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1.
Biopolymers ; 102(3): 260-72, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24615557

RESUMO

We previously characterized α3, a polypeptide that has a three times repeated sequence of seven amino acids (abcdefg: LETLAKA) and forms fibrous assemblies composed of amphipathic α-helices. Upon comparison of the amino acid sequences of α3 with other α-helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven α3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The α-helix forming abilities of the substituted polypeptides were less than that of α3. The polypeptides with amino acid substitutions at position g and the polypeptide KEα3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed ß-sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies.


Assuntos
Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Vermelho Congo , Microscopia Eletrônica de Transmissão , Microscopia de Polarização , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Cloreto de Sódio/química , Espectroscopia de Infravermelho com Transformada de Fourier , Ultracentrifugação
2.
Biochemistry ; 52(16): 2810-20, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23530905

RESUMO

Polypeptide α3 (21 residues), with three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms an amphipathic α-helix and a long fibrous assembly. Here, we investigated the ability of α3-series polypeptides (with 14-42 residues) of various chain lengths to form α-helices and fibrous assemblies. Polypeptide α2 (14 residues), with two same-sequence repeats, did not form an α-helix, but polypeptide α2L (15 residues; α2 with one additional leucine residue on its carboxyl terminal) did form an α-helix and fibrous assembly. Fibrous assembly formation was associated with polypeptides at least as long as polypeptide α2L and with five leucine residues, indicating that the C-terminal leucine has a critical element for stabilization of α-helix and fibril formation. In contrast, polypeptides α5 (35 residues) and α6 (42 residues) aggregated easily, although they formed α-helices. A 15-35-residue chain was required for fibrous assembly formation. Electron microscopy and X-ray fiber diffraction showed that the thinnest fibrous assemblies of polypeptides were about 20 Å and had periodicities coincident with the length of the α-helix in a longitudinal direction. These results indicated that the α-helix structures were orientated along the fibrous axis and assembled into a bundle. Furthermore, the width and length of fibrous assemblies changed with changes in the pH value, resulting in variations in the charged states of the residues. Our results suggest that the formation of fibrous assemblies of amphipathic α-helices is due to the assembly of bundles via the hydrophobic faces of the helices and extension with hydrophobic noncovalent bonds containing a leucine.


Assuntos
Peptídeos/química , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Peptídeos/metabolismo , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
3.
J Biochem ; 139(2): 271-8, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16452315

RESUMO

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.


Assuntos
Aminoácidos/química , Peptídeos/química , Estrutura Secundária de Proteína , Dicroísmo Circular , Engenharia Genética/métodos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Peptídeos/genética , Cloreto de Potássio/química , Conformação Proteica , Sensibilidade e Especificidade , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
4.
Protein Sci ; 24(5): 883-94, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25694229

RESUMO

We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)3, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)3, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)3, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)3; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation.


Assuntos
Peptídeos/química , Estrutura Secundária de Proteína , Sequências Repetitivas de Aminoácidos , Dicroísmo Circular , Concentração de Íons de Hidrogênio
5.
FEBS Lett ; 562(1-3): 155-9, 2004 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-15044017

RESUMO

We report a morphological study of functioning ribosomes in a efficient and robust cell-free protein synthesis system prepared from wheat embryos. Sucrose density gradient analysis of translated mixtures programmed with luciferase mRNAs having different 5' and 3' untranslated regions showed formation of large polysomes. Electron microscopic examination of translation mixtures programmed with those of capped and polyadenylated mRNA revealed that ribosomes assemble into a circular-type polysome in vitro. Furthermore, a series of experiments using mRNAs lacking either cap, poly(A) tail or both also resulted in the formation of circular polysomes, which are indistinguishable from those with the original mRNA. The wheat germ cell-free system may provide a good experimental system for understanding functional ribosomes at the molecular level.


Assuntos
Sistema Livre de Células , Proteínas de Plantas/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , Triticum/fisiologia , Proteínas de Plantas/genética , Polirribossomos/ultraestrutura , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Triticum/citologia
6.
J Comp Neurol ; 472(2): 246-56, 2004 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-15048691

RESUMO

In the course of evolution, the vomeronasal organ (VNO) first appeared in amphibians. To understand the relationship between the VNO and the vomeronasal receptors, we isolated and analyzed the expression of the vomeronasal receptor genes of Xenopus laevis. We identified genes of the Xenopus V2R receptor family, which are predominantly expressed throughout the sensory epithelium of the VNO. The G-protein Go, which is coexpressed with V2Rs in the rodent VNO, was also extensively expressed throughout the vomeronasal sensory epithelium. These results strongly suggest that the V2Rs and Go are coexpressed in the vomeronasal receptor cells. The predominant expression of the Xenopus V2R families and the coexpression of the V2Rs and Go imply that V2Rs play important roles in the sensory transduction of Xenopus VNO. We found that these receptors were expressed not only in the VNO, but also in the posterolateral epithelial area of the principal cavity (PLPC). Electron microscopic study revealed that the epithelium of the PLPC is more like that of the VNO than that of the principal and the middle cavity. These results suggest that in adult Xenopus the V2Rs analyzed so far are predominantly expressed in the vomeronasal and vomeronasal-like epithelium. The analysis of V2R expression in Xenopus larvae demonstrates that V2Rs are predominantly expressed in the VNO even before metamorphosis.


Assuntos
Receptores de Feromônios/biossíntese , Receptores de Feromônios/genética , Órgão Vomeronasal/metabolismo , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Larva/genética , Larva/metabolismo , Dados de Sequência Molecular , Mucosa Olfatória/metabolismo , Mucosa Olfatória/ultraestrutura , Especificidade de Órgãos/genética , Receptores de Feromônios/ultraestrutura , Órgão Vomeronasal/ultraestrutura , Proteínas de Xenopus/ultraestrutura , Xenopus laevis
7.
Biochem Biophys Res Commun ; 331(2): 577-82, 2005 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-15850799

RESUMO

The alpha3-peptide, which comprises three repeats of the sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala and forms an amphipathic alpha-helix, is unique among various alpha-helix-forming peptides in that it assembles into fibrous structures that can be observed by transmission electron microscopy. As part of our investigation of the structure-stability relationships of the alpha3-peptide, we synthesized the r3-peptide, whose amino acid sequence is the reverse of that of the alpha3-peptide, and we investigated the effects of sequence reversal on alpha-helix stability and the formation of fibrous structures. Unexpectedly, the r3-peptide formed a more-stable alpha-helix and longer fibers than did the alpha3-peptide. The stability of the r3-peptide helix decreased when the ionic strength of the buffer was increased and when the pH of the buffer was adjusted to 2 or 12. These results suggest that the r3-peptide underwent a "magnet-like" oligomerization and that an increase in the charge-distribution inequality may be the driving force for the formation of fibrous structures.


Assuntos
Peptídeos/química , Peptídeos/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Soluções Tampão , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Concentração Osmolar , Peptídeos/genética , Estrutura Quaternária de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Relação Estrutura-Atividade , Termodinâmica
8.
Chromosoma ; 111(6): 359-68, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12644951

RESUMO

The dicyemid mesozoans are simple multicellular parasites with a long cylindrical axial cell surrounded by a single outer layer of 20 to 30 ciliated peripheral somatic cells. Their larval development proceeds within the axial cell. Here we demonstrate the appearance of extrachromosomal circular DNAs and their fate during early embryogenesis in Dicyema japonicum. These DNAs are highly heterogeneous in sequence, suggesting that they consist of unique--not repetitive--elements. Potential open reading frames were not evident in the elements, so these DNAs are unlikely to have a protein-encoding function. In situ hybridization revealed that the circular DNA elements were restricted to the early embryonic larvae and gradually faded out as larvae approached maturity. Furthermore Southern blot analysis and polymerase chain reaction analysis using a high molecular weight DNA as a template provided evidence that the extrachromosomal DNA circles are originally present in chromosomes. These observations suggest DNA elimination--or selective replication--of the elements from chromosomes during early embryogenesis in dicyemid mesozoans.


Assuntos
DNA Circular/biossíntese , DNA de Protozoário/biossíntese , Invertebrados/embriologia , Invertebrados/genética , Animais , Cromossomos/genética , Replicação do DNA , DNA Circular/química , DNA Circular/ultraestrutura , DNA de Protozoário/química , DNA de Protozoário/ultraestrutura , Invertebrados/crescimento & desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Peso Molecular , Octopodiformes/parasitologia , Fatores de Tempo
9.
Chem Senses ; 27(3): 207-13, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11923183

RESUMO

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.


Assuntos
Células Quimiorreceptoras/metabolismo , Mucosa Olfatória/metabolismo , Órgão Vomeronasal/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Quimiorreceptoras/química , Clonagem Molecular , Primers do DNA , Cabras , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
10.
Exp Cell Res ; 287(2): 219-27, 2003 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-12837278

RESUMO

Nck-interacting kinase (NIK)-related kinase (NRK)/NIK-like embryo-specific kinase (NESK) is a protein kinase that belongs to the germinal center kinase family, and activates the c-Jun N-terminal kinase (JNK) signaling pathway. In this study, we examined the effect of NRK/NESK on actin cytoskeletal organization. Overexpression of NRK/NESK in COS7 cells induced accumulation of polymerized actin at the perinuclear. Phosphorylation of cofilin, an actin-depolymerizing factor, was increased in NRK/NESK-expressing HEK 293T cells. In addition, in vitro phosphorylation of cofilin was observed on NRK/NESK immunoprecipitates from HEK 293T cells expressing the kinase domain of NRK/NESK. The cofilin phosphorylation occurred at the serine residue of position 3 (Ser-3). Since the phosphorylation at Ser-3 inactivates the actin-depolymerizing activity of cofilin, these results suggest that NRK/NESK induces actin polymerization through cofilin phosphorylation. The cofilin phosphorylation did not appear to be mediated through activation of LIM-kinasel, a cofilin-phosphorylating kinase, or through the activation of JNK. Thus, cofilin is likely to be a direct substrate of NRK/NESK. NRK/NESK is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. Thus, NRK/NESK may be involved in the regulation of actin cytoskeletal organization in skeletal muscle cells through cofilin phosphorylation.


Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Despolimerização de Actina , Animais , Biopolímeros/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Centro Germinativo/metabolismo , Quinases do Centro Germinativo , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Serina/metabolismo
11.
Development ; 129(10): 2367-79, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11973269

RESUMO

In the mouse, the definitive endoderm is derived from the epiblast during gastrulation, and, at the early organogenesis stage, forms the primitive gut tube, which gives rise to the digestive tract, liver, pancreas and associated visceral organs. The transcription factors, Sox17 (a Sry-related HMG box factor) and its upstream factors, Mixer (homeobox factor) and Casanova (a novel Sox factor), have been shown to function as endoderm determinants in Xenopus and zebrafish, respectively. However, whether the mammalian orthologues of these genes are also involved with endoderm formation is not known. We show that Sox17(-/-) mutant embryos are deficient of gut endoderm. The earliest recognisable defect is the reduced occupancy by the definitive endoderm in the posterior and lateral region of the prospective mid- and hindgut of the headfold-stage embryo. The prospective foregut develops properly until the late neural plate stage. Thereafter, elevated levels of apoptosis lead to a reduction in the population of the definitive endoderm in the foregut. In addition, the mid- and hindgut tissues fail to expand. These are accompanied by the replacement of the definitive endoderm in the lateral region of the entire length of the embryonic gut by cells that resemble the visceral endoderm. In the chimeras, although Sox17-null ES cells can contribute unrestrictedly to ectodermal and mesodermal tissues, few of them could colonise the foregut endoderm and they are completely excluded from the mid- and hindgut endoderm. Our findings indicate an important role of Sox17 in endoderm development in the mouse, highlighting the idea that the molecular mechanism for endoderm formation is likely to be conserved among vertebrates.


Assuntos
Proteínas de Ligação a DNA , Sistema Digestório/embriologia , Endoderma/patologia , Proteínas de Grupo de Alta Mobilidade , Proteínas/genética , Fatores de Transcrição , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Apoptose/genética , Anormalidades do Sistema Digestório , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Mutantes , Proteínas/metabolismo , Fatores de Transcrição SOXF , Vísceras/embriologia , Vísceras/patologia
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