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1.
Scrapie-associated fibrils (SAF) are unique structures characteristic of the group of unconventional slow infections which includes scrapie and Creutzfeldt-Jakob disease. A major component of hamster fibrils has been described as a protease-resistant glycoprotein with an apparent mol. wt of 27,000-30,000 (PrP27-30). However, we report here that if fibrils are prepared by procedures designed to minimise proteolysis the PrP proteins co-purifying with hamster SAF have mol. wts of 33,000-35,000 (PrP33-35) and 26,000-29,000 (PrP26-29). We find a Lys-Lys-Arg-Pro-Lys sequence at the amino terminus of these SAF proteins, that is absent from PrP27-30, and which has recently been predicted to be the N-terminal sequence of the native PrP protein of uninfected brain. The major SAF protein (PrP33-35) and its normal brain homologue are shown to have the same apparent mol. wt and ionic charge distribution by two-dimensional gel analysis, silver staining and immunoblotting. These results support our view that PrP33-35 and the normal brain PrP protein may have the same covalent structure, and that the PrP protein is recruited into these amyloid-like SAF or into association with a non-protein component of SAF by an irreversible event initiated directly or indirectly by scrapie infection.  相似文献   

2.
We identified fibrils from non-transmissible systemic and cerebral amyloidosis using the purification method of scrapie-associated fibrils (SAF). The fibrils possessed the same nature of congophilia, filamentous structures and molecular weights as amyloid fibrils, and were resistant to Proteinase K digestion. This SAF method makes for a rapid extraction from amyloid-laden tissues. The method, therefore, may purify nontransmissible amyloids alone or together with SAF proteins.  相似文献   

3.
Scrapie-associated fibrils (SAF) are an infection-specific structure observed in the unconventional-agent diseases. Polyclonal antisera raised to scrapie proteins were used to test the antigenic relationship between purified fibrils and SAF isolated from non-protease-treated synaptosomal-mitochondrial preparations. The experimental design utilized fibrils from scrapie strain 263K-infected hamsters, scrapie strain 139A-infected mice, and scrapie strain ME7-infected mice. Preparations were examined by negative-stain immune electron microscopy and Western blot analysis of the polypeptides. Fibrils and polypeptides from each preparation reacted with a rabbit antiserum raised to each of the following: hamster 263K prion protein (PrP 27-30), hamster 263K SAF protein, and mouse ME7 SAF protein. Immune electron microscopy and Western blot analysis revealed similar antigenic relationships among the three scrapie antisera. Thus, fibrils and polypeptides can be considered to be the same in each preparation. No reactivity of the fibrils was observed with antisera raised to Alzheimer neurofibrillary tangles or a synthetic peptide of cerebrovascular amyloid. Thus, the fibrils observed in purified preparations share structural and antigenic similarities plus biochemically related peptides with SAF present in non-protease-treated preparations.  相似文献   

4.
Scrapie-associated fibrils (SAF) are disease-specific structures found in extracts of the brains of animals affected with scrapie. These structures are pathological aggregates of a normal host protein (PrP). Abnormal post-translational modification of PrP has been suggested to explain its aberrant properties in scrapie-affected brains and although there is a form of PrP in SAF indistinguishable in size from the protein in uninfected brain, lower-molecular-mass variants of PrP are also found in SAF fractions. We report the characterisation of the multiple forms of PrP found in SAF fractions purified from mouse brain affected by the ME7 strain of scrapie. The quantitatively major forms of PrP in SAF prepared without the use of proteinase K have the amino-terminal sequence Lys-Lys-Arg-Pro-Lys-Pro-Gly-Gly-, identical to that predicted for the amino-terminus of normal mouse brain PrP. However N-terminal cleavage of some PrP does occur in vivo within a domain of repetitive sequences at sites similar to but distinct from those cut by proteinase K in vitro. This suggests the conformation of the protein in aggregates in vivo does not differ extensively from that in detergent-treated SAF in vitro. We conclude that the size diversity of PrP in SAF is only partly due to N-terminal proteolysis and is independent of the proteolysis that occurs if proteinase K is used in the purification of SAF. Apart from proteolytic changes in the structure of PrP, we found a novel, as yet unidentified, amino-acid derivative of the arginine residue at position 3 in mouse PrP, which may predispose PrP to form SAF.  相似文献   

5.
Scrapie: concept of a virus-induced amyloidosis of the brain.   总被引:10,自引:0,他引:10  
After an intraperitoneal infection disease-specific incorporation of [3H]leucine into protein and [3H]uridine into RNA in the brain precede clinical scrapie in hamsters. Onset of both incorporations are the earliest measurable events in the disease. Infectivity and subsequent clinical symptoms appear only after this biochemical activity has ceased. The disease-specific [3H]protein co-purifies with scrapie-associated fibrils (SAF) and infectivity during differential centrifugation and buffer extraction. SDS-PAGE shows that the [3H]protein is not SAF protein but a protein with an apparently higher mol. wt. The [3H]RNA is metabolically stable and separates from SAF and the main portion of infectivity in the last step of the purification. The appearance of SAF-protein is a late event and correlates with severe clinical symptoms. SAF seems to be derived from a brain protein turning over slowly. Our data are consistent with early pre-clinical virus replication. In this case treatment aimed at suppressing virus replication in the clinical phase of the human Creutzfeldt-Jakob disease is unlikely to produce any beneficial effect.  相似文献   

6.
High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by various authors. We describe a fibril morphology analysis method that allows for quantitative comparison of features of amyloid fibrils of any amyloidogenic protein measured by atomic force microscopy. We have used tapping mode atomic force microscopy in liquid to measure the morphology of fibrillar aggregates of human wild-type alpha-synuclein and the disease-related mutants A30P, E46K, and A53T. Analysis of the images shows that fibrillar aggregates formed by E46K alpha-synuclein have a smaller diameter (9.0 +/- 0.8 nm) and periodicity (mode at 55 nm) than fibrils of wild-type alpha-synuclein (height 10.0 +/- 1.1 nm; periodicity has a mode at 65 nm). Fibrils of A30P have smaller diameter still (8.1 +/- 1.2 nm) and show a variety of periodicities. This quantitative analysis procedure enables comparison of the results with existing models for assembly of amyloid fibrils.  相似文献   

7.
Multiple assembly pathways underlie amyloid-beta fibril polymorphisms   总被引:1,自引:0,他引:1  
The amyloid beta-protein transiently forms low and high molecular mass oligomers and protofibrils in vitro, and after longer incubation times assembles into polymorphic mature fibrils. The precursor-to-product relationship of these species remains to be understood. Protofibrils are up to approximately 600 nm in length and have mass-per-lengths of 19(+/-2) kDa/nm measured by scanning transmission electron microscopy. Two predominant mature fibril types, several microns in length and with mass-per-lengths of 18(+/-3) and 27(+/-3) kDa/nm, are identified after longer incubation times. The difference of approximately 9 kDa/nm between the two fibril types indicates a bona fide elementary protofilament subunit of this mass-per-length. Fibrils in the 18(+/-3) kDa/nm group often exhibited distinct coiling with axial cross-over spacings of approximately 25 nm. Although strikingly different in morphology, the mass-per-length (MPL) of these coiled fibrils is equivalent to that measured for protofibrils. They could therefore arise from a conformational change in the protofibril concurrent with coiling and rapid elongation. Alternatively, we cannot rule out an assembly pathway not directly related to protofibrils. In contrast, the 27(+/-3) kDa/nm fibrils correspond to a MPL of approximately 1.5 x the protofibril and thus can neither arise from a simple conformational transition nor from lateral association of 19 kDa/nm protofibril precursors. Twisted ribbons with axial periodicities ranging from approximately 80 nm to 130 nm were prominent in the 27(+/-3) kDa/nm group as well as more tightly coiled fibrils. Individual fibril ribbons had elongation rates of 20(+/-12) nm/min when imaged by time-lapse atomic force microscopy. Protofibrils exhibited growth rates approximately 15 x slower at 1.3(+/-0.5) nm/min. The data support a model where concurrent multiple assembly pathways give rise to the various polymorphic fibril types.  相似文献   

8.
The conversion of the alpha-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , 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 PrP Sc from PrP C and 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 beta-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, beta-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 SHaPrP C . 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 PrP C were 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.  相似文献   

9.
The fraction (P4) containing scrapie infectivity was obtained by treatment of scrapie-infected mouse brains with the detergent sarcosyl, differential centrifugation, and proteolytic enzyme digestion. Scrapie infectivity in the P4 fraction was purified 239-2,390 times with respect to protein. Similar fractions were also prepared from the brain of a sheep naturally infected with scrapie. Morphological observation of the P4 fractions revealed that the main components were unique rods of 3-5 X 60-200 nm, which resembled scrapie-associated fibrils (SAF) or prion rods. The P4 fractions formed three major broad bands of polypeptides with molecular weights (MWs) of about 24.5K, 21K, and 17K dalton (Kd) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some low MW polypeptides were also present in the fraction. Rabbits immunized with this fraction prepared from mouse brains raised antibodies against the three major polypeptides.  相似文献   

10.
The diameter of collagen fibrils was measured in different regions of the antimesometrial endometrium of mice on days 5, 6, and 7 of pregnancy as well as in the endometrium of virgin mice. The average diameter of fibrils of virgin mice was 39.18 nm (range: 20-80). In the region of fully decidualized cells, the averages and ranges were 45.32 nm (30-170), 89.39 nm (30-270), and 125.88 nm (20-370), respectively, on days 5, 6, and 7 of pregnancy. Thick fibrils larger than 70 nm had irregular profiles. Our results show that the increase in diameter is associated with the decidualization of the mouse endometrium.  相似文献   

11.
Scrapie-associated fibril protein (SAF-protein) extracted from infectious scrapie-associated fibrils (SAF) isolated from scrapie hamster brains is not infectious. SAF-protein is composed of various mol. wt. species of glycoproteins differing in carbohydrate content rather than amino acid composition. The N-linked carbohydrate chains represent approximately 40-60% of the mol. wt. of SAF-protein. The deglycosylated SAF-protein has a surprisingly low mol. wt. of approximately 7 kd, representing approximately 55 amino acid residues. This size and chemical analyses indicate that SAF-protein is an amyloid-type of protein. The simplest explanation for the available data is that SAF-polypeptide is very likely not to be part of the scrapie agent but that it is, like other amyloid proteins, derived from host-encoded proteins and not infectious. It is suggested that the infectivity of fractions rich in SAF is due to co-purification of scrapie virus and SAF caused by the high carbohydrate content of SAF-protein.  相似文献   

12.
By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.  相似文献   

13.
Gerstmann-Sträussler-Scheinker (GSS) disease is a familial neurological disorder pathologically characterized by amyloid deposition in the cerebrum and cerebellum. The GSS amyloid is immunoreactive to antisera raised against the hamster prion protein (PrP) 27-30. This is a proteinase K-resistant glycoprotein of 27-30 kd that is derived from an abnormal isoform of a neuronal glycoprotein of 33-35 kd designated PrPSc and is a molecular marker of amyloid fibrils isolated from animals with scrapie and humans with related disorders. We have purified and characterized proteins extracted from amyloid plaque cores isolated from two patients of the Indiana kindred of GSS disease. We found that the major component of GSS amyloid is an 11 kd degradation product of PrP, whose N-terminus corresponds to the glycine residue at position 58 of the amino acid sequence deduced from the human PrP cDNA. In addition, amyloid fractions contained larger PrP fragments with apparently intact N-termini and amyloid P component. These findings suggest that the disease process leads to proteolytic cleavage of PrP, generating an amyloidogenic peptide that polymerizes into insoluble fibrils. The N-terminal cleavage of PrP in GSS disease occurs at a tryptophan-glycine peptide bond identical to that cleaved by proteinase K in vitro to generate PrP 27-30 from hamster PrPSc at codon 90. Since no mutations of the structural PrP gene have been found in the Indiana family of GSS disease, it is conceivable that factors other than the primary structure of PrP play a crucial role in the process of amyloid formation and the development of clinical neurologic dysfunction.  相似文献   

14.
Amyloid fibrils are present in the extracellular space of various tissues in neurodegenerative and protein misfolding diseases. Amyloid fibrils may be used in nanotechnology applications, because of their self-assembly properties and stability, if their growth and orientation can be controlled. Recently, we have shown that amyloid beta 25-35 (A beta 25-35) forms a highly oriented, K(+)-dependent network on mica. Here, we analyzed the properties of A beta 25-35_N27C, the cysteine residue of which may be used for subsequent chemical modifications. We find that A beta 25-35_N27C forms epitaxially growing fibrils on mica, which evolve into a trigonally oriented branched network. The binding is apparently more sensitive to cation concentration than that of the wild-type peptide. By nanomanipulating A beta 25-35_N27C fibrils with a gold-coated AFM tip, we show that the sulfhydryl of Cys27 is reactive and accessible from the solution. The oriented network of A beta 25-35_N27C fibrils can therefore be specifically labeled and may be used for constructing nanobiotechnological devices.  相似文献   

15.
We have examined the chemical dissection and subsequent reassembly of fibrils formed by a ten-residue peptide to probe the forces that drive the formation of amyloid. The peptide, TTR(10-19), encompasses the A strand of the inner beta-sheet structure that lines the thyroid hormone binding site of the human plasma protein transthyretin. When dissolved in water under low pH conditions the peptide readily forms amyloid fibrils. Electron microscopy of these fibrils indicates the presence of long (>1000 nm) rigid structures of uniform diameter (approximately 14 nm). Addition of urea (3 M) to preformed fibrils disrupts these rigid structures. The partially disrupted fibrils form flexible ribbon-like arrays, which are composed of a number of clearly visible protofilaments (3-4 nm diameter). These protofilaments are highly stable, and resist denaturation in 6 M urea at 75 degrees C over a period of hours. High concentrations (>50%, v/v) of 2,2,2-trifluoroethanol also dissociate TTR(10-19) fibrils to the constituent protofilaments, but these slowly dissociate to monomeric, soluble peptides with extensive alpha-helical structure. Dilution of the denaturant or co-solvent at the stage when dissociation to protofilaments has occurred results in the efficient reassembly of fibrils. These results indicate that assembly of fibrils from protofilaments involves relatively weak and predominantly hydrophobic interactions, whereas assembly of peptides into protofilaments involves both electrostatic and hydrophobic forces, resulting in a highly stable and compact structures.  相似文献   

16.
An accurate estimate of the prevalence of scrapie infection in the Great Britain (GB) sheep flock is essential when assessing any potential risk to human health through exposure to sheep transmissible spongiform encephalopathies (TSEs). One method for assessing the prevalence is to sample sheep intended for human consumption using a diagnostic test capable of detecting infected animals prior to the onset of clinical signs. An abattoir survey conducted in Great Britain in 1997-1998 tested brain samples from 2809 apparently healthy sheep of which none was found to be positive for scrapie by histopathology or immunohistochemistry (IHC) although 10 were positive for scrapie-associated fibrils (SAF). Subsequently, the tonsils from a subset of the animals sampled were examined using IHC, one of which tested positive. To interpret these results we use a likelihood-based approach, which accounts for the variation in the prevalence of infection with age and test sensitivity and specificity with stage of infection. Combining the results for all of the diagnostic tests yields an estimate of the prevalence of scrapie infection in the GB sheep flock of 0.22% (95% confidence interval: 0.01-0.97%). Moreover, our analysis suggests that all of the diagnostic tests used are very specific (greater than 99%). Indeed, only SAF detection yields a specificity estimate of less than 100%, which helps to account for the high number of samples found to be positive for SAF.  相似文献   

17.
Scrapie-associated fibrils (SAFs) are abnormal filamentous structures that are uniquely associated with unconventional slow virus diseases. The antigenic relationships of SAFs from animals infected with four biologically distinct scrapie strains were investigated by using antisera raised to purified SAF proteins. Rabbit antisera were raised to SAFs isolated from mice infected with the ME7 scrapie strain and to SAFs isolated from hamsters infected with the 263K scrapie strain. A strong antigenic relationship was shown among SAF proteins (PrPs) isolated from all scrapie-infected animals (ME7, 139A, and 87V in mice and 263K in hamsters), and this relationship was demonstrable regardless of which antiserum was used. SAF proteins were antigenically distinct from those of paired helical filaments or amyloid isolated from patients with Alzheimer disease. Distinct Western blot profiles were demonstrated for SAFs isolated from animals infected with each scrapie strain. Differences seen among SAFs were independent, at least in part, of host species or genotype, implying that certain specific structural and molecular properties of SAFs are mediated by the strain of scrapie agent.  相似文献   

18.
The self-assembly in films dried from aqueous solutions of a modified amyloid beta peptide fragment is studied. We focus on sequence Abeta(16-20), KLVFF, extended by two alanines at the N-terminus to give AAKLVFF. Self-assembly into twisted ribbon fibrils is observed, as confirmed by transmission electron microscopy (TEM). Dynamic light scattering reveals the semi-flexible nature of the AAKLVFF fibrils, while polarized optical microscopy shows that the peptide fibrils crystallize after an aqueous solution of AAKLVFF is matured over 5 days. The secondary structure of the fibrils is studied by FT-IR, circular dichroism and X-ray diffraction (XRD), which provide evidence for beta-sheet structure in the fibril. From high resolution TEM it is concluded that the average width of an AAKLVFF fibril is (63+/-18) nm, indicating that these fibrils comprise beta-sheets with multiple repeats of the unit cell, determined by XRD to have b and c dimensions 1.9 and 4.4 nm with an a axis 0.96 nm, corresponding to twice the peptide backbone spacing in the antiparallel beta-sheet.  相似文献   

19.
We have determined that a protein of the fodrin-spectrin-TW260/240 (FST) family is a component of the thin fibrils (~5 nm wide, 100–200 nm long) that cross-link bundles of actin filaments to adjacent actin bundles and to the plasma membrane in the terminal web of the brush border of the intestinal epithelium. When isolated brush borders were incubated with anti-fodrin antibodies and prepared for electron microscopy by the quick-freeze, deepetch technique, these ~5 nm fibrils were specifically decorated with the antibody. In addition, these cross-linking fibrils disappeared when the anti-fodrin-reactive proteins were extracted from the brush border. We conclude that FST is a component of a cross-linking system composed of ~5 nm fibrils that are morphologically distinct from the ~8 nm myosin-containing fibrils which were identified by antimyosin decoration. In addition to linking actin bundles to adjacent actin bundles and to the plasma membrane, these FST fibrils may mediate actinvesicle, actin-intermediate filament and vesicle-plasma membrane linkages.  相似文献   

20.
Elucidation of the structure of PrP(Sc) continues to be one of the most important and difficult challenges in prion research. This task, essential for gaining an understanding of the basis of prion infectivity, has been hampered by the insoluble, aggregated nature of this molecule. We used a combination of chemical cross-linking, proteolytic digestion, and mass spectrometry (MALDI-TOF and nanoLC-ESI-QqTOF), in an attempt to gain structural information about PrP 27-30 purified from the brains of Syrian hamsters infected with scrapie. The rationale of this approach is to identify pairs of specific amino acid residues that are close enough to each other to react with a bifunctional reagent of a given chain length. We cross-linked PrP 27-30 with the amino-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), obtaining dimers, trimers, and higher-order oligomers that were separated by SDS-PAGE. In-gel digestion followed by mass spectrometric analysis showed that BS(3) reacted preferentially with Gly90. A cross-link involving two Gly90 amino termini was found in cross-linked PrP 27-30 dimers, but not in intramolecularly cross-linked monomers or control samples. This observation indicates the spatial proximity of Gly90 amino termini in PrP 27-30 fibrils. The Gly90-Gly90 cross-link is consistent with a recent model of PrP 27-30, based on electron crystallographic data, featuring a fiber composed of stacked trimers of PrP monomers; specifically, it is compatible with cross-linking of monomers stacked vertically along the fiber axis but not those adjacent to each other horizontally in the trimeric building block. Our results constitute the first measured distance constraint in PrP(Sc).  相似文献   

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