RESUMEN
Prion diseases are caused by misfolding of the cellular prion protein, PrPC. In vitro studies have shown that PrP binds copper via the octarepeat region lying within the unstructured N-terminal segment of the protein, but the significance of copper in PrP metabolism remains unclear. Here, six specific antibodies recognizing different epitope regions of PrP were used to measure the effect of copper on the conformation of the molecule at the cell surface. Binding of an antibody, E149, to an epitope within the octarepeat domain of PrP is halved in the presence of copper, whereas binding of antibodies recognizing epitope motifs C-terminal to residue 90 of PrP remain relatively unaltered under equivalent conditions. These experiments strongly suggest that copper induces localized conformational change within the N-terminal portion of cell-surface PrPC.
Asunto(s)
Cobre/farmacología , Proteínas PrPC/química , Proteínas PrPC/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Células CHO , Cricetinae , Mapeo Epitopo , Epítopos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Datos de Secuencia Molecular , Proteínas PrPC/metabolismoRESUMEN
To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.
Asunto(s)
Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Empalme Alternativo/genética , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caveolas/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Endopeptidasa K/metabolismo , Formaldehído/metabolismo , Sustancias Macromoleculares , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Moléculas de Adhesión de Célula Nerviosa/genética , Neuroblastoma/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Fosfatidilinositol Diacilglicerol-Liasa , Proteínas PrPC/genética , Proteínas PrPSc/farmacología , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Sitios de Empalme de ARN/genética , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismoRESUMEN
Doppel (Dpl) is a paralog of the mammalian prion protein (PrP); it is abundant in testes but expressed at low levels in the adult central nervous system. In two Prnp-deficient (Prnp(0/0)) mouse lines (Ngsk and Rcm0), Dpl overexpression correlated with ataxia and death of cerebellar neurons. To determine whether Dpl overexpression, rather than the dysregulation of genes neighboring the Prn gene complex, was responsible for the ataxic syndrome, we placed the mouse Dpl coding sequence under the control of the Prnp promoter and produced transgenic (Tg) mice on the Prnp(0/0)-ZrchI background (hereafter referred to as ZrchI). ZrchI mice exhibit neither Dpl overexpression nor cerebellar degeneration. In contrast, Tg(Dpl)ZrchI mice showed cerebellar granule and Purkinje cell loss; the age of onset of ataxia was inversely proportional to the levels of Dpl protein. Crosses of Tg mice overexpressing wild-type PrP with two lines of Tg(Dpl)ZrchI mice resulted in a phenotypic rescue of the ataxic syndrome, while Dpl overexpression was unchanged. Restoration of PrP expression also rendered the Tg(Dpl) mice susceptible to prion infection, with incubation times indistinguishable from non-Tg controls. Whereas the rescue of Dpl-induced neurotoxicity by coexpression of PrP argues for an interaction between the PrP and Dpl proteins in vivo, the unaltered incubation times in Tg mice overexpressing Dpl in the central nervous system suggest that Dpl is unlikely to be involved in prion formation.
Asunto(s)
Cerebelo/patología , Priones/fisiología , Animales , Ataxia/genética , Cerebelo/anatomía & histología , Proteínas Ligadas a GPI , Ratones , Ratones Transgénicos , Fenotipo , Priones/genética , Regiones Promotoras GenéticasRESUMEN
Huntington disease (HD) is a common autosomal dominant neurodegenerative disease with early adult-onset motor abnormalities and dementia. Many studies of HD show that huntingtin (CAG)n repeat-expansion length is a sensitive and specific marker for HD. However, there are a significant number of examples of HD in the absence of a huntingtin (CAG)n expansion, suggesting that mutations in other genes can provoke HD-like disorders. The identification of genes responsible for these "phenocopies" may greatly improve the reliability of genetic screens for HD and may provide further insight into neurodegenerative disease. We have examined an HD phenocopy pedigree with linkage to chromosome 20p12 for mutations in the prion protein (PrP) gene (PRNP). This reveals that affected individuals are heterozygous for a 192-nucleotide (nt) insertion within the PrP coding region, which encodes an expanded PrP with eight extra octapeptide repeats. This reveals that this HD phenocopy is, in fact, a familial prion disease and that PrP repeat-expansion mutations can provoke an HD "genocopy." PrP repeat expansions are well characterized and provoke early-onset, slowly progressive atypical prion diseases with an autosomal dominant pattern of inheritance and a remarkable range of clinical features, many of which overlap with those of HD. This observation raises the possibility that an unknown number of HD phenocopies are, in fact, familial prion diseases and argues that clinicians should consider screening for PrP mutations in individuals with HD-like diseases in which the characteristic HD (CAG)n repeat expansions are absent.
Asunto(s)
Cromosomas Humanos Par 20/genética , Enfermedad de Huntington/fisiopatología , Mutagénesis Insercional/genética , Enfermedades por Prión/genética , Enfermedades por Prión/fisiopatología , Priones/genética , Secuencia de Aminoácidos , Mapeo Cromosómico , Análisis Mutacional de ADN , Pruebas Genéticas/métodos , Humanos , Enfermedad de Huntington/genética , Escala de Lod , Datos de Secuencia Molecular , Fenotipo , Priones/química , Alineación de SecuenciaRESUMEN
The secondary structure of a 55-residue fragment of the mouse prion protein, MoPrP(89-143), was studied in randomly aggregated (dried from water) and fibrillar (precipitated from water/acetonitrile) forms by (13)C solid-state NMR. Recent studies have shown that the fibrillar form of the P101L mutant of MoPrP(89-143) is capable of inducing prion disease in transgenic mice, whereas unaggregated or randomly aggregated samples do not provoke disease. Through analysis of (13)C chemical shifts, we have determined that both wild-type and mutant sequence MoPrP(89-143) form a mixture of beta-sheet and alpha-helical conformations in the randomly aggregated state although the beta-sheet content in MoPrP(89-143, P101L) is significantly higher than in the wild-type peptide. In a fibrillar state, MoPrP(89-143, P101L) is completely converted into beta-sheet, suggesting that the formation of a specific beta-sheet structure may be required for the peptide to induce disease. Studies of an analogous peptide from Syrian hamster PrP verify that sequence alterations in residues 101-117 affect the conformation of aggregated forms of the peptides.
Asunto(s)
Fragmentos de Péptidos/química , Proteínas PrPC/química , Priones/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Isótopos de Carbono , Marcaje Isotópico/métodos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Enfermedades por Prión , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
[URE3] is a non-Mendelian genetic element in Saccharomyces cerevisiae, which is caused by a prion-like, autocatalytic conversion of the Ure2 protein (Ure2p) into an inactive form. The presence of [URE3] allows yeast cells to take up ureidosuccinic acid in the presence of ammonia. This phenotype can be used to select for the prion state. We have developed a novel reporter, in which the ADE2 gene is controlled by the DAL5 regulatory region, which allows monitoring of Ure2p function by a colony color phenotype. Using this reporter, we observed induction of different [URE3] prion variants ("strains") following overexpression of the N-terminal Ure2p prion domain (UPD) or full-length Ure2p. Full-length Ure2p induced two types of [URE3]: type A corresponds to conventional [URE3], whereas the novel type B variant is characterized by relatively high residual Ure2p activity and efficient curing by coexpression of low amounts of a UPD-green fluorescent protein fusion protein. Overexpression of UPD induced type B [URE3] but not type A. Both type A and B [URE3] strains, as well as weak and strong isolates of type A, were shown to stably maintain different prion strain characteristics. We suggest that these strain variants result from different modes of aggregation of similar Ure2p monomers. We also demonstrate a procedure to counterselect against the [URE3] state.
Asunto(s)
Priones/química , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Endopeptidasa K/metabolismo , Proteínas Fúngicas/metabolismo , Genes Reporteros , Técnicas Genéticas , Glutatión Peroxidasa , Guanidina/farmacología , Modelos Genéticos , Fenotipo , Plásmidos/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The neurochemical alterations associated with neurodegeneration in prion diseases are not well defined. It is therefore of interest to study the influence of prion infection on messenger molecules and their receptors. In the present study we have analyzed the possible involvement of NMDA receptors in prion-infected mice using ligand binding autoradiography and iodinated MK-801, a noncompetitive NMDA antagonist. The results show a reduced binding of MK-801 in discrete regions of hippocampus at 110 days after infection, that is before the appearance of behavioral symptoms. In addition, early transient increases in MK-801 binding were observed in several layers. The exact neuroanatomical correlate of these changes in MK-801 binding, as well as its functional significance in relation to prion symptomatology, remain to be analyzed.
Asunto(s)
Maleato de Dizocilpina/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Hipocampo/metabolismo , Scrapie/metabolismo , Animales , Autorradiografía , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Hipocampo/patología , Masculino , Ratones , Ratones Endogámicos , Degeneración Nerviosa/metabolismo , Priones/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Scrapie/patologíaRESUMEN
Prion diseases in humans and animals are invariably fatal. Prions are composed of a disease-causing isoform (PrP(Sc)) of the normal host prion protein (PrP(C)) and replicate by stimulating the conversion of PrP(C) into nascent PrP(Sc). We report here that tricyclic derivatives of acridine and phenothiazine exhibit half-maximal inhibition of PrP(Sc) formation at effective concentrations (EC(50)) between 0.3 microM and 3 microM in cultured cells chronically infected with prions. The EC(50) for chlorpromazine was 3 microM, whereas quinacrine was 10 times more potent. A variety of 9-substituted, acridine-based analogues of quinacrine were synthesized, which demonstrated variable antiprion potencies similar to those of chlorpromazine and emphasized the importance of the side chain in mediating the inhibition of PrP(Sc) formation. Thus, our studies show that tricyclic compounds with an aliphatic side chain at the middle ring moiety constitute a new class of antiprion reagents. Because quinacrine and chlorpromazine have been used in humans for many years as antimalarial and antipsychotic drugs, respectively, and are known to pass the blood-brain barrier, we suggest that they are immediate candidates for the treatment of Creutzfeldt-Jakob disease and other prion diseases.
Asunto(s)
Acridinas/uso terapéutico , Fenotiazinas/uso terapéutico , Proteínas PrPSc/efectos de los fármacos , Enfermedades por Prión/tratamiento farmacológico , Acridinas/química , Acridinas/farmacología , Animales , Células Cultivadas/efectos de los fármacos , Clorpromazina/química , Clorpromazina/farmacología , Ácidos Grasos/química , Humanos , Ratones , Neuroblastoma/patología , Fenotiazinas/química , Fenotiazinas/farmacología , Conformación Proteica/efectos de los fármacos , Quinacrina/química , Quinacrina/farmacología , Seguridad , Relación Estructura-Actividad , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.
Asunto(s)
Fragmentos de Inmunoglobulinas/inmunología , Priones/inmunología , Animales , Especificidad de Anticuerpos , Bioensayo , Epítopos de Linfocito B/inmunología , Escherichia coli , Ratones , Proteínas PrPC/inmunología , Priones/antagonistas & inhibidores , Priones/biosíntesis , Proteínas Recombinantes , Células Tumorales CultivadasRESUMEN
The conversion of the alpha-helical, protease sensitive and noninfectious form of the prion protein (PrP(C)) into an insoluble, protease resistant, predominantly beta-sheeted and infectious form (PrP(Sc)) is the fundamental event in prion formation. In the present work, two soluble and stable intermediate structural states are newly identified for recombinant Syrian hamster PrP(90-231) (recPrP), a dimeric alpha-helical state and a tetra- or oligomeric, beta-sheet rich state. In 0.2% SDS at room temperature, recPrP is soluble and exhibits alpha-helical and random coil secondary structure as determined by circular dichroism. Reduction of the SDS concentration to 0.06% leads first to a small increase in alpha-helical content, whereas further dilution to 0.02% results in the aquisition of beta-sheet structure. The reversible transition curve is sigmoidal within a narrow range of SDS concentrations (0.04 to 0.02%). Size exclusion chromatography and chemical crosslinking revealed that the alpha-helical form is dimeric, while the beta-sheet rich form is tetra- or oligomeric. Both the alpha-helical and beta-sheet rich intermediates are soluble and stable. Thus, they should be accessible to further structural and mechanistic studies. At 0.01% SDS, the oligomeric intermediates aggregated into large, insoluble structures as observed by fluorescence correlation spectroscopy. Our results are discussed with respect to the mechanism of PrP(Sc) formation and the propagation of prions.
Asunto(s)
Proteínas PrPSc/química , Priones/química , Pliegue de Proteína , Tampones (Química) , Cromatografía en Gel , Dicroismo Circular , Reactivos de Enlaces Cruzados/química , Polímeros/química , Proteínas PrPSc/patogenicidad , Conformación Proteica , Proteínas Recombinantes , Solubilidad , Espectrometría de FluorescenciaRESUMEN
Prion diseases are diseases of protein conformation. Structure-dependent antibodies have been sought to probe conformations of the prion protein (PrP) resulting from environmental changes, such as differences in pH. Despite the absence of such antibodies for full-length PrP, a recombinant Fab (D13) and a Fab derived from mAb 3F4 showed pH-dependent reactivity toward epitopes within the N-terminus of N-terminally truncated PrP(90-231). Refolding and maintaining this protein at pH > or =5.2 before immobilization on an ELISA plate inhibited reactivity relative to protein exposed to pH < or =4.7. The reactivity was not affected by pH changes after immobilization, showing retention of conformation after binding to the plate surface, although guanidine hydrochloride at 1.5-2 M was able to expose the cryptic epitopes after immobilization at pH > or =5.2. The alpha-helical CD spectrum of PrP(90-231) refolded at pH 5.5 was reduced somewhat by these pH changes, with a minor shift toward beta-sheet at pH 4 and then toward coil at pH 2. No covalent changes were caused by the pH differences. This pH dependence suggests titration of an acidic region that might inhibit the N-terminal epitopes. A similar pH dependence for a monoclonal antibody reactive to the central region identified an acidic region incorporating Glu152 as a significant participant.
Asunto(s)
Epítopos/química , Epítopos/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Priones/química , Priones/genética , Secuencia de Aminoácidos , Animales , Reacciones Antígeno-Anticuerpo , Dicroismo Circular , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Guanidina/química , Concentración de Iones de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Mesocricetus , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Priones/inmunología , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29-231). Using Cu2+/ascorbate, we oxidized SHaPrP(29-231) in vitro. Oxidation was demonstrated by liquid chromatography/mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29-101 region, which includes the histidine-containing octarepeats, is particularly affected by oxidation. Oxidation was time- and copper concentration-dependent and was evident with copper concentrations as low as 1 microM. Concomitant with oxidation, SHaPrP(29-231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37 degrees C with a 6-fold molar excess of Cu2+. These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.
Asunto(s)
Cobre , Priones/química , Priones/metabolismo , Secuencia de Aminoácidos , Ácido Ascórbico , Catálisis , Cromatografía Líquida de Alta Presión , Humanos , Cinética , Espectrometría de Masas , Modelos Químicos , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/química , Priones/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraAsunto(s)
Enfermedades Neurodegenerativas , Enfermedades por Prión , Factores de Edad , Diagnóstico Diferencial , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/epidemiología , Enfermedades Neurodegenerativas/terapia , Enfermedades por Prión/diagnóstico , Enfermedades por Prión/epidemiología , Enfermedades por Prión/prevención & control , Enfermedades por Prión/transmisión , Priones/química , Priones/clasificación , Priones/patogenicidadRESUMEN
It is hypothesized that infectious prions are generated as the cellular form of the prion protein (PrP(C)) undergoes pronounced conformational change under the direction of an infectious PrP(Sc) template. Conversion to the infectious conformer is particularly associated with major structural rearrangement in the central portion of the protein (residues 90-120), which has an extended flexible structure in the PrP(C) isoform. Using a panel of recombinant antibodies reactive with different parts of PrP, we show that equivalent major structural rearrangements occur spontaneously in this region of PrP immobilized on a surface. In contrast, regions more towards the termini of the protein remain relatively unaltered. The rearrangements occur even under conditions where individual PrP molecules should not contact one another. The propensity of specific unstructured regions of PrP to spontaneously undergo large and potentially deleterious conformational changes may have important implications for prion biology.
Asunto(s)
Proteínas PrPC/química , Proteínas PrPSc/química , Animales , Anticuerpos Monoclonales/inmunología , Ratones , Proteínas PrPC/inmunología , Proteínas PrPSc/inmunología , Conformación Proteica , Factores de TiempoRESUMEN
The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric alpha-helical or oligomeric beta-sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the beta-rich isoform of MoPrP is thermodynamically more stable than the native alpha-helical isoform. The conformational transition from the alpha-helical to beta-rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the alpha-helical isoform and folds directly to the thermodynamically more stable beta-rich isoform. Our data demonstrate that the folding of the prion protein to its native alpha-helical monomeric conformation is under kinetic control.
Asunto(s)
Priones/química , Dicroismo Circular , Cinética , Conformación Proteica , Pliegue de Proteína , TermodinámicaRESUMEN
The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The large size of the N-linked sugars, together with their dynamic properties, enables them to shield two orthogonal faces of the protein almost completely. Thus, the sugars can protect large regions of the protein surface from proteases and from nonspecific protein-protein interactions. Immunoprecipitation of prion protein with calnexin suggests that in the ER the oligosaccharides may provide a route for protein folding via the calnexin pathway. Major questions relate to the relevance of the glycoform distribution (as defined by glycan site occupancy) to strain type and disease transmission. Glycan analysis has shown that prion protein contains at least 52 different sugars, that these consist of a subset of brain sugars, and that there is site specific glycan processing. PrP(Sc) from the brains of Syrian hamsters contains the same set of glycans as PrP(C), but a higher proportion of tri- and tetra-antennary sugars. This may be attributed to a decrease in the activity of GnTIII. The GPI anchor, which is modified with sialic acid, may allow the prion protein to be mobile in the lipid bilayer. Potentially, this provides a possible means for translocating the prions from one cell to another.
Asunto(s)
Carbohidratos/fisiología , Glicoproteínas/química , Glicoproteínas/fisiología , Priones/química , Priones/fisiología , Termodinámica , Animales , Conformación de Carbohidratos , Carbohidratos/química , Humanos , Modelos Moleculares , Enfermedades por Prión/metabolismo , Relación Estructura-ActividadRESUMEN
A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.
Asunto(s)
Proteínas PrPC/química , Termodinámica , Animales , Anisotropía , Cricetinae , Difusión , Mesocricetus , Modelos Químicos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Proteínas PrPSc/química , Enfermedades por Prión/virología , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , SolucionesRESUMEN
Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrP(Sc), the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529-14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 microg of PPI generation 4.0/ml for 4 weeks not only reduced PrP(Sc) to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the beta-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrP(Sc) to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrP(Sc) from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrP(Sc) from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrP(Sc) clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.
Asunto(s)
Poliaminas/farmacología , Enfermedades por Prión/tratamiento farmacológico , Priones/metabolismo , Humanos , Neuroblastoma/patología , Poliaminas/metabolismo , Poliaminas/uso terapéutico , Conformación Proteica , Desnaturalización Proteica , Especificidad de la Especie , Células Tumorales CultivadasRESUMEN
Protein refolding/misfolding to an alternative form plays an aetiologic role in many diseases in humans, including Alzheimer's disease, the systemic amyloidoses, and the prion diseases. Here we have discovered that such refolding can occur readily for a simple lattice model of proteins in a propagatable manner without designing for any particular alternative native state. The model uses a simple contact energy function for interactions between residues and does not consider the peculiarities of polypeptide geometry. In this model, under conditions where the normal (N) native state is marginally stable or unstable, two chains refold from the N native state to an alternative multimeric energetic minimum comprising a single refolded conformation that can then propagate itself to other protein chains. The only requirement for efficient propagation is that a two-faced mode of packing must be in the ground state as a dimer (a higher-energy state for this packing leads to less efficient propagation). For random sequences, these ground-state dimeric configurations tend to have more beta-sheet-like extended structure than almost any other sort of dimeric ground-state assembly. This implies that propagating states (such as for prions) are beta-sheet rich because the only likely propagating forms are beta-sheet rich. We examine the details of our simulations to see to what extent the observed properties of prion propagation can be predicted by a simple protein folding model. The formation of the alternative state in the present model shows several distinct features of amyloidogenesis and of prion propagation. For example, an analog of the phenomenon of conformationally distinct strains in prions is observed. We find a parallel between 'glassy' behavior in liquids and the formation of a propagatable state in proteins. This is the first report of simulation of conformational propagation using any heteropolymer model. The results imply that some (but not most) small protein sequences must maintain a sequence signal that resists refolding to propagatable alternative native states and that the ability to form such states is not limited to polypeptides (or reliant on regular hydrogen bonding per se) but can occur for other protein-like heteropolymers.
Asunto(s)
Amiloide/química , Modelos Moleculares , Priones/química , Pliegue de Proteína , Secuencia de Aminoácidos , Amiloide/metabolismo , Fenómenos Químicos , Química Física , Mutación Puntual , Priones/metabolismo , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrP(Sc)). To explore this postulate, we examined the properties of PrP(Sc) from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrP(Sc) molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrP(Sc) as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have approximately 75-d incubation periods; a concentration of 1.08 M was found for the DY strain with a approximately 170-d incubation period and approximately 1.25 M for the SHa(RML) and 139H isolates with approximately 180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a approximately 320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrP(Sc) conformers encipher the biological properties of prion strains.