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
Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.
Asunto(s)
Citocinas/química , Glicina/química , Proteínas de Insectos/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Bioensayo , Proliferación Celular , Citocinas/metabolismo , Eliminación de Gen , Proteínas de Insectos/metabolismo , Insectos , Lepidópteros , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Péptidos/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Sustitución de Aminoácidos , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/metabolismo , Prealbúmina/química , Prealbúmina/genética , Amiloide/genética , Amiloide/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Variación Genética , Histidina/genética , Humanos , Concentración de Iones de Hidrógeno , Prealbúmina/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Cuaternaria de Proteína/genética , Serina/genética , Espectrometría de Fluorescencia , Triptófano/química , Tirosina/genética , Urea/químicaRESUMEN
Transthyretin is an amyloidogenic protein that causes human amyloid polyneuropathy and senile systemic amyloidosis as a result of the deposition of normal and/or mutant transthyretin in the form of amyloid fibrils. A high-expression plasmid of human transthyretin was constructed in order to facilitate the study of amyloid fibril formation of this protein. The transthyretin gene was constructed by an assembly of eight chemically synthesized oligonucleotides and amplified by polymerase chain reaction, and the amplified gene was inserted into an Escherichia coli expression vector. The expression plasmid was transformed into M15 cells and the gene product was expressed as a polyhistidine-tagged fusion protein. Purified recombinant transthyretin was obtained by one-step nickel chelation affinity chromatography and the production level of the protein was 130mg per 1L of culture. Furthermore, the expressed protein showed the same characteristics in terms of tetramer formation at neutral pH and amyloid formation at acidic pH as did the authentic human transthyretin. This system will enable biophysical and structural studies of this protein to be advanced.
Asunto(s)
Escherichia coli , Genes Sintéticos/genética , Prealbúmina/genética , Prealbúmina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Expresión Génica , Ingeniería Genética , Histidina , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Prealbúmina/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.
