RESUMEN
Characterization of small oligomers formed at an early stage of amyloid formation is critical to understanding molecular mechanism of pathogenic aggregation process. Here we identified and characterized cytotoxic oligomeric intermediates populated during transthyretin (TTR) aggregation process. Under the amyloid-forming conditions, TTR initially forms a dimer through interactions between outer strands. The dimers are then associated to form a hexamer with a spherical shape, which serves as a building block to self-assemble into cytotoxic oligomers. Notably, wild-type (WT) TTR tends to form linear oligomers, while a TTR variant (G53A) prefers forming annular oligomers with pore-like structures. Structural analyses of the amyloidogenic intermediates using circular dichroism (CD) and solid-state NMR reveal that the dimer and oligomers have a significant degree of native-like ß-sheet structures (35-38%), but with more disordered regions (~60%) than those of native TTR. The TTR variant oligomers are also less structured than WT oligomers. The partially folded nature of the oligomeric intermediates might be a common structural property of cytotoxic oligomers. The higher flexibility of the dimer and oligomers may also compensate for the entropic loss due to the oligomerization of the monomers.
Asunto(s)
Prealbúmina/metabolismo , Prealbúmina/toxicidad , Agregación Patológica de Proteínas , Desnaturalización Proteica , Multimerización de Proteína , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Prealbúmina/química , Conformación ProteicaRESUMEN
Single point mutations in the transthyretin (TTR) gene may cause a hereditary neurodegenerative disease termed familial amyloidotic polyneuropathy (FAP) due to accelerated deposition of amyloid fibrils, resulting in peripheral and autonomic nervous system dysfunction. Recently, we found a Chinese FAP family involving a TTR V30A mutation. To understand the pathogenic mechanisms of this V30A TTR, we investigated the effects of this mutation on TTR quaternary and tertiary structural stabilities and cytotoxicities against neuroblastoma cells along with the most common variant V30M TTR and the wild-type (WT) TTR. Our results showed that the V30A mutation impaired the thermodynamic and kinetic stabilities of the TTR protein by increasing the extent and rate of tetramer dissociation and unfolded monomer and amyloid fibril formation, even to a greater extent than the V30M mutation under several experimental conditions. Further, an obviously cytotoxic effect of the V30A TTR on the human neuroblastoma cell line, IMR-32, was observed. The V30A TTR induced apoptosis and autophagy concomitant with the accumulation of reactive oxygen species (ROS) and DNA double-strand breaks, reflected in the induction of phosphor-H2A.X. These results suggest that the V30A mutation in the TTR gene promotes the formation of unfolded monomers and amyloid fibrils, which potentially contribute to the increased neurotoxicity and the pathology associated with this FAP family.
Asunto(s)
Prealbúmina/química , Amiloide/metabolismo , Apoptosis , Autofagia , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Histonas/metabolismo , Humanos , Cinética , Simulación de Dinámica Molecular , Mutación , Neuroblastoma , Fosforilación , Prealbúmina/genética , Prealbúmina/toxicidad , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Desplegamiento Proteico , Especies Reactivas de Oxígeno/metabolismo , TermodinámicaRESUMEN
The transthyretin amyloidoses are diseases of protein misfolding characterized by the extracellular deposition of fibrils and other aggregates of the homotetrameric protein transthyretin (TTR) in peripheral nerves, heart, and other tissues. Age is the major risk factor for the development of these diseases. We hypothesized that an age-associated increase in the level of protein oxidation could be involved in the onset of the senile forms of the TTR amyloidoses. To test this hypothesis, we have produced and characterized relevant age-related oxidative modifications of the wild type (WT) and the Val122Ile (V122I) TTR variant, both involved in cardiac TTR deposition in the elderly. Our studies show that methionine/cysteine-oxidized TTR and carbonylated TTR from either the WT or the V122I variant are thermodynamically less stable than their nonoxidized counterparts. Moreover, carbonylated WT and carbonylated V122I TTR have a stronger propensity to form aggregates and fibrils than WT and V122I TTR, respectively, at physiologically attainable pH values. It is well-known that TTR tetramer dissociation, the limiting step for aggregation and amyloid fibril formation, can be prevented by small molecules that bind the TTR tetramer interface. Here, we report that carbonylated WT TTR is less amenable to resveratrol-mediated tetramer stabilization than WT TTR. All the oxidized forms of TTR tested are cytotoxic to a human cardiomyocyte cell line known to be a target for cardiac-specific TTR variants. Overall, these studies demonstrate that age-related oxidative modifications of TTR can contribute to the onset of the senile forms of the TTR amyloidoses.
Asunto(s)
Amiloide/química , Amiloide/metabolismo , Amiloidosis/metabolismo , Prealbúmina/química , Prealbúmina/metabolismo , Factores de Edad , Envejecimiento , Amiloide/genética , Amiloide/toxicidad , Amiloidosis/epidemiología , Amiloidosis/genética , Amiloidosis/patología , Benzotiazoles , Línea Celular , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Oxidación-Reducción , Mutación Puntual , Prealbúmina/genética , Prealbúmina/toxicidad , Carbonilación Proteica , Multimerización de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Resveratrol , Estilbenos/farmacología , Tiazoles/metabolismoRESUMEN
The transthyretin amyloidoses are a subset of protein misfolding diseases characterized by the extracellular deposition of aggregates derived from the plasma homotetrameric protein transthyretin (TTR) in peripheral nerves and the heart. We have established a robust disease-relevant human cardiac tissue culture system to explore the cytotoxic effects of amyloidogenic TTR variants. We have employed this cardiac amyloidosis tissue culture model to screen 23 resveratrol analogs as inhibitors of amyloidogenic TTR-induced cytotoxicity and to investigate their mechanisms of protection. Resveratrol and its analogs kinetically stabilize the native tetramer preventing the formation of cytotoxic species. In addition, we demonstrate that resveratrol can accelerate the formation of soluble non-toxic aggregates and that the resveratrol analogs tested can bring together monomeric TTR subunits to form non-toxic native tetrameric TTR.
Asunto(s)
Amiloidosis/metabolismo , Antioxidantes/química , Antioxidantes/farmacología , Miocitos Cardíacos/efectos de los fármacos , Prealbúmina/antagonistas & inhibidores , Estilbenos/química , Estilbenos/farmacología , Línea Celular , Humanos , Miocitos Cardíacos/metabolismo , Prealbúmina/metabolismo , Prealbúmina/toxicidad , ResveratrolRESUMEN
Familial amyloidotic polyneuropathy (FAP) is linked to destabilising point mutations in the human plasma protein transthyretin (TTR). Consistent with similar amyloid disorders, low molecular weight TTR oligomers have been shown to exert the major cytotoxic effect. The amyloid structure of TTR contains non-native inter-molecular disulphide linkages via the cysteine at position 10 (Cys10). Moreover, substitution of Cys10 in a mouse model for TTR-amyloidosis abolishes TTR deposits, indicating an important role of Cys10 in FAP pathogenesis. However, the role of disulphide bridges in TTR cytotoxicity has not been elucidated. By probing Cys10Ser TTR variants to the human neuroblastoma SH-SY5Y cell line, we have addressed this question, and our results clearly show that formation of an inter-molecular disulphide bridge is not a pre-requisite for TTR cytotoxicity. This finding suggests that prevention of inter-molecular TTR disulphide bridges as a therapeutic intervention will not impair the cytotoxic potential of TTR.
Asunto(s)
Amiloide/química , Prealbúmina/química , Sustitución de Aminoácidos , Amiloide/toxicidad , Neuropatías Amiloides Familiares/etiología , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cisteína/química , Disulfuros/química , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/toxicidad , Mutagénesis Sitio-Dirigida , Prealbúmina/genética , Prealbúmina/toxicidad , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidadRESUMEN
We report the amyloid-like properties of Escherichia coli transthyretin-like protein (TLP). TLP is 32% homologous to human transthyretin (hTTR), and is also tetrameric. In contrast to hTTR, TLP does not bind thyroxine. TLP orthologues are found in several prokaryotes, lower eukaryotes and vertebrates. TLP carries a signal peptide that targets the protein to the periplasmic space. We found that TLP and hTTR tetramers dissociate into monomers under similar conditions, although TLP monomers have different association properties. Like hTTR, TLP forms aggregates, small fibrillar structures of 8 nm width, and annular structures of 8 nm diameter which present amyloid-like properties and are toxic to cells.
Asunto(s)
Amiloide/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Prealbúmina/química , Amiloide/toxicidad , Línea Celular Tumoral , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/toxicidad , Evolución Molecular , Humanos , Neuroblastoma , Prealbúmina/genética , Prealbúmina/toxicidad , Tiroxina/químicaRESUMEN
The amyloidoses are the extracellular subset of a group of diseases in which in vivo protein misfolding leads to a pathologic gain of function, i.e., aggregation leading to protein deposition, with subsequent tissue damage. Wild-type and mutant transthyretins (TTR) are the etiologic agents in prototypic systemic amyloidoses. We describe a cell-based assay that measures the cytotoxicity of physiologic concentrations of the amyloidogenic Val30Met TTR variant (V30M TTR) using cells of the same lineage as the in vivo tissue target of amyloid deposition. We have utilized the assay to screen small molecules for their capacity to inhibit the TTR-induced cell damage. We compared the inhibitory activity of each compound with its ability to prevent TTR fibril formation in vitro. Our results emphasize the importance of screening compounds under physiologic conditions. Moreover, if a common conformational intermediate is responsible for cell death in all the amyloid diseases, the cell-based assay has the potential to aid in the discovery of compounds useful in the treatment of amyloidoses caused by other misfolded proteins as well as those caused by TTR.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Prealbúmina/antagonistas & inhibidores , Prealbúmina/metabolismo , Amiloidosis/tratamiento farmacológico , Amiloidosis/genética , Amiloidosis/metabolismo , Línea Celular Tumoral , Linaje de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diflunisal/análogos & derivados , Diflunisal/farmacología , Evaluación Preclínica de Medicamentos , Humanos , Metionina/genética , Microfibrillas/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Prealbúmina/genética , Prealbúmina/toxicidad , Resveratrol , Estilbenos/farmacología , Valina/genéticaRESUMEN
Familial amyloidotic polyneuropathy is a hereditary autosomal-dominant disease in which the deposited transthyretin fibrils are derived from amyloidogenic mutation. We investigated structure and stability of a human Ser112Ile transthyretin variant and showed that the Ser112Ile variant exists as a dimer having nonnative tertiary structure at physiological pH. In addition, the dimeric Ser112Ile assembles into a spherical aggregate and exerts cytotoxicity in a human neuroblastoma cell line. Our results suggest the importance of an unstable dimeric structure in forming spherical aggregates that will induce cell death.
Asunto(s)
Variación Genética , Neurotoxinas/metabolismo , Prealbúmina/metabolismo , Procesamiento Proteico-Postraduccional , Acrilamida/química , Sustitución de Aminoácidos/genética , Naftalenosulfonatos de Anilina/química , Naftalenosulfonatos de Anilina/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cromatografía en Gel , Dimerización , Humanos , Isoleucina/genética , Microscopía de Fuerza Atómica , Nefelometría y Turbidimetría , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/toxicidad , Prealbúmina/genética , Prealbúmina/toxicidad , Prealbúmina/ultraestructura , Unión Proteica/genética , Conformación Proteica , Procesamiento Proteico-Postraduccional/genética , Estructura Cuaternaria de Proteína/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Serina/genética , Espectrometría de Fluorescencia/métodos , Termodinámica , Triptófano/químicaRESUMEN
In diseases like Alzheimer's disease and familial amyloidotic polyneuropathy (FAP) amyloid deposits co-localize with areas of neurodegeneration. FAP is associated with mutations of the plasma protein transthyretin (TTR). We can here show an apoptotic effect of amyloidogenic mutants of TTR on a human neuroblastoma cell line. Toxicity could be blocked by catalase indicating a free oxygen radical dependent mechanism. The toxic effect was dependent on the state of aggregation and unexpectedly mature fibrils from FAP-patients who failed to exert an apoptotic response. Morphological studies revealed a correlation between toxicity and the presence of immature amyloid. Thus, we can show that toxicity is associated with early stages of fibril formation and propose that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.
Asunto(s)
Neuropatías Amiloides Familiares/metabolismo , Péptidos beta-Amiloides/metabolismo , Apoptosis , Neuroblastoma/metabolismo , Fragmentos de Péptidos/metabolismo , Prealbúmina/toxicidad , Enfermedad de Alzheimer/metabolismo , Neuropatías Amiloides Familiares/genética , Péptidos beta-Amiloides/toxicidad , Apoptosis/efectos de los fármacos , Catalasa/farmacología , Citoprotección/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Etiquetado Corte-Fin in Situ , Mutagénesis Sitio-Dirigida , Neuroblastoma/patología , Fragmentos de Péptidos/toxicidad , Prealbúmina/genética , Prealbúmina/ultraestructura , Unión Proteica/fisiología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
We have expressed transthyretin (TTR) mutants which have significantly destabilised tetramers that aggregate into amyloid fibrils via a series of intermediates. We used atomic force microscopy to follow the morphology of aggregates during fibril formation. Initially, amorphous aggregates are formed that subsequently mature into fibrillar structures. This observation is interpreted as an optimisation of beta-strand registers. The rate of aggregation and maturation is highly temperature-dependent suggesting that entropic forces significantly contribute to stability. In addition, we identified a correlation between the presence of early formed aggregates of TTR and cytotoxicity. The toxic response was mediated via an apoptotic mechanism. The fact that early formed amorphous aggregates, but not more mature fibrils, exert a toxic response suggests that the rate of fibril formation may be a critical parameter. We propose that a slow rate of aggregation facilitates an increased concentration of a toxic intermediate.