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1.
Prion diseases are conformational diseases, many factors are involved in altering the conformation of prion, such as RNA,
DNA, pH, and copper etc. However the neurotoxic mechanism of prion diseases is not clear yet. The aim of this study is to
investigate the effect of the nucleoprotein complex of RNA and recombinant ovine prion protein (OvPrP C) on the cultured rat cortical neurons in vitro. Our previous study revealed that the nucleoprotein complex (OvPrP C-RNA) is characterized with high β sheet conformation and proteinase K resistance. Here we found that the OvPrP C-RNA induced marked neuronal cell death by the MTT (3-(4,5-dimethyl-thiazole -2-yl)-2,5-diphenyl –tetrazolium bromide) and
TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and the neurotoxic effects were confirmed by testing the content
of Bcl-2 Associated X protein (Bax) in the immunoprecipitation assay and Western blot assay. Compared to the control group,
there is no significant difference of active Bax or total Bax after RNA alone treatment or OvPrP C alone treatment, but the OvPrP C-RNA induced significant increases of active Bax level, while the contents of total Bax had no obvious changes after OvPrP C-RNA treatment. The results suggested that OvPrP C-RNA is neurotoxic in vitro, which added further evidence to the current understanding of mechanism of cellular injury by
RNA molecules for transformation of the PrP C to PrP Sc. 相似文献
2.
Conversion of the normal soluble form of prion protein, PrP (PrP C), to proteinase K-resistant form (PrP Sc) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change
from α-helix to β-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational
conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still
elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111–135) of Syrian hamster PrP penetrates
into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane
insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free
mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn,
expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates
the membrane binding mode of PrP by controlling the motional freedom of the TMD. 相似文献
3.
The mechanism that underlies a multitude of human disorders, including type II diabetes, Parkinson’s, Huntington’s and Alzheimer’s,
and the prion encephalopathies, is β-structure expansion through a pathogenic aggregation-prone monomeric form. β-sheet expansion
disorders share intermolecular association as a common determinant, being therefore collectively identified as conformational
diseases, but little is known about the underlying mechanism. Transmissible spongiform encephalopathies, also known as prion
diseases, are all characterised by progressive neuronal degeneration associated to marked extracellular accumulation of an
amyloidogenic conformer of the normal cellular prion protein (PrP C), referred to as the scrapie isoform (PrP Sc), which is thought to be responsible for the disease symptoms. PrP C is a ubiquitous 231-amino acid glycoprotein, whose physiological role is still elusive. It is organised as an N-terminal
disordered region and a compact C-terminal domain, where secondary structure elements consist of three α-helices (α1, α2 and
α3), with an α2-α3 disulphide bridge, and two short β-strands (β1 and β2). Evidence accumulated so far suggests that the protein
possesses one or several ‘spots’ of intrinsic conformational weakness, which may trigger generic folding, leading the whole
architecture to adopt aggregation-prone conformations. One of such spots is suspected to be the C-terminal side of the α-helix
2, which has recently gained the attention of several investigations because it gathers several disease-associated point mutations,
can be strongly fibrillogenic and toxic to neuronal cells, and possesses chameleon conformational behaviour. This paper briefly
reviews recent literature on α-2 domain-derived model peptides. 相似文献
4.
1. Prion diseases include kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker disease (GSS), and fatal familia insomnia (FFI) of humans, as well as scrapie and bovine spongiform encephalopathy (BSE) of animals.2. All these disorders involve conversion of the normal, cellular prion protein (PrP C) into the corresponding scrapie isoform (PrP Sc). PrP C adopts a structure rich in -helices and devoid of -sheet, in contrast to PrP Sc, which has a high -sheet content and is resistant to limited digestion by proteases. That a conformational transition features in the conversion of PrP C into PrP Sc implies that prion diseases are disorders of protein conformation.3. This concept has been extended by our studies with heat shock proteins (Hsp), many of which are thought to function as molecular chaperones. We found that the induction of some Hsps but not others was profoundly altered in scrapie-infected cells and that the distribution of Hsp73 is unusual in these cells.4. Whether the conversion of PrP C into PrP Sc is assisted by molecular chaperones, or if the accumulation of the abnormally folded PrP Sc is complexed with Hsps remains to be established. 相似文献
5.
Prion disease is a neurodegenerative disorder with progressive neurologic symptoms and accelerated cognitive decline. The causative protein of prion disease is the prion protein (PrP), and structural transition of PrP from the normal helix rich form (PrPC) to the abnormal β-sheet rich form (PrPSc) occurs in prion disease. While so far numerous therapeutic agents for prion diseases have been developed, none of them are still useful. A fluorinated alcohol, hexafluoro isopropanol (HFIP), is a precursor to the inhalational anesthetic sevoflurane and its metabolites. HFIP is also known as a robust α-helix inducer and is widely used as a solvent for highly aggregated peptides. Here we show that the α-helix-inducing activity of HFIP caused the conformational transformation of the fibrous structure of PrP into amorphous aggregates in vitro. HFIP added to the ScN2a cell medium, which continuously expresses PrPSc, reduced PrPSc protease resistance after 24-h incubation. It was also clarified that ScN2a cells are more susceptible to HFIP than any of the cells being compared. Based on these findings, HFIP is expected to develop as a therapeutic agent for prion disease. 相似文献
6.
Reagents that can precipitate the disease-associated prion protein (PrP Sc) are vital for the development of high sensitivity tests to detect low levels of this disease marker in biological material.
Here, a range of minerals are shown to precipitate both ovine cellular prion protein (PrP C) and ovine scrapie PrP Sc. The precipitation of prion protein with silicon dioxide is unaffected by PrP Sc strain or host species and the method can be used to precipitate bovine BSE. This method can reliably concentrate protease-resistant
ovine PrP Sc (PrP res) derived from 1.69 μg of brain protein from a clinically infected animal diluted into either 50 ml of buffer or 15 ml of
plasma. The introduction of a SiO 2 precipitation step into the immunological detection of PrP res increased detection sensitivity by over 1,500-fold. Minerals such as SiO 2 are readily available, low cost reagents with generic application to the concentration of diseases-associated prion proteins. 相似文献
8.
The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrP C from PrP Sc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of
animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala and Met, residues
that are absent from the protease accessible aminoterminal region of PrP Sc. Therefore, although thermolysin readily digests PrP c into small protein fragments, full-length PrP Sc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrP Sc species is produced, PrP 27–30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay
sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant
PrP Sc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrP Sc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents
and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary
tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrP Sc to disease pathogenesis. 相似文献
9.
The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP C into its pathogenic isoform PrP Sc 1. PrP C is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP Sc forms detergent-insoluble aggregates and is partially resistant to PK 2-6. The conversion of PrP C to PrP Sc is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP Sc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain 7.Using a combination of biophysical and biochemical approaches, we identified insoluble PrP C aggregates (designated iPrP C) from uninfected mammalian brains and cultured neuronal cells 8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms 10, and PK-treatment. The combination of these approaches isolates not only insoluble PrP Sc and PrP C aggregates but also soluble PrP C oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrP Sc from infected brains and iPrP C from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrP C are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrP Sc that shares the immunoreactive behavior and fragmentation with iPrP C 11, 12. Moreover, we recently demonstrated that iPrP C is the main species that interacts with amyloid-β protein in Alzheimer disease 13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer''s disease 13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders. 相似文献
10.
The conformational conversion of the cellular prion protein (PrP C) to the β-rich infectious isoform PrP Sc is considered a critical and central feature in prion pathology. Although PrP Sc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrP C to proteinase K resistant PrP Sc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrP C conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrP C to PrP Sc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232). 相似文献
11.
Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP C, for “cellular prion protein”) into an abnormal state (PrP Sc, for “scrapie prion protein”). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP C. In contrast to its homologue PrP C, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP Sc-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP C (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the α-helical monomer forms soluble β-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy. 相似文献
12.
Structures of the infectious form of prion protein ( e.g. PrP Sc or PrP-Scrapie) remain poorly defined. The prevalent structural models of PrP Sc retain most of the native α-helices of the normal, noninfectious prion protein, cellular prion protein (PrP C), but evidence is accumulating that these helices are absent in PrP Sc amyloid. Moreover, recombinant PrP C can form amyloid fibrils in vitro that have parallel in-register intermolecular β-sheet architectures in the domains originally occupied by helices 2 and 3. Here, we provide solid-state NMR evidence that the latter is also true of initially prion-seeded recombinant PrP amyloids formed in the absence of denaturants. These results, in the context of a primarily β-sheet structure, led us to build detailed models of PrP amyloid based on parallel in-register architectures, fibrillar shapes and dimensions, and other available experimentally derived conformational constraints. Molecular dynamics simulations of PrP(90–231) octameric segments suggested that such linear fibrils, which are consistent with many features of PrP Sc fibrils, can have stable parallel in-register β-sheet cores. These simulations revealed that the C-terminal residues ∼124–227 more readily adopt stable tightly packed structures than the N-terminal residues ∼90–123 in the absence of cofactors. Variations in the placement of turns and loops that link the β-sheets could give rise to distinct prion strains capable of faithful template-driven propagation. Moreover, our modeling suggests that single PrP monomers can comprise the entire cross-section of fibrils that have previously been assumed to be pairs of laterally associated protofilaments. Together, these insights provide a new basis for deciphering mammalian prion structures. 相似文献
13.
The mammalian prions replicate by converting cellular prion protein (PrP C) into pathogenic conformational isoform (PrP Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP Sc on conversion of PrP C in vitro using PrP Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP Sc. The tight correlation between conversion potency of small oligomers of human sPrP Sc observed in vitro and duration of the disease suggests that sPrP Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease. 相似文献
14.
Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrP Sc) converted from a normal host cellular prion protein (PrP C). Experimental studies suggest that PrP C is enriched with α-helical structure, whereas PrP Sc contains a high proportion of β-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrP C leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface. 相似文献
15.
Background Protein misfolding is the main cause of a group of fatal neurodegenerative diseases in humans and animals. In particular,
in Prion-related diseases the normal cellular form of the Prion Protein PrP ( PrP
C
) is converted into the infectious PrP
Sc
through a conformational process during which it acquires a high β-sheet content. Doppel is a protein that shares a similar
native fold, but lacks the scrapie isoform. Understanding the molecular determinants of these different behaviours is important
both for biomedical and biophysical research. 相似文献
16.
Conversion of native cellular prion protein (PrP c) from an α-helical structure to a toxic and infectious β-sheet structure (PrP Sc) is a critical step in the development of prion disease. There are some indications that the formation of PrP Sc is preceded by a β-sheet rich PrP (PrP β) form which is non-infectious, but is an intermediate in the formation of infectious PrP Sc. Furthermore the presence of lipid cofactors is thought to be critical in the formation of both intermediate-PrP β and lethal, infectious PrP Sc. We previously discovered that the endotoxin, lipopolysaccharide (LPS), interacts with recombinant PrP c and induces rapid conformational change to a β-sheet rich structure. This LPS induced PrP β structure exhibits PrP Sc-like features including proteinase K (PK) resistance and the capacity to form large oligomers and rod-like fibrils. LPS is a large, complex molecule with lipid, polysaccharide, 2-keto-3-deoxyoctonate (Kdo) and glucosamine components. To learn more about which LPS chemical constituents are critical for binding PrP c and inducing β-sheet conversion we systematically investigated which chemical components of LPS either bind or induce PrP conversion to PrP β. We analyzed this PrP conversion using resolution enhanced native acidic gel electrophoresis (RENAGE), tryptophan fluorescence, circular dichroism, electron microscopy and PK resistance. Our results indicate that a minimal version of LPS (called detoxified and partially de-acylated LPS or dLPS) containing a portion of the polysaccharide and a portion of the lipid component is sufficient for PrP conversion. Lipid components, alone, and saccharide components, alone, are insufficient for conversion. 相似文献
17.
In prion diseases, the cellular form of the prion protein, PrP C, undergoes a conformational conversion to the infectious isoform, PrP Sc. PrP C associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI) anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs). We show that heparin displaces PrP C from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP C. We then utilised a transmembrane-anchored form of PrP (PrP-TM), which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP C to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP C from rafts, promoting its endocytosis. Glypican-1 and PrP C colocalised on the cell surface and both PrP C and PrP Sc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP Sc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP C on the β-secretase cleavage of the Alzheimer''s amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP C and PrP Sc in lipid rafts. 相似文献
18.
BackgroundPrion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP C) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP Sc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP C molecules. A great deal of effort has been devoted to developing protocols for purifying PrP Sc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP Sc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases. Principal FindingsWe developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP Sc and mutant PrP aggregates at electrophoretic homogeneity. PrP Sc purified from prion-infected mice was able to seed misfolding of PrP C in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons. SignificanceThe immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates. 相似文献
19.
Summary 1. Vaccination-induced anti-prion protein antibodies are presently regarded as a promising approach toward treatment of prion
diseases. Here, we investigated the ability of five peptides corresponding to three different regions of the bovine prion
protein (PrP) to elicit antibodies interfering with PrP Sc propagation in prion-infected cells.
2. Rabbits were immunized with free nonconjugated peptides. Obtained immune sera were tested in enzyme-linked immunosorbent
assay (ELISA) and immunoblot for their binding to recombinant PrP and cell-derived pathogenic isoform (PrP Sc) and normal prion protein (PrP c), respectively. Sera positive in all tests were chosen for PrP Sc inhibition studies in cell culture.
3. All peptides induced anti-peptide antibodies, most of them reacting with recombinant PrP. Moreover, addition of the serum
specific to peptide 95–123 led to a transient reduction of PrP Sc levels in persistently prion-infected cells.
4. Thus, anti-PrP antibodies interfering with PrP Sc propagation were induced with a prion protein peptide nonconjugated to a protein carrier. These results point to the potential
application of the nonconjugated peptide 95–123 for the treatment of prion diseases. 相似文献
20.
Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrP Sc) that occur throughout the brain. Formation of synthetic or in vitro PrP Sc can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrP Sc. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrP c (spanning residues 90–231). We then obtained two-dimensional solution NMR spectra of the PrP c monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation ( 1H– 15N) studies revealed that, in comparison to monomeric PrP c, the oligomer has intense amide peak signals in the N-terminal (residues 90–114) and C-terminal regions (residues 226–231). Furthermore, a core region with decreased mobility is revealed from residues ~127 to 225. Within this core oligomer region with decreased mobility, there is a pocket of increased amide peak signal corresponding to the middle of α-helix 2 and the loop between α-helices 2 and 3 in the PrP c monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP. 相似文献
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