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Surachai Supattapone

Publications and source records attributed to Surachai Supattapone.

17 recordsLinked to original sources
PubMed2006

Accelerated accumulation of misfolded prion protein and spongiform degeneration in a Drosophila model of Gerstmann-Sträussler-Scheinker syndrome.

Prion diseases are CNS disorders that can occur in sporadic, infectious, and inherited forms. Although all forms of prion disease are associated with the accumulation of pathogenic conformers of the prion protein, collectively termed PrP(Sc), the mechanisms by which PrP(Sc) molecules form and cause neuronal degeneration are unknown. Using the bipartite galactosidase-4-upstream activating sequence expression system, we generated transgenic Drosophila melanogaster heterologously expressing either wild-type (WT) or mutant, disease-associated (P101L) mouse PrP molecules in cholinergic neurons. Transgenic flies expressing neuronal P101L PrP molecules exhibited severe locomotor dysfunction and premature death as larvae and adults. These striking clinical abnormalities were accompanied by age-dependent accumulation of misfolded PrP molecules, intracellular PrP aggregates, and neuronal vacuoles. In contrast, transgenic flies expressing comparable levels of WT PrP displayed no clinical, pathological, or biochemical abnormalities. These results indicate that transgenic Drosophila expressing neuronal P101L PrP specifically exhibit several hallmark features of human Gerstmann-Sträussler-Scheinker (GSS) syndrome. Because the rates of abnormal PrP accumulation and clinical progression are highly accelerated in Drosophila compared with the rates of these processes in rodents or humans, the P101L mutant may be used for future genetic and pharmacologic studies as a novel invertebrate model of GSS.

Animals↗
PubMed2006

The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro.

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrPC molecules are required to propagate mouse RML prions, whereas diglycosylated PrPC molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrPC molecules. Thus, interactions between different PrPC glycoforms appear to control the efficiency of prion formation in a species-specific manner.

Animals↗
PubMed2006

On the horizon: a blood test for prions.

A recent paper by Castilla et al. reported the detection of prions in the blood of scrapie-infected hamsters using a biochemical amplification method that is conceptually analogous to the polymerase chain reaction. This technological advance raises the possibility that prion diseases in humans and livestock could soon be diagnosed using blood samples if several technical limitations can be overcome.

Animals↗
PubMed2006

Copper (II) ions potently inhibit purified PrPres amplification.

The structural conversion of a host protein, PrP(C), into a protease-resistant isoform, PrPres, is the central event in the pathogenesis of infectious prion diseases. Purification of native PrP(C) molecules from hamster brain by either cation exchange or immobilized chelator chromatographic resins yielded preparations that supported efficient amplification of scrapie-induced PrPres in vitro. Using these purified preparations, we determined that in vitro PrPres amplification was inhibited by CuCl2 and ZnCl2 at IC50 concentrations of approximately 400 nm and 10 microM, respectively. In contrast, 100 microM MnCl2 did not directly inhibit PrPres amplification or block Cu2+-mediated inhibition. Additionally, the inhibition of PrPres amplification by Cu2+ ions could be reversed by addition of either neocuproine or imidazole. Cu2+ inhibited PrPres amplification in both the presence and absence of stimulatory polyanion molecules. These biochemical findings support the hypothesis that Cu2+ ions might regulate the pathogenesis of prion diseases in vivo.

Animals↗
PubMed2006

Inactivation of prions by acidic sodium dodecyl sulfate.

Prompted by the discovery that prions become protease-sensitive after exposure to branched polyamine dendrimers in acetic acid (AcOH) (S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay, F. C. Szoka, F. E. Cohen, S. B. Prusiner, and M. R. Scott, J. Virol. 75:3453-3461, 2001), we investigated the inactivation of prions by sodium dodecyl sulfate (SDS) in weak acid. As judged by sensitivity to proteolytic digestion, the disease-causing prion protein (PrPSc) was denatured at room temperature by SDS at pH values of < or =4.5 or > or =10. Exposure of Sc237 prions in Syrian hamster brain homogenates to 1% SDS and 0.5% AcOH at room temperature resulted in a reduction of prion titer by a factor of ca. 10(7); however, all of the bioassay hamsters eventually developed prion disease. When various concentrations of SDS and AcOH were tested, the duration and temperature of exposure acted synergistically to inactivate both hamster Sc237 prions and human sporadic Creutzfeldt-Jakob disease (sCJD) prions. The inactivation of prions in brain homogenates and those bound to stainless steel wires was evaluated by using bioassays in transgenic mice. sCJD prions were more than 100,000 times more resistant to inactivation than Sc237 prions, demonstrating that inactivation procedures validated on rodent prions cannot be extrapolated to inactivation of human prions. Some procedures that significantly reduced prion titers in brain homogenates had a limited effect on prions bound to the surface of stainless steel wires. Using acidic SDS combined with autoclaving for 15 min, human sCJD prions bound to stainless steel wires were eliminated. Our findings form the basis for a noncorrosive system that is suitable for inactivating prions on surgical instruments, as well as on other medical and dental equipment.

Animals↗
PubMed2005

Protease-resistant prion protein amplification reconstituted with partially purified substrates and synthetic polyanions.

Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27-30 and PrPC molecules at a molar ratio of 1:250 yielded approximately 2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to approximately 10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27-30, PrPC, and a stimulatory polyanionic compound.

Animals↗
PubMed2004

Ionic strength and transition metals control PrPSc protease resistance and conversion-inducing activity.

The essential component of infectious prions is a misfolded protein termed PrPSc, which is produced by conformational change of a normal host protein, PrPC. It is currently unknown whether PrPSc molecules exist in a unique conformation or whether they are able to undergo additional conformational changes. Under commonly used experimental conditions, PrPSc molecules are characteristically protease-resistant and capable of inducing the conversion of PrPC molecules into new PrPSc molecules. We describe the effects of ionic strength, copper, and zinc on the conformation-dependent protease resistance and conversion-inducing activity of PrPSc molecules in scrapie-infected hamster brains. In the absence of divalent cations, PrPSc molecules were > 20-fold more sensitive to proteinase K digestion in low ionic strength buffers than in high ionic strength buffers. Addition of micromolar concentrations of copper or zinc ions restored the protease resistance of PrPSc molecules under conditions of low ionic strength. These transition metals also controlled the conformation of purified truncated PrP-(27-30) molecules at low ionic strength, confirming that the N-terminal octapeptide repeat region of PrPSc is not required for binding to copper or zinc ions. The protease-sensitive and protease-resistant conformations of PrPSc were reversibly interchangeable, and only the protease-resistant conformation of PrPSc induced by high ionic strength was able to induce the formation of new protease-resistant PrP (PrPres) molecules in vitro. These findings show that PrPSc molecules are structurally interconvertible and that only a subset of PrPSc conformations are able to induce the conversion of other PrP molecules.

Animals↗
PubMed2004

Prion protein conversion in vitro.

The infectious agents of prion diseases are composed primarily of an infectious protein designated PrPSc. In cells infected with prions, a host glycoprotein termed PrPC undergoes induced conformational change to PrPSc, but the molecular mechanism underlying this structural transition occurs remains unknown. The prion-seeded conversion of PrPC to protease-resistant PrPSc-like molecules (PrPres) has been studied both in crude and purified in vitro systems in order to investigate the mechanism of protein conformational change in prion disease. Conversion of purified PrPC into PrPres is specific with respect to species-dependent and polymorphic differences in PrP sequence as well as biophysical variations between prion strains, recapitulating the specificity of prion propagation in vitro. The protein misfolding cyclic amplification (PMCA) technique, which utilizes crude brain homogenates, produces much higher yields of PrPres than conversion of purified PrP molecules, suggesting that additional cellular factors may stimulate PrPres formation. In a modified version of the PMCA technique, PrPres from diluted prion-infected brain homogenate can be amplified > ten-fold when mixed with normal brain homogenate without sonication or the anionic detergent sodium dodecyl sulfate (SDS). Under these conditions, PrPres amplification in vitro depends upon both time and temperature, has a neutral pH optimum, and does not require divalent cations. In vitro PrPres amplification is inhibited by both reversible and irreversible thiol blockers, indicating that the conformational change from PrPC to PrPres requires a thiol-containing factor. Stoichiometric transformation of PrPC to PrPres in vitro also requires specific RNA molecules, suggesting that host-encoded catalytic RNA molecules may play a role in the pathogenesis of prion disease. Heparan sulfate stimulates conversion of purified PrPC into PrPres in vitro, and heparan sulfate proteoglycan molecules are required for efficient PrPres formation in prion-infected cells. Future studies using in vitro PrPres conversion and amplification assays promise to provide new mechanistic insights about the PrP conversion process, and to generate clinically useful tools.

Animals↗
PubMed2004

In vitro prion protein conversion in detergent-solubilized membranes.

A fundamental event in the pathogenesis of prion disease is the conversion of PrP(C), a normal glycophosphatidyl-anchored glycoprotein, into an infectious isoform designated PrP(Sc). In a modified version of the protein misfolding cyclic amplification (PMCA) technique [Saborio et al. (2001) Nature 411, 810-813], protease-resistant PrP(Sc)-like molecules (PrPres) can be amplified in vitro in a species- and strain-specific manner from crude brain homogenates, providing a biochemical model of the prion conversion reaction [Lucassen et al. (2003) Biochemistry 42, 4127-4135]. In this study, we investigated the ability of enriched membrane subsets and detergent-solubilized membrane preparations to support PrPres amplification. Membrane fractionation experiments showed that purified synaptic plasma membrane preparations enriched in PrP(C) but largely depleted of late endosomal and lysosomal markers were sufficient to support PrPres amplification. Detergent solubilization experiments showed that a small group of select detergents could be used to produce soluble preparations that contain PrP(C) and fully support PrPres amplification. The stability of PrPres amplification ability in detergent-solubilized supernatants was dependent on detergent concentration. These results lead to the surprising conclusion that membrane attachment is not required for PrP(C) to convert efficiently into PrPres in vitro and also indicate that biochemical purification of PrPres amplification factors from brain homogenates is a feasible approach.

Animals↗
PubMed2003

RNA molecules stimulate prion protein conversion.

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

Animals↗
PubMed2003

In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups.

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).

Animals↗
PubMed2003

Post-transcriptional suppression of pathogenic prion protein expression in Drosophila neurons.

A wealth of evidence supports the view that conformational change of the prion protein, PrPC, into a pathogenic isoform, PrPSc, is the hallmark of sporadic, infectious, and inherited forms of prion disease. Although the central role played by PrPSc in the pathogenesis of prion disease is appreciated, the cellular mechanisms that recognize PrPSc and modulate its production, clearance, and neural toxicity have not been elucidated. To address these questions, we used a tissue-specific expression system to express wild-type and disease-associated PrP molecules heterologously in Drosophila melanogaster. Our results indicate that Drosophila brain possesses a specific and saturable mechanism that suppresses the accumulation of PG14, a disease-associated insertional PrP mutant. We also found that wild-type PrP molecules are maintained in a detergent-soluble conformation throughout life in Drosophila brain neurons, whereas they become detergent-insoluble in retinal cells as flies age. PG14 protein expression in Drosophila eye did not cause retinal pathology. Our work reveals the presence of mechanisms in neurons that specifically counterbalance the production of misfolded PrP conformations, and provides an opportunity to study these processes in a model organism amenable to genetic analysis.

Age Factors↗
PubMed2002

Pharmacological approaches to prion research.

The "protein-only" mechanism by which infectious agents of prion diseases such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy replicate remains undetermined. The identification of several distinct classes of prion inhibitors has created an opportunity to investigate the mechanism of prion formation using pharmacological tools. These new inhibitors include substituted tricyclic derivatives, tetrapyrrole compounds, cysteine protease inhibitors, branched polyamines, and specific antibodies. Each inhibitor class contains at least one active compound that inhibits prion propagation in cell culture at sub-micromolar concentrations and several structurally related, inactive compounds. Work with branched polyamines and specific antibodies has already provided insight into the kinetics and cell biology of endogenous prion clearance mechanisms. Other anti-prion compounds do not appear to bind directly to the prion protein. Detailed investigation of the mechanism of drug action of these compounds may lead to the identification of novel prion propagation factors.

Animals↗
PubMed2002

Structural studies of the scrapie prion protein by electron crystallography.

Because the insolubility of the scrapie prion protein (PrP(Sc)) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP(Sc) (PrP 27-30) and a miniprion (PrP(Sc)106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 A resolution. By comparing projection maps of PrP 27-30 and PrP(Sc)106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP(Sc). Only models featuring parallel beta-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.

Animals↗
PubMed1991

Endothelin Opens Potassium Channels in Glial Cells.

Endothelin-1 (ET-1), an autocrine hormone synthesized by astrocytes, and endothelin-3 (ET-3), a highly homologous peptide produced by neurons, have both been shown previously to cause proliferation of these astrocytes in culture [Supattapone et al. (1989) Biochem. Biophys. Res. Commun., 165, 1115 - 1122; MacCumber et al. (1990) Proc. Natl. Acad. Sci. USA, 87, 2359 - 2363]. We now demonstrate, using 86Rb+ influx assays and single channel patch-clamp recording, that both endothelins-ET-3 and ET-1-can also open a charybdotoxin-sensitive, calcium-activated K+ channel of 15 - 40 pS in glial cells. The opening of this channel may be important for the regulation of [K+] in the brain microenvironment. Thus, the endothelins may be a general mediator of astroglial response to neuronal injury.

Journal Article↗