Although increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by inf... more Although increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by infectious prion particles is still unresolved. Return refocusing gel electrophoresis was developed to detect homogeneous
By far most in vitro studies on the prion problem are carried out with recombinant prion protein ... more By far most in vitro studies on the prion problem are carried out with recombinant prion protein (recPrP). RecPrP is available in large quantities, in high purity, from many species and with or without mutations. Although it can be used for many studies, it cannot be used for all studies. Since the cellular PrP (PrPC) carries the post-translational modifications, i. e., the N-glycosylation at the two sites Asn 181 and Asn 197 (in hamster) and the C-terminal glycosylphosphatidylinositol anchor, whereas recombinant PrP is lacking those modifications, both molecules are chemically different, and a series of studies is either directed to the differences or may be affected by the differences.
Assembly of natural and recombinant prion protein into fibrils
Biological Chemistry, Jun 1, 2005
The conversion of the α-helical, cellular isoform of the prion protein (PrPC) to the insoluble, β... more The conversion of the α-helical, cellular isoform of the prion protein (PrPC) to the insoluble, β-sheet-rich, infectious, disease-causing isoform (PrPSc) is the fundamental event in the prion diseases. The C-terminal fragment of PrPSc(PrP 27–30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrPSc, PrP 27–30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrPScfrom PrPCand the assembly of rods from PrP 27–30, we solubilized Syrian hamster (sol SHa) PrP 27–30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27–30 adopted a β-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27–30 in a soluble, β-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27–30, but also with native SHaPrPC. Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90–231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27–30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27–30 or PrPCwere bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89–230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.
8. The Scarpie Isoform of the Prion Protein PrPSe Compared to the Celluar Isoform PrPC
De Gruyter eBooks, Dec 21, 2006
46. Prevention of Prion Diseases in the Production of Medicinal Products, Medical Devices, and Cosmetics
De Gruyter eBooks, Dec 21, 2006
Protein Aggregation Associated with alzhiemer and Prion Diseases
Springer eBooks, 2001
The brains of patients with Alzheimer's disease (AD), Creutzfeldt-Jakob disease (CJD), Kuru a... more The brains of patients with Alzheimer's disease (AD), Creutzfeldt-Jakob disease (CJD), Kuru and others are characterized by an abundance of amyloid deposits. As long as the amyloid deposit could be described only by its histopathological morphology, these and other diseases appeared to have common molecular grounds, although the clinical pictures were different. Rudolf Virchow was the first who described the deposits in Alzheimer patients brains as “Eiweiss-Starke”(protein-starch) and therefore gave the name “amyloid”. Due to the work of Beyreuther and colleagues (see [11.1]) and Prusiner and colleagues (see [11.2]) it became known that the Alzheimer deposits consisted predominantly of the p-amyloid-peptide and those of CJD and Kuru of the prion protein, respectively, and that both proteins are encoded by a host nuclear gene. The molecular mechanism, cellular location and disease etiology, however, are quite different. Besides CJD and Kuru more human diseases like Gestmann-Straussler-Scheinker disease and familial fatal insomnia, but also animal diseases like scrapie in sheep and bovine spongiform encephalopathy (BSE) are closely connected with a pathological form of the prion protein, therefore denoted generally as “prion-diseases”. Most strikingly, and in contrast to AD, prion-diseases are transmissible, which led also to the name prion as proteinaceous infectious agent [11.3]. It is not yet known whether formation of insoluble deposits is a prerequisite of infectivity, but it is generally accepted that a pathological isoform of the prion protein, the so called Scrapie-isoform, PrPS c , is the major component of the brain deposits as well as of the infectious agent. During the infection event P r P S c is formed by a post-translational process from the cellular isoform P r P c. The relationship between the aggregate formation, the conformational transition of P r P c into PrPS c , and generation of infectivity and pathogenicity will be one of the major subjects of this chapter.
Extending the recent analysis of the safety of industrial bovine fat-derived products for human c... more Extending the recent analysis of the safety of industrial bovine fat-derived products for human consumption (Mu ¨ller, H., Stitz, L., and Riesner, D. (2006) Eur. J. Lip. Sci. Technol. 108, 812-826), we investigated systematically the effects of fat, fatty acids, and glycerol on the heat destruction of prions. Prion destruction was qualitatively and quantitatively evaluated in PrP 27-30, or prion rods, by the inactivation of infectivity as well as by the degradation of the polypeptide backbone. Under all conditions analyzed, inactivation of prion infectivity was achieved more efficiently than backbone degradation by several orders of magnitude. The presence of fat enhanced prion inactivation and offers a mild treatment for prion decontamination. In contrast, the presence of fat, fatty acids, and especially glycerol protected the PrP 27-30 backbone against heat-induced degradation. Glycerol also protected against heat-induced inactivation of prion infectivity. A phase distribution analysis demonstrated that prions migrated to the interphase of a fat/water mixture at room temperature and accumulated in the water phase at higher temperatures. In a systematic study of the mechanism of prion destruction, we found an intermediate structure of PrP that has fewer fibrils in -sheet formation, lower resistance to protease digestion, greater aggregation, and reduced solubility compared with PrP 27-30 but retains residual infectivity. These findings suggest that prion infectivity depends on -sheet-rich fibrillar structure and that inactivation proceeds in a stepwise manner, which explains the tailing effect frequently observed during inactivation. The prion diseases include bovine spongiform encephalopathy (BSE), 3 scrapie in goat and sheep, chronic wasting disease in
Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The age... more Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.
The Structural Transition of the Prion Protein into its Pathogenic Conformation is Induced by Unmasking Hydrophobic Sites
Journal of Molecular Biology, Nov 1, 2004
A series of structural intermediates in the putative pathway from the cellular prion protein PrP(... more A series of structural intermediates in the putative pathway from the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was established by systematic variation of low concentrations (<0.1%) of the detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial chaperonin GroEL. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were obtained with full-length recombinant PrP, hamster PrP 27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES, but always at neutral pH and room temperature, different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and to molecular mass, as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation: alpha-helical monomers, soluble alpha-helical dimers, soluble but beta-structured oligomers of a minimal size of 12-14 PrP molecules, and insoluble multimers were observed. A high activation barrier was found between the alpha-helical dimers and beta-structured oligomers. The numbers of SDS-molecules bound to PrP in different conformations were determined: Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and beta-structured multimers show very strong binding of five SDS molecules per PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads to fast formation of beta-structures followed by irreversible aggregation. It is discussed that strongest binding of SDS has an effect identical with or similar to the interaction with GroEL thereby inducing identical or very similar transitions. The interaction with GroEL/ES stabilizes the soluble, alpha-helical conformation. The structure and their stabilities and particularly the induction of transitions by interaction of hydrophobic sites of PrP are discussed in respect to their biological relevance.
Prion Rods Contain Small Amounts of Two Host Sphingolipids as Revealed by Thin-Layer Chromatography and Mass Spectrometry
Biological Chemistry, Jun 1, 1998
Sphingolipids were detected in prions, the agents of transmissible spongiform encephalopathies. T... more Sphingolipids were detected in prions, the agents of transmissible spongiform encephalopathies. The analysis was carried out on highly purified, infectious prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein, so-called PrP 27-30. Lipid classes were quantified by high performance thin-layer chromatography with a detection limit of 25-50 ng per lipid class. Matrix-assisted laser desorption/ionization mass spectrometry was applied for the first time to lipid analysis in complex biological samples. A newly developed preparation technique improved the sensitivity to 1-20 pg per molecular species. Only the sphingolipids, galactosylceramide and sphingomyelin, were consistently observed in chloroform/methanol (2:1 v/v) extracts of prion rods. The molar ratio of PrP to the sphingolipids was between 2:1 and 40:1, depending on the purity of the prion preparation. The same lipids were also present in the low density fraction of a gradient centrifugation of prion-rods after sonication in 0.2% SDS. From the two alternatives, that the sphingolipids are either required for prion function or are relics from the cellular location of PrP in caveolae, the second alternative appears more plausible since the preparation of highest specific infectivity contained the lowest amount of sphingolipids.
44. Chemical Disinfection and Inactivation of Prions
De Gruyter eBooks, Dec 21, 2006
Acid inactivation of prions: efficient at elevated temperature or high acid concentration
Journal of General Virology, May 1, 2006
Scrapie prion rods isolated from hamster and non-infectious aggregates of the corresponding recom... more Scrapie prion rods isolated from hamster and non-infectious aggregates of the corresponding recombinant protein rPrP(90–231) were incubated with hydrochloric acid. The amount of PrP and of infectivity that survived incubation in HCl at varying times, acid concentrations and temperatures was quantified by Western blot densitometry and bioassays, respectively. Prion rods and rPrP aggregates showed similar HCl hydrolysis kinetics of PrP, indicating structural homology. For 1 M HCl and 25 °C, the rate of PrP hydrolysis follows first-order kinetics at 0·014 h−1; the rate of infectivity inactivation is 0·54 h−1. Hydrolysis for 1 h at 25 °C was only slightly proportional to HCl concentration up to 5 M, but complete loss of infectivity and PrP reduction to <2 % was observed at 8 M HCl. The temperature dependence of unhydrolysed PrP, as well as infectivity at 1 M HCl for 1 h, showed a slight decrease up to 45 °C, but a sigmoidal decrease by several orders of magnitude at higher temperatures. The slow hydrolysis of PrP and inactivation of infectivity by acid treatment at room temperature are attributed to solvent inaccessibility of prion rods and rPrP aggregates, respectively. The more effective hydrolysis and inactivation at temperatures above 45 °C are interpreted as thermally induced disaggregation with an activation energy of 50–60 kJ mol−1. Most importantly, infectivity was always inactivated faster or to a higher extent than PrP was hydrolysed at several incubation times, HCl concentrations and temperatures.
Prion diseases are transmissible spongiform encephalopathies in humans and animals, including scr... more Prion diseases are transmissible spongiform encephalopathies in humans and animals, including scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer, and Creutzfeldt-Jakob disease (CJD) in humans. The hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP C ) to its pathological isoform PrP Sc , which is accompanied by PrP fibrillation. Transmission is not restricted within one species, but can also occur between species. In some cases a species barrier can be observed that results in limited or unsuccessful transmission. The mechanism behind interspecies transmissibility or species barriers is not completely understood. To analyse this process at a molecular level, we previously established an in vitro fibrillation assay, in which recombinant PrP (recPrP) as substrate can be specifically seeded by PrP Sc as seed. Seeding with purified components, with no additional cellular components, is a direct consequence of the ''prion-protein-only'' hypothesis. We therefore hypothesise, that the species barrier is based on the interaction of PrP C and PrP Sc . Whereas in our earlier studies, the interspecies transmission in animal systems was analysed, the focus of this study lies on the transmission from animals to humans. We therefore combined seeds from species cattle, sheep and deer (BSE, scrapie, CWD) with human recPrP. Homologous seeding served as a control. Our results are consistent with epidemiology, other in vitro aggregation studies, and bioassays investigating the transmission between humans, cattle, sheep, and deer. In contrast to CJD and BSE seeds, which show a seeding activity we can demonstrate a species barrier for seeds from scrapie and CWD in vitro. We could show that the seeding activity and therewith the molecular interaction of PrP as substrate and PrP Sc as seed is sufficient to explain the phenomenon of species barriers. Therefore our data supports the hypothesis that CWD is not transmissible to humans.
Prion Rods Contain an Inert Polysaccharide Scaffold
Biological Chemistry, Jan 17, 1999
A polysaccharide consisting of mainly 1,4-linked glucose units was found associated with prion ro... more A polysaccharide consisting of mainly 1,4-linked glucose units was found associated with prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein (PrP 27-30) exhibiting the ultrastructural and tinctorial properties of amyloid. The polysaccharide differs in composition from the Asn-linked oligosaccharides and the GPI-anchor of the prion protein. Prion rods were prepared from scrapie-infected hamster brains using two different purification protocols. Prolonged digestion of rods with proteinase K reduced PrP by a factor of at least 500, leaving about 10% (w/w) of the sample as an insoluble remnant. Only glucose was obtained by acid hydrolysis of the remnant and methylation analysis showed 80% 1,4-, 15% 1,6- and 5% 1,4,6-linked glucose units. The physical and chemical properties as well as the absence of terminal glucose units indicate a very high molecular mass of the polysaccharide. No evidence was found for covalent bonds between PrP and the polysaccharide. The polysaccharide certainly contributes to the unusual chemical and physical stability of prion rods, acting like a scaffold. A potential structural and/or functional relevance of the polysaccharide scaffold is discussed.
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