RESUMEN
Significant outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (C for cellular), to a toxic and infectious form, PrP(Sc) (Sc for scrapie). None of the prionoses currently have an effective treatment. Some forms of prion disease are thought to be spread by oral ingestion of PrP(Sc), such as chronic wasting disease and variant Creutzfeldt-Jakob disease. Attempts to obtain an active immunization in wild-type animals have been hampered by auto-tolerance to PrP and potential toxicity. Previously, we demonstrated that it is possible to overcome tolerance and obtain a specific anti-PrP antibody response by oral inoculation of the PrP protein expressed in an attenuated Salmonella vector. This past study showed that 30% of vaccinated animals were free of disease more than 350 days post-challenge. In the current study we have both optimized the vaccination protocol and divided the vaccinated mice into low and high immune responder groups prior to oral challenge with PrP(Sc) scrapie strain 139A. These methodological refinements led to a significantly improved therapeutic response. 100% of mice with a high mucosal anti-PrP titer immunoglobulin (Ig) A and a high systemic IgG titer, prior to challenge, remained without symptoms of PrP infection at 400 days (log-rank test P<0.0001 versus sham controls). The brains from these surviving clinically asymptomatic mice were free of PrP(Sc) infection by Western blot and histological examination. These promising findings suggest that effective mucosal vaccination is a feasible and useful method for overcoming tolerance to PrP and preventing prion infection via an oral route.
Asunto(s)
Anticuerpos/sangre , Priones/inmunología , Scrapie/prevención & control , Vacunas/administración & dosificación , Administración Oral , Animales , Western Blotting , Encéfalo/patología , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina A/sangre , Inmunoglobulina A/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Scrapie/patología , Vacunación/métodos , Vacunas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Gerstmann-Sträussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Sträussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.
Asunto(s)
Amiloide/genética , Enfermedad de Gerstmann-Straussler-Scheinker/etiología , Fragmentos de Péptidos/aislamiento & purificación , Priones/patogenicidad , Precursores de Proteínas/genética , Adulto , Alelos , Corteza Cerebral/patología , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Heterocigoto , Humanos , Masculino , Metionina/genética , Proteínas Priónicas , Priones/aislamiento & purificación , Análisis de Secuencia de Proteína , Síndrome , Valina/genéticaRESUMEN
BACKGROUND: Transmissible spongiform encephalopathies are associated with a structural transition in the prion protein that results in the conversion of the physiological PrPc to pathological PrP(Sc). We investigated whether this conformational transition can be inhibited and reversed by peptides homologous to the PrP fragments implicated in the abnormal folding, which contain specific residues acting as beta-sheet blockers (beta-sheet breaker peptides). METHODS: We studied the effect of a 13-residue beta-sheet breaker peptide (iPrP13) on the reversion of the abnormal structure and properties of PrP(Sc) purified from the brains of mice with experimental scrapie and from human beings affected by sporadic and variant Creutzfeldt-Jakob disease. In a cellular model of familial prion disease, we studied the effect of the peptide in the production of the abnormal form of PrP in intact cells. The influence of the peptide on prion infectivity was studied in vivo by incubation time assays in mice with experimental scrapie. FINDINGS: The beta-sheet breaker peptide partly reversed in-vitro PrP(Sc) to a biochemical and structural state similar to that of PrPc. The effect of the peptide was also detected in intact cells. Treatment of prion infectious material with iPrP13 delayed the appearance of clinical symptoms and decreased infectivity by 90-95% in mice with experimental scrapie. INTERPRETATION: Beta-sheet breaker peptides reverse PrP conformational changes implicated in the pathogenesis of spongiform encephalopathies. These peptides or their derivatives provide a useful tool to study the role of PrP conformation and might represent a novel therapeutic approach for prion-related disorders.
Asunto(s)
Priones/efectos de los fármacos , Proteínas/farmacología , Animales , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Enfermedades por Prión/metabolismo , Priones/química , Estructura Secundaria de Proteína , Proteínas/química , Scrapie/metabolismoRESUMEN
We have investigated the proteolytic cleavage of the cellular (PrPC) and pathological (PrPSc) isoforms of the human prion protein (PrP) in normal and prion-affected brains and in tonsils and platelets from neurologically intact individuals. The various PrP species were resolved after deglycosylation according to their electrophoretic mobility, immunoreactivity, Sarkosyl solubility, and, as a novel approach, resistance to endogenous proteases. First, our data show that PrPC proteolysis in brain originates amino-truncated peptides of 21 to 22 and 18 (C1) kd that are similar in different regions and are not modified by the PrP codon 129 genotype, a polymorphism that affects the expression of prion disorders. Second, this proteolytic cleavage of PrPC in brain is blocked by inhibitors of metalloproteases. Third, differences in PrPC proteolysis, and probably in Asn glycosylation and glycosylphosphatidylinositol anchor composition, exist between neural and non-neural tissues. Fourth, protease-resistant PrPSc cores in sporadic Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker F198S disease brains all have an intact C1 cleavage site (Met111-His112), which precludes disruption of a domain associated with toxicity and fibrillogenesis. Fifth, the profile of endogenous proteolytic PrPSc peptides is characteristic of each disorder studied, thus permitting the molecular classification of these prion diseases without the use of proteinase K and even a recognition of PrPSc heterogeneity within type 2 CJD patients having different codon 129 genotype and neuropathological phenotype. This does not exclude the role of additional factors in phenotypic expression; in particular, differences in glycosylation that may be especially relevant in the new variant CJD. Proteolytic processing of PrP may play an important role in the neurotropism and phenotypic expression of prion diseases, but it does not appear to participate in disease susceptibility.
Asunto(s)
Enfermedad de Alzheimer/patología , Síndrome de Creutzfeldt-Jakob/patología , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Neuronas/química , Proteínas PrPC/análisis , Proteínas PrPSc/análisis , Anciano , Enfermedad de Alzheimer/genética , Plaquetas/química , Encéfalo/patología , Química Encefálica , Codón , Síndrome de Creutzfeldt-Jakob/genética , Genotipo , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Glicosilación , Humanos , Metaloendopeptidasas/metabolismo , Persona de Mediana Edad , Tonsila Palatina/química , Fragmentos de Péptidos/análisis , Mapeo PeptídicoRESUMEN
In hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), a genetic variant (E22Q) of amyloid beta (Abeta) accumulates predominantly in the small vessels of leptomeninges and cerebral cortex, leading to fatal strokes in the fifth or sixth decade of life. Abeta deposition in the neuropil occurs mainly in the form of preamyloid, Congo red negative deposits, while mature neuritic plaques and neurofibrillary tangles, hallmark lesions in Alzheimer's disease (AD), are characteristically absent. A recent hypothesis regarding the pathogenesis of AD states that Abeta extending to residues 42-43 (as opposed to shorter species) can seed amyloid formation and trigger the development of neuritic plaques followed by neuronal damage in AD. We characterized biochemically and immunohistochemically Abeta from three cases of HCHWA-D to determine its length in vascular and parenchymal deposits. Mass spectrometry of formic acid-soluble amyloid, purified by size-exclusion gel chromatography, showed that Abeta 1-40 and its carboxyl-terminal truncated derivatives were the predominant forms in leptomeningeal and cortical vessels. Abeta 1-42 was a minor component in these amyloid extracts. Immunohistochemistry with antibodies S40 and S42, specific for Abeta ending at Val-40 or Ala-42, respectively, were consistent with the biochemical data from vascular amyloid. In addition, parenchymal preamyloid lesions were specifically stained with S42 and were not labeled by S40, in agreement with the pattern reported for AD, Down's syndrome, and aged dogs. Our results suggest that in HCHWA-D the carboxyl-terminal Abeta heterogeneity is due to limited proteolysis in vivo. Moreover, they suggest that Abeta species ending at Ala-42 may not be critical for the seeding of amyloid formation and the development of AD-like neuritic changes.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/fisiología , Amiloidosis/complicaciones , Hemorragia Cerebral/complicaciones , Fragmentos de Péptidos/fisiología , Animales , Western Blotting , Cromatografía en Gel , Perros , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de Masas , Meninges/químicaRESUMEN
Gerstmann-Sträussler-Scheinker (GSS) disease is a cerebral prion protein (PrP) amyloidosis associated with mutations in the PrP gene (PRNP). A GSS disease variant with mutation at codon 198 (F198S) has been studied in a large Indiana kindred. Biochemical investigations showed that the amyloid protein consists of 11 and 7 kDa fragments of PrP. Immunohistochemical studies showed that in addition to amyloid, these patients accumulate PrP deposits which are neither fluorescent nor birefringent when stained with thioflavin S and Congo red. In the present paper, we analyzed proteinase-K (PK)-resistant PrP in 7 patients with GSS F198S disease. Immunoblots of PK-treated brain extracts show prominent bands of ca. 27-29, 18-19, and 8 kDa. Immunohistochemistry and thioflavin-S-fluorescence show that the amyloid deposits are conspicuous in the cerebellum but sparse in the caudate nucleus. However, immunoblot analysis reveals PK-resistant PrP bands of similar intensity in both regions. Treatment with PK and PNGase F generates a pattern similar to that of PK alone. Our findings suggest that brain extracts from GSS F198S disease contain 3 prominent nonglycosylated PK-resistant PrP fragments forming a pattern not previously described in other prion diseases, which may in part explain the pathology of this GSS disease variant.
Asunto(s)
Endopeptidasa K/farmacología , Enfermedad de Gerstmann-Straussler-Scheinker/metabolismo , Priones/efectos de los fármacos , Priones/metabolismo , Adulto , Anciano , Amiloide/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Resistencia a Medicamentos , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Humanos , Immunoblotting , Isomerismo , Persona de Mediana Edad , Fracciones Subcelulares/metabolismoRESUMEN
Prion diseases are neurodegenerative disorders characterized by the accumulation of abnormal isoforms of prion protein (PrPSc) in the central nervous system. PrPSc isoforms differ from their normal homologue (PrPC), in that they possess increased beta-sheet conformation, are partially protease resistant and may be associated with amyloid deposition. Amyloid proteins are thought to derive from soluble precursors or fragments thereof, present in biological fluids, which in the disease state undergo conformational change leading to aggregation and deposition in target tissues. We report here that platelets carry PrP mRNA and release PrPC, a sialoglycoprotein bound to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Soluble PrPC, and a N-terminal truncated PrPC isoform starting at position 90 are secreted by resting and agonist-stimulated platelets and are detectable after partial deglycosylation of releasates. N-terminal sequence analysis of the soluble 27-30 kDa isoform, GQGGGTHSQ(W)NKP, revealed homology to scrapie PrP27-30, the protease resistant core derived from PrPSc. These findings indicate that in addition to PrPC, platelets process a soluble PrP27-30 isoform. Whether this isoform can be converted in scrapie PrP27-30 remains to be determined.
Asunto(s)
Plaquetas/fisiología , Proteína PrP 27-30/sangre , Priones/sangre , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/química , Reacción en Cadena de la Polimerasa , Proteína PrP 27-30/química , Priones/química , ARN/sangre , Scrapie , Homología de Secuencia de AminoácidoRESUMEN
The prion protein (PrP) plays an essential role in the pathogenesis of a group of sporadic, genetically determined and infectious fatal degenerative diseases, referred to as "prion diseases", affecting the central nervous system of humans and other mammals. The cellular PrP is encoded by a single copy gene, highly conserved across mammalian species. In prion diseases, PrP undergoes conformational changes involving a shift from alpha-helix to beta-sheet structure. This conversion is important for PrP amyloidogenesis, which occurs to the highest degree in the genetically determined Gerstmann-Sträussler-Scheinker disease (GSS) and prion protein cerebral amyloid angiopathy (PrP-CAA), while it is less frequently seen in other prion diseases. GSS and PrP-CAA are associated with point mutations of the prion protein gene (PRNP); these conditions show a broad spectrum of clinical presentation, the main signs being ataxia, spastic paraparesis, extrapyramidal signs and dementia. In GSS, parenchymal amyloid may be associated with spongiform changes or neurofibrillary lesions; in PrP-CAA, vascular amyloid is associated with neurofibrillary lesions. A major component of the amyloid fibrils in the two diseases is a 7 kDa peptide, spanning residues 81-150 of PrP.
Asunto(s)
Amiloidosis/patología , Encefalopatías/genética , Encéfalo/patología , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Enfermedades por Prión/patología , Priones/química , Priones/genética , Secuencia de Aminoácidos , Amiloidosis/genética , Animales , Secuencia de Bases , Encefalopatías/patología , Codón , Secuencia Conservada , Femenino , Genotipo , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Humanos , Masculino , Mamíferos , Linaje , Mutación Puntual , Enfermedades por Prión/genética , Estructura Secundaria de Proteína , Scrapie/genética , Scrapie/patologíaRESUMEN
Alzheimer's amyloid beta-protein (A beta) is a modified, pathogenic form of a constitutive host protein, soluble amyloid beta-protein (sA beta). Both are conformational isomers encoded by the gene for the beta-amyloid precursor protein (APP), located on chromosome 21. sA beta and A beta have identical sequence but are thought to differ in their secondary structure and physicochemical properties, hence they are conformational isomers. sA beta is easily degraded, while A beta is particularly resistant. A beta has a high beta-pleated sheet content, while sA beta is thought to be more random-coil and/or alpha-helical. A beta, unlike sA beta, adopts an amyloidogenic conformation, forms aggregates and gives rise to fibrils. Most early-onset forms of Alzheimer's disease (AD) have been linked to mutations of the presenilin 1, presenilin 2 or APP genes, located on chromosomes 14, 1 and 21, respectively. Their relationship to amyloidogenesis is being investigated. On the other hand, the major risk factor for the most common form, sporadic and familial late-onset AD, is the presence of the apoE epsilon 4 allele. Recent studies have shown that a 10 kDa C-terminal fragment of apoE is complexed to A beta in neuritic plaques and that apoE isoforms can modulate amyloid formation in vitro. Moreover, thrombin cleavage of apoE generates a similar C-terminal fragment that can form amyloid-like fibrils. Thus neuritic plaques may contain both A beta and apoE amyloid fibrils. AD can be neuropathologically defined by the presence of several interacting proteins that can adopt an amyloidogenic conformation. This has led us to hypothesize that in AD, amyloidosis may be reactive rather than causative.
Asunto(s)
Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/metabolismo , Amiloidosis/etiología , Apolipoproteínas E/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Péptidos beta-Amiloides/genética , Amiloidosis/genética , Amiloidosis/metabolismo , Apolipoproteínas E/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Prealbúmina/genética , Prealbúmina/metabolismoRESUMEN
Deposition of PrP amyloid in cerebral vessels in conjunction with neurofibrillary lesions is the neuropathologic hallmark of the dementia associated with a stop mutation at codon 145 of PRNP, the gene encoding the prion protein (PrP). In this disorder, the vascular amyloid in tissue sections and the approximately 7.5-kDa fragment extracted from amyloid are labeled by antibodies to epitopes located in the PrP sequence including amino acids 90-147. Amyloid-laden vessels are also labeled by antibodies against the C terminus, suggesting that PrP from the normal allele is involved in the pathologic process. Abundant neurofibrillary lesions are present in the cerebral gray matter. They are composed of paired helical filaments, are labeled with antibodies that recognize multiple phosphorylation sites in tau protein, and are similar to those observed in Alzheimer disease. A PrP cerebral amyloid angiopathy has not been reported in diseases caused by PRNP mutations or in human transmissible spongiform encephalopathies; we propose to name this phenotype PrP cerebral amyloid angiopathy (PrP-CAA).
Asunto(s)
Angiopatía Amiloide Cerebral/patología , Demencia/etiología , Mutación , Priones/genética , Adulto , Amiloide/química , Vasos Sanguíneos/patología , Corteza Cerebral/patología , Femenino , Humanos , Japón/etnología , Neuronas/patología , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Priones/inmunologíaRESUMEN
Apolipoprotein E (apoE) has been found in association with several different types of systemic and cerebral amyloid deposits and the presence of the epsilon 4 allele constitutes a risk factor for Alzheimer's disease. It has been shown that apoE binds and promotes the fibrillogenesis in vitro of Alzheimer's amyloid beta-peptide, suggesting an important role for apoE in the modulation of amyloidogenesis. Due to the co-localization of apoE with several biochemically distinct amyloid deposits, it has been proposed that apoE plays a general role modulating and/or participating in amyloidosis. In the present study, we show for the first time that apoE, isolated from human plasma, increases fibril formation of synthetic peptides comprising the amyloidogenic sequences of gelsolin amyloid related to familial amyloidosis Finnish type, and amyloid A found in secondary amyloidosis and familial Mediterranean fever. Our results suggest that apoE acts as a general pathological chaperone in various amyloidoses by enhancing the transition from soluble peptides into amyloid-forming, pathological molecules.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Apolipoproteínas E/farmacología , Gelsolina/farmacología , Neurofibrillas/efectos de los fármacos , Proteína Amiloide A Sérica/farmacología , Secuencia de Aminoácidos , Gelsolina/química , Humanos , Sustancias Macromoleculares , Microscopía Electrónica , Datos de Secuencia Molecular , Neurofibrillas/ultraestructura , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Proteína Amiloide A Sérica/química , Espectrometría de FluorescenciaRESUMEN
Apolipoprotein E (ApoE) immunoreactivity is consistently present in the senile plaques and neurofibrillary tangles of Alzheimer's disease (AD) brain. In vitro, apoE, and in particular its apoE4 isoform, can bind to and promote fibrillogenesis of the amyloid A beta peptide, the main constituent of senile plaques. These findings, together with the strong genetic association between late onset AD and the E4 allele of apoE, have strengthened the hypothesis that apoE may have a central role in the pathogenesis of AD by modulating A beta cerebral accumulation. However, apoE immunoreactivity is present in all cerebral and systemic amyloidoses tested, and tryptic apoE fragments have been identified in association with amyloid A (AA). In order to further elucidate the interaction between apoE and amyloids, we purified AA and amyloid L (AL) fibrils from patients with familial Mediterranean fever and primary amyloidosis, respectively, and studied the association of apoE with AA and AL proteins. In each case, apoE fragments, detected by Western blot, co-purified with the amyloid fibrils. Microsequencing analysis identified COOH-terminal fragments of apoE, similar to the 10-kDa fragment produced by thrombin digestion that contains the purported binding region to A beta. In vitro co-incubation of AA with purified human apoE resulted in the formation of an SDS-resistant AA.apoE complex and a higher degree of polymerization of the AA peptide. These findings and similar results obtained from AD senile plaques suggest that 1) the carboxyl-terminal fragment of apoE is complexed to amyloid fibrils and resists proteolysis in vivo and 2) apoE may promote amyloidogenesis through a conformation-dependent interaction regardless of the primary structure of the amyloid precursors.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Amiloide/química , Apolipoproteínas E/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Amiloide/biosíntesis , Humanos , Datos de Secuencia MolecularRESUMEN
A central event in Alzheimer's disease is the conformational change from normally circulating soluble amyloid beta peptides (A beta) and tau proteins into amyloid fibrils, in the form of senile plaques and neurofibrillary tangles respectively. The apolipoprotein E (apoE) gene locus has recently been associated with late-onset Alzheimer's disease. It is not know whether apoE plays a direct role in the pathogenesis of the disease. In the present work we have investigated whether apoE can affect the known spontaneous in vitro formation of amyloid-like fibrils by synthetic A beta analogues using a thioflavine-T assay for fibril formation, electron microscopy and Congo Red staining. Our results show that, under the conditions used, apoE directly promotes amyloid fibril formation, increasing both the rate of fibrillogenesis and the total amount of amyloid formed. ApoE accelerated fibril formation of both wild-type A beta-(1-40) and A beta-(1-40A), an analogue created by the replacement of valine with alanine at residue 18, which alone produces few amyloid-like fibrils. However, apoE produced only a minimal effect on A beta-(1-40Q), found in the Dutch variant of Alzheimer's disease. When recombinant apoE isoforms were used, apoE4 was more efficient than apoE3 at enhancing amyloid formation. These in vitro observations support the hypothesis that apoE acts as a pathological chaperone, promoting the beta-pleated-sheet conformation of soluble A beta into amyloid fibres, and provide a possible explanation for the association of the apoE4 genetic isoform with Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Apolipoproteínas E/farmacología , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Apolipoproteínas E/química , Apolipoproteínas E/genética , Rojo Congo , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Conformación Proteica , Proteínas Recombinantes/farmacología , Coloración y EtiquetadoRESUMEN
Gerstmann-Sträussler-Scheinker (GSS) disease is a cerebral amyloidosis linked to mutations of the PRNP gene. We previously reported that the amyloid protein in the Indiana kindred of GSS is an internal fragment of prion protein (PrP). To investigate whether this fragment originates only from mutant or from both mutant and wild-type PrP, we have characterized amyloid proteins purified from patients of the Indiana and Swedish GSS families. These patients were heterozygous for the Met-Val polymorphism at PRNP codon 129 and carried a mutation at PRNP codon 198 (Phe-->Ser) and codon 217 (Gln-->Arg), respectively. The smallest amyloid subunit was a 7 kDa peptide spanning residues approximately 81 to approximately 150 in the Indiana patient and approximately 81 to approximately 146 in the Swedish patient. In both patients, only Val was present at position 129. Since Val-129 was in coupling phase with Ser-198 and Arg-217, our findings indicate that only the mutant PrP is involved in amyloid formation in both kindreds.
Asunto(s)
Amiloide/biosíntesis , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Mutación Puntual , Priones/biosíntesis , Priones/genética , Secuencia de Aminoácidos , Amiloide/genética , Amiloide/aislamiento & purificación , Secuencia de Bases , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Codón/genética , Cartilla de ADN , Genotipo , Enfermedad de Gerstmann-Straussler-Scheinker/metabolismo , Humanos , Immunoblotting , Indiana , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Priones/aislamiento & purificación , SueciaRESUMEN
Gerstmann-Sträussler-Scheinker disease (GSS) is a prion-related encephalopathy pathologically characterized by massive deposition of prion protein (PrP) amyloid in the central nervous system. The major component of amyloid fibrils isolated from patients of the Indiana kindred of GSS (GSS-Ik) is an 11-kDa fragment of PrP spanning residues 58 to approximately 150. These patients carry a missense mutation of the PRNP gene, causing a Phe-->Ser substitution at codon 198. We investigated fibrillogenesis in vitro by using synthetic peptides homologous to consecutive segments of GSS-Ik amyloid protein (residues 57-64, 89-106, 106-126, and 127-147) as well as peptides from the PrP region with the GSS-Ik mutation (residues 191-205 and 181-205, both wild type and mutant). Peptide PrP-(106-126) formed straight fibrils similar to those extracted from GSS brains, whereas peptide PrP-(127-147) formed twisted fibrils resembling scrapie-associated fibrils isolated from subjects with transmissible spongiform encephalopathies. Congo red staining and x-ray fibril diffraction showed that both straight and twisted fibrils had tinctorial and conformational properties of native amyloid. Conversely, the other peptides did not form amyloid-like fibrils under similar conditions. These findings suggest that the sequence spanning residues 106-147 of PrP is central to amyloid fibril formation in GSS and related encephalopathies.
Asunto(s)
Amiloide/química , Proteínas del Tejido Nervioso/química , Priones/química , Secuencia de Aminoácidos , Neuropatías Amiloides/patología , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Microscopía Electrónica , Datos de Secuencia Molecular , Péptidos/química , Polímeros , Proteínas PrPSc , SolubilidadRESUMEN
Gerstmann-Sträussler-Scheinker disease in the Indiana kindred is pathologically characterized by prion protein amyloid deposits and neurofibrillary tangles (NFT) with paired helical filaments (PHF). Using antibodies to various domains of the tau molecule, we investigated the composition of PHF in this family by immunocytochemistry and immunoblot analysis. The results indicate that A68 is a component of NFT in this family as it is in Alzheimer's disease, and suggest that post-translational modifications of tau leading to formation of A68 are not unique to Alzheimer's disease.
Asunto(s)
Corteza Cerebral/patología , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Proteínas del Tejido Nervioso/análisis , Ovillos Neurofibrilares/patología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Corteza Cerebral/ultraestructura , Enfermedad de Gerstmann-Straussler-Scheinker/genética , Humanos , Immunoblotting , Inmunohistoquímica , Indiana , Persona de Mediana Edad , Ovillos Neurofibrilares/ultraestructura , Proteínas tauRESUMEN
The amino acid sequence corresponding to the V region H chain gene used by three monoclonal IgM directed to the myelin-associated glycoprotein (MAG) is presented. They all belonged to the VHIII variability subgroup, but each may well represent a new member of this family inasmuch as their homology with previously sequenced VHIII genes was less than 80%. Strikingly, there was no greater homology between the H chain V regions of the anti-MAG IgM. Partial amino acid sequence data indicated that these V regions were joined to as yet unidentified DH segments; however, two H chains used very similar DH, possibly indicating that this sequence was involved in the fine specificity of the IgM for MAG. All H chains included a JHIV region. These data, together with results obtained from the sequence of the three kappa L chains of the same IgM molecules (Mihaesco, E., H. Ayadi, N. Congy, M. C. Gendron, J. P. Roy, H. Heyermann, B. Frangione, and J. C. Brouet. 1989. J. Biol. Chem. 264:21481), indicate that the repertoire of VL and VH gene segments used by anti-MAG IgM is quite diverse, in contrast to previous structural data obtained for other human monoclonal IgM autoantibodies. Possibly, these differences reflect distinct pathogenesis.
Asunto(s)
Anticuerpos Monoclonales/genética , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/genética , Región Variable de Inmunoglobulina/genética , Proteínas de la Mielina/inmunología , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Glicoproteína Asociada a Mielina , Homología de Secuencia de Ácido NucleicoRESUMEN
The cellular prion protein (PrPc) is a 33-35 kDa sialoglycoprotein anchored to the external surface of neural and non-neural cells by a glycosyl phosphatidylinositol moiety. In addition, a secretory form of PrPc has been found in cell-free translation systems and in cell cultures. On this basis, we investigated human cerebrospinal fluid for the presence of soluble PrP and identified a protein whose molecular weight, antigenic determinants, N-terminal amino acid sequence and sensitivity to protease digestion corresponded to those of PrPc. In prion-related encephalopathies of humans and animals, the secretory form of PrPc might be converted into the abnormal isoform PrPSc and play a role in the dissemination of the disease process and amyloid formation.