as therapeutic or disinfecting reagents for prion diseases. Ongoing epidemics of new variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy (BSE) in the United Kingdom highlight the urgency of this task.We recently reported that branched polyamines could purge scrapie-infected neuroblastoma (ScN2a) cells of PrP Sc , the disease-causing isoform of the prion protein (33). The ability of these compounds to eliminate PrP Sc from ScN2a cells depended upon a highly branched structure and a high surface density of primary amino groups. The most potent compounds identified were generation 4.0 polyamidoamine (PAMAM) and polypropyleneimine (PPI) dendrimers. Dendrimers are branched polyamines manufactured by a repetitive divergent growth technique, allowing the synthesis of successive, welldefined "generations" of homodisperse structures. In the current study, we demonstrate that branched polyamines cure prion-infected cells and identify the site and mechanism of polyamine-mediated prion clearance. We also demonstrate that these compounds can be employed in a rapid and simple assay to discriminate between different prion strains in vitro. MATERIALS AND METHODSChemical compounds. High-molecular-weight polyethyleneimine (PEI) was purchased from Fluka. SuperFect transfection reagent was purchased from Qiagen. All other polyamines were purchased from Sigma-Aldrich. Fluoresceinlabeled PPI was synthesized by mixing 30 mg of fluorescein isothiocyanate (FITC) with 1 mg of PPI generation 4.0 in 2 ml of ethanol overnight at 4°C. Labeled PPI was separated from residual, unreacted FITC using a Sephadex P-2 column.Cultured cells. Cultures of ScN2a cells were maintained as described previously (33). Cytotoxicity after treatment with polyamines was assessed in ScN2a cells by the following four methods: (i) examination of morphology under phase contrast microscopy, (ii) observation of growth curves and cell counts for 3 weeks after treatment, (iii) vital staining of living cells with 0.4% trypan blue (SigmaAldrich), and (iv) assay of dehydrogenase enzymes with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich). For ScN2a cells treated with either PAMAM or PPI generation 4.0 continuously for 1 week, the 50% toxic dose was ϳ50 g/ml.To prepare samples for infectivity assays, 100-mm-diameter plates (Falcon) of confluent cells were washed three times with 5 ml of phosphate-buffered saline, scraped into 2 ml of phosphate-buffered saline, and homogenized by repeated extrusion through a 26-gauge needle. Prion infectivity was determined by intracerebral inoculation of 30 l of cell homogenate into Tg(MoPrP)4053 mice. Mice were observed for clinical signs of scrapie, and a subset of diagnoses were confir...

Prions containing misfolded prion protein (PrP Sc ) can be formed with cofactor molecules using the technique of serial protein misfolding cyclic amplification. However, it remains unknown whether cofactors materially participate in maintaining prion conformation and infectious properties. Here we show that withdrawal of cofactor molecules during serial propagation of purified recombinant prions caused adaptation of PrP Sc structure accompanied by a reduction in specific infectivity of >10 5 -fold, to undetectable levels, despite the ability of adapted "protein-only" PrP Sc molecules to self-propagate in vitro. We also report that changing only the cofactor component of a minimal reaction substrate mixture during serial propagation induced major changes in the strain properties of an infectious recombinant prion. Moreover, propagation with only one functional cofactor (phosphatidylethanolamine) induced the conversion of three distinct strains into a single strain with unique infectious properties and PrP Sc structure. Taken together, these results indicate that cofactor molecules can regulate the defining features of mammalian prions: PrP Sc conformation, infectivity, and strain properties. These findings suggest that cofactor molecules likely are integral components of infectious prions.phospholipid | bioassay | repertoire | convergence | diversity

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrP Sc ) can be produced de novo from a mixture of the normal conformer (PrP C ) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nucleaseresistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrP Sc molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrP Sc formation in the absence of RNA. PE facilitated the propagation of PrP Sc molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrP Sc formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrP Sc molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions.PrP | scrapie P rions are mechanistically unique infectious agents that contain a misfolded, membrane-bound, glycoprotein (PrP Sc ) formed by the conformational change of a host-encoded conformer (PrP C ) (1). Conversion of PrP C into PrP Sc is the central event in the formation of infectious prions, but the molecular mechanism underlying conformational change remains poorly understood. In particular, the number and identity of endogenous factors other than PrP required for prion formation has not been determined (2).Cell culture and biochemical studies have implicated several classes of macromolecules such as GAGs, nucleic acids, proteins, and lipids as potential cofactors for prion formation (3). Reconstitution experiments with defined substrates (in which purified PrP molecules are mixed with Prnp 0/0 brain homogenate or purified cofactors that facilitate its conversion to PrP Sc ) have suggested that the conversion mechanism may be relatively simple, requiring only a few components (4, 5). Wild-type hamster prions possessing specific infectivity levels similar to those associated with natural scrapie have been formed de novo by using a defined mixture of purified native PrP C , copurified lipid, and RNA molecules (4), and infectious prions have also been formed de novo from bacterially expressed, recombinant PrP substrate in a reaction facilitated by synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA molecules (5, 6). In summary, infectious prions have not yet been produced either with a single cofactor or in the abs...