RESUMEN
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
The familial amyloidotic polyneuropathy is strictly associated with point mutations in the coding region of the transthyretin gene. Here, we focused on the mutations in the monomer-monomer and dimer-dimer interaction site of the transthyretin tetramer. The naturally occurring amyloidogenic Tyr114His (Y114H) and Tyr116Ser (Y116S) variants formed more amyloid fibrils than the wild-type transthyretin, nonamyloidogenic Tyr116Val (Y116V) variant, and other amyloidogenic variants in previous studies. The secondary, tertiary, and quaternary structural stabilities of the Y114H and Y116S variants were compared with those of the wild-type transthyretin and nonamyloidogenic Y116V variant. The unfolding data indicated that the amyloidogenic Y114H and Y116S mutations reduced the stability of the secondary, tertiary, and quaternary structure. Our results also indicated that the unfolding of Y114H and Y116S is less cooperative than that of the wild-type transthyretin. Moreover, the tetramer of the amyloidogenic variants dissociated to the monomer even at pH 7.0, indicating the importance of Tyr114 and Tyr116 in strengthening the contacts between monomers and/or dimers of the transthyretin molecule.