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B Caughey

Publications and source records attributed to B Caughey.

At least 37 records · Page 2Linked to original sources
PubMed2000

Formation of protease-resistant prion protein in cell-free systems.

In transmissible spongiform encephalopathies (TSE) or prion diseases, the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to an abnormal pathogenic form that has a characteristic partial protease resistance (PrP-res). Studies with cell-free reactions indicate that the PrP-res itself can directly induce this conversion of PrP-sen. This PrP-res induced conversion reaction is highly specific in ways that might account at the molecular level for TSE species barriers, polymorphism barriers, and strains. Not only has this reaction been observed using mostly purified PrP-sen and PrP-res reactants, but also in TSE-infected brain slices. The conversion mechanism appears to involve both the binding of PrP-sen to polymeric PrP-res and a conformational change that results in incorporation into the PrP-res polymer.

Animals↗
PubMed1999

Prion protein interconversions and the transmissible spongiform encephalopathies.

Autocatalytic changes in the conformation and aggregation state of prion protein appear to be fundamental to transmissible spongiform encephalopathies or prion diseases. Here we review the considerable progress that has been made in describing the normal properties of prion protein and the changes that occur during these devastating neurodegenerative diseases.

Animals↗
PubMed1999

Specific binding of normal prion protein to the scrapie form via a localized domain initiates its conversion to the protease-resistant state.

In the transmissible spongiform encephalopathies, normal prion protein (PrP-sen) is converted to a protease-resistant isoform, PrP-res, by an apparent self-propagating activity of the latter. Here we describe new, more physiological cell-free systems for analyzing the initial binding and subsequent conversion reactions between PrP-sen and PrP-res. These systems allowed the use of antibodies to map the sites of interaction between PrP-sen and PrP-res. Binding of antibodies (alpha219-232) to hamster PrP-sen residues 219-232 inhibited the binding of PrP-sen to PrP-res and the subsequent generation of PK-resistant PrP. However, antibodies to several other parts of PrP-sen did not inhibit. The alpha219-232 epitope itself was not required for PrP-res binding; thus, inhibition by alpha219-232 was likely due to steric blocking of a binding site that is close to, but does not include the epitope in the folded PrP-sen structure. The selectivity of the binding reaction was tested by incubating PrP-res with cell lysates or culture supernatants. Only PrP-sen was observed to bind PrP-res. This highly selective binding to PrP-res and the localized nature of the binding site on PrP-sen support the idea that PrP-sen serves as a critical ligand and/or receptor for PrP-res in the course of PrP-res propagation and pathogenesis in vivo.

Animals↗
PubMed1999

Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems. An update.

Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease-sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.

Animals↗
PubMed1998

Strain-dependent differences in beta-sheet conformations of abnormal prion protein.

Strain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm-1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629-1630 cm-1 and stronger band intensity at both 1616 and 1694-1695 cm-1. Because absorbances from 1616 to 1636 cm-1 of protein infrared spectra are attributed primarily to beta-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with beta-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.

Animals↗
PubMed1998

Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines.

A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5-1 microM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies.

Cell-Free System↗
PubMed1998

The importance of the disulfide bond in prion protein conversion.

The conversion of normal, protease sensitive prion protein (PrP-sen) to the abnormal protease-resistant form (PrP-res) is of central importance in the pathogenesis of scrapie and other transmissible spongiform encephalopathies. In the present study, the effects of reduction of the disulfide bond on the PrP-sen to PrP-res conversion in a cell-free system were examined. The addition of the disulfide reducing agent dithiothreitol inhibited the cell-free conversion reaction with an IC50 of 2-2.5 mM. Separate pretreatment of either PrP-sen or PrP-res with dithiothreitol and an alkylating agent also inhibited the conversion reaction. Results of this study show that preservation of the disulfide bond is important in the conversion of PrP-sen to PrP-res.

Alkylation↗
PubMed1998

Measurement of altered aspartyl residues in the scrapie associated form of prion protein.

In transmissible spongiform encephalopathies (TSE), the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to a pathologic form (PrP-res) that has greatly enhanced proteinase K resistance, insolubility, and beta sheet content. To investigate the possibility that alterations at aspartyl or asparaginyl residues in the form of D-aspartate and/or L-isoaspartate could play a role in either the formation or stabilization of PrP-res in TSE-infected animals, we assayed for the presence of these abnormal residues in PrP-res. Protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) was used to methylate and radiolabel altered aspartyl residues, which were detected in PrP-res, but at low levels (0.5 mole%). The scarcity of D-aspartyl and/or L-isoaspartyl groups in PrP-res suggests that this modification is unlikely to be primarily responsible for the differences between PrP-res and PrP-sen. However, it remains possible that such modifications in substoichiometric numbers of PrP molecules could help to initiate the PrP-res formation or stabilize PrP-res polymers in vivo.

Animals↗
PubMed1998

Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides.

The transmissible spongiform encephalopathies are characterized by the conversion of the protease-sensitive prion protein (PrPsen) into a protease-resistant isoform (PrPres) associated with the neuropathogenic process in vivo. Recently, PrPres has been shown to be capable of directly inducing the conversion of PrPsen to PrPres in a cell-free in vitro system. In the present experiments, various PrP peptides were studied for their ability to enhance or inhibit this cell-free conversion reaction. None of the synthetic peptides was able to confer protease-resistance to the labeled PrPsen molecules on their own. On the contrary, peptides from the central part of the hamster PrP sequence from 106 to 141 could completely inhibit the conversion induced by preformed PrPres. The presence of residues 119 and 120 from the highly hydrophobic sequence AGAAAAGA (position 113 to 120) was crucial for an efficient inhibitory effect. Fourier transform infrared spectroscopy analysis indicated that inhibitory peptides formed high beta-sheet aggregates under the conditions of the conversion reaction, but this was also true of certain peptides that were not inhibitory. Thus, the potential to form beta-sheeted aggregates may be necessary, but not sufficient, for peptides to act as inhibitors of PrPres formation. Clearly, the amino acid sequence of the peptide is also important for inhibition. The sequence specificity of the inhibition is consistent with the idea that residues in the vicinity of positions 106-141 of PrPres and/or PrPsen are critically involved in the intermolecular interactions that lead to PrPres formation.

Amino Acid Sequence↗
PubMed1998

Structural aspects of Congo red as an inhibitor of protease-resistant prion protein formation.

Congo red (CR) has been shown to inhibit the accumulation in scrapie-infected cells of prion protein (PrP) in the abnormal protease-resistant form (PrP-res). However, it was not clear if this effect was due to a direct interaction of CR with either PrP-res or its protease-sensitive precursor (PrP-sen) or to a less direct effect on living cells. Here we show that CR inhibits PrP-res formation in a simple cell-free reaction composed predominantly of purified PrP-res and PrP-sen. Structurally modified CR analogues were also compared in both the cell-free conversion reaction and scrapie-infected neuroblastoma cells. Methylation of the central phenyl groups at the 2,2' positions diminished the inhibitory potency by > or = 10-fold. In contrast, there was little effect of 3,3' methylation of the phenyls, deletion of one phenyl, or addition of an amido group between the phenyls. The relative activities of these compounds were well correlated in both cellular and acellular systems. Molecular modeling indicated that CR and 3,3'-methyl-CR have little rotational restriction about the biphenyl bond and can readily adopt a planar conformation, as can phenyl-CR and amido-CR. In contrast, 2,2'-methyl-CR is restricted to a nonplanar conformation of the biphenyl group. Thus, planarity and/or torsional mobility of the central phenyl rings of CR and its analogues is probably important for inhibition of PrP-res formation. On the other hand, variations in the intersulfonate distance in these molecules had little effect on PrP-res inhibition. These results indicated a high degree of structural specificity in the inhibition of PrP-res formation by CR and related compounds.

Animals↗
PubMed1997

Chaperone-supervised conversion of prion protein to its protease-resistant form.

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet. The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.

Adenosine Triphosphate↗
PubMed1997

Molecular assessment of the potential transmissibilities of BSE and scrapie to humans.

More than a million cattle infected with bovine spongiform encephalopathy (BSE) may have entered the human food chain. Fears that BSE might transmit to man were raised when atypical cases of Creutzfeldt-Jakob disease (CJD), a human transmissible spongiform encephalopathy (TSE), emerged in the UK. In BSE and other TSE diseases, the conversion of the protease-sensitive host prion protein (PrP-sen) to a protease-resistant isoform (PrP-res) is an important event in pathogenesis. Biological aspects of TSE diseases are reflected in the specificities of in vitro PrP conversion reactions. Here we show that there is a correlation between in vitro conversion efficiencies and known transmissibilities of BSE, sheep scrapie and CJD. On this basis, we used an in vitro system to gauge the potential transmissibility of scrapie and BSE to humans. We found limited conversion of human PrP-sen to PrP-res driven by PrP-res associated with both scrapie (PrP[Sc]) and BSE (PrP[BSE]). The efficiencies of these heterologous conversion reactions were similar but much lower than those of relevant homologous conversions. Thus the inherent ability of these infectious agents of BSE and scrapie to affect humans following equivalent exposure may be finite but similarly low.

Animals↗
PubMed1997

In situ formation of protease-resistant prion protein in transmissible spongiform encephalopathy-infected brain slices.

The transmissible spongiform encephalopathies (TSEs) comprise a group of fatal neurodegenerative diseases that are characterized by the conversion of the normal host cellular prion protein (PrPC), to the abnormal protease-resistant prion protein isoform (PrP-res). It has been proposed, though not proven, that the infectious TSE agent consists solely of PrP-res and that PrP-res-induced conformational conversion of PrPC to additional PrP-res represents agent replication. In this study we demonstrate in situ conversion of protease-sensitive PrPC to PrP-res in TSE-infected brain slices. One step in this process is the binding of soluble PrPC to endogenous PrP-res deposits. The newly formed PrP-res associated with the slices in a pattern that correlated with the pre-existing brain distribution of PrP-res. Punctate in situ PrP conversion was observed in brain regions containing PrP-res amyloid plaques, and a more dispersed conversion product was detected in areas containing diffuse PrP-res deposits. These studies provide direct evidence that PrP-res formation involves the incorporation of soluble PrPC into both nonfibrillar and fibrillar PrP-res deposits in TSE-infected brain. Our findings suggest that the in situ PrP conversion reaction leads to additional polymerization of endogenous PrP-res aggregates and is analogous to the process of PrP-res fibril and subfibril growth in vivo.

Animals↗
PubMed1997

Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms.

Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrPSc) of the host-encoded cellular prion protein (PrPC) mainly in the central nervous system. Polymorphisms in the PrP gene are linked to differences in susceptibility for prion diseases. The mechanisms underlying these effects are still unknown. Here we describe studies of the influence of sheep PrP polymorphisms on the conversion of PrPC into protease-resistant forms. In a cell-free system, sheep PrPSc induced the conversion of sheep PrPC into protease-resistant PrP (PrP-res) similar or identical to PrPSc. Polymorphisms present in either PrPC or PrPSc had dramatic effects on the cell-free conversion efficiencies. The PrP variant associated with a high susceptibility to scrapie and short survival times of scrapie-affected sheep was efficiently converted into PrP-res. The wild-type PrP variant associated with a neutral effect on susceptibility and intermediate survival times was converted with intermediate efficiency. The PrP variant associated with scrapie resistance and long survival times was poorly converted. Thus the in vitro conversion characteristics of the sheep PrP variants reflect their linkage with scrapie susceptibility and survival times of scrapie-affected sheep. The modulating effect of the polymorphisms in PrPC and PrPSc on the cell-free conversion characteristics suggests that, besides the species barrier, polymorphism barriers play a significant role in the transmissibility of prion diseases.

Alleles↗
PubMed1997

Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies.

Denaturation studies with guanidine HCl (GdnHCl) were performed to test the relationship between scrapie infectivity and properties of scrapie-associated prion protein (PrP(Sc)). Large GdnHCl-induced reductions in infectivity were associated with the irreversible elimination of both the proteinase K resistance and apparent self-propagating converting activity of PrP(Sc). In intermediate GdnHCl concentrations that stimulate converting activity and partially disaggregate PrP(Sc), both scrapie infectivity and converting activity were associated with residual partially protease-resistant multimers of PrP(Sc).

Animals↗
PubMed1996

Partial unfolding and refolding of scrapie-associated prion protein: evidence for a critical 16-kDa C-terminal domain.

The conversion of the normal form of prion protein (PrPC) to a disease-specific form (PrPSc) is a central event in scrapie and other transmissible spongiform encephalopathies. PrPSc is distinguished from PrPC by its insolubility and its resistance to proteolysis. PrPSc is also capable of converting 35S-PrPC, in vitro, into a form which is indistinguishable from PrPSc with respect to its protease-sensitivity. Both the "converting activity" and the protease-resistance of isolated hamster PrPSc can be at least partially eliminated by denaturation and recovered by renaturation, provided that the concentration of denaturant does not exceed a threshhold. This study was undertaken in order to localize the regions of native PrPSc structure that must remain intact to allow refolding. Proteinase K was used to digest exposed, denatured PrPSc sequences, and the residual fragments were characterized using anti-PrP antibodies directed toward four PrP epitopes. A 16-kDa fragment marked by an epitope within residues 143-156 remained protease-resistant under conditions which at least partially unfolded epitopes within residues 90-115 and 217-232. However, dilution of denaturant restored protease-resistance to these epitopes. This reversible unfolding was observed with both purified PrPSc and PrPSc in crude brain homogenates. Size fractionation of partially GdnHCl-solubilized PrPSc revealed that only the insoluble aggregates retained the ability to refold, consistent with the hypothesis that native PrPSc is an ordered aggregate. When the threshold denaturant concentration was exceeded, both protease-resistance of the 16-kDa C-terminal domain and converting activity were irreversibly destroyed. These results suggest that the in vitro converting activity requires ordered, protease-resistant PrPSc aggregates and that a critical aspect of the PrPSc structure is the folding of a particularly stable approximately 16-kDa C-terminal domain.

Animals↗