Biomedical subjects

Shin-ichi Sato

Publications and source records attributed to Shin-ichi Sato.

11 recordsLinked to original sources

PubMed2005

Covalent blocking of fibril formation and aggregation of intracellular amyloidgenic proteins by transglutaminase-catalyzed intramolecular cross-linking.

Two different types of physical bonding have been proposed to involve in the formation of neuronal inclusions of patients with neurodegenerative diseases such as Alzheimer's, Parkinson's, and polyglutamine diseases. One is the noncovalent bonding that stabilizes the amyloid-type fibrous aggregates, and the other is the covalent cross-linking catalyzed by tissue transglutaminase. The cross-linking is subdivided into the inter- and intramolecular cross-linking. Little attention has been paid to the pathological roles of the intramolecular cross-linking. To elucidate the possible interplay between the intramolecular cross-linking and the amyloid-type fibril formation, we performed an in vitro aggregation analysis of three intracellular amyloidgenic proteins (a domain of tau protein, alpha-synuclein, and truncated yeast prion Sup35) in the presence of tissue transglutaminase. The analysis was performed in low concentrations of the proteins using techniques including thioflavin T binding and mass spectrometry. The results demonstrated that the amyloid-type fibril formation was strongly inhibited by the transglutaminase-catalyzed intramolecular cross-linking, which blocked both the nucleation and the fiber extension steps of the amyloid formation. Far-UV CD spectroscopy indicated that the cross-linking slightly altered the backbone conformation of the proteins. It is likely that conformational restriction imposed by the intramolecular cross-links has impaired the ordered assembly of the amyloidgenic proteins. Nonamyloid type aggregation was also suppressed by the intramolecular cross-links. On the basis of the results, we proposed that tissue transglutaminase is a modulator for the protein aggregation and can act defensively against the fibril deposition in neurons.

Adult↗

PubMed2005

Stepwise molding of a highly selective ribonucleopeptide receptor.

The structural characteristics of RNA-peptide (RNP) complexes are suitable for molding of a ligand-binding pocket of the RNP complex in a stepwise manner. The first step involves molding of the RNA subunit by in vitro selection of an RNP pool originating from an RNA library and the peptide, as previously reported for the construction of an ATP-binding RNP complex from an RRE RNA-Rev peptide complex. The second step involves selection from an RNP library consisting of Rev peptides with randomized amino acid residues and the RNA subunit selected in the first molding. The ATP-binding pocket produced by sequential molding of RNA and peptide subunits shows higher affinity to ATP and a distinct specificity for ATP versus dATP as compared to the ATP-binding RNP receptor in which only the RNA subunit has been molded. The second step selection from the peptide-based RNP library allows expansion of the ATP recognition surface, consisting of both RNA and peptide subunits, to enhance the affinity and selectivity to discriminate ATP against dATP. Our approach of stepwise molding offers the advantage of increasing the diversity of the RNP library by utilizing characteristics of different biopolymers. The ribonucleopeptide-based, multi-subunit approach is also extendable to other biomacromolecular assemblies, which may yield artificial receptors and enzymes with increased specificity and more diverse chemical activities.

Adenosine Triphosphate↗

PubMed2004

Ribonucleopeptides: functional RNA-peptide complexes.

Selection of functional RNAs from randomized pool of RNA molecules successfully affords RNA aptamers that specifically bind to small molecules, and that have catalytic activities. Recent structural analyses of the ribosomal RNA complex suggest that the RNA-protein complex would be a new structural candidate for the design of tailor-made receptors and enzymes. We have designed an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure-based design approach and successive in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA-binding peptide. The randomized nucleotide region was placed next to the HIV-1 Rev response element to enable the formation of "ribonucleopeptide" pools in the presence of the Rev peptide. In vitro selection of RNA oligonucleotides from the randomized pool afforded a ribonucleopeptide receptor specific for ATP. The ATP-binding ribonucleopeptide did not share the known consensus nucleotide sequence for ATP aptamers, and completely lost its ATP-binding ability in the absence of the Rev peptide. The ATP-binding activity of the ribonucleopeptide was increased by a substitution of the N-terminal amino acid of the Rev peptide. These results demonstrate that the peptide stabilizes the functional structure of RNA and suggest that amino acids outside the RNA binding region of the peptide participate in the ATP binding. Our approach would provide a new strategy for the design of tailor-made ribonucleopeptide receptors.

Adenosine Triphosphate↗

PubMed2004

Design of sensing ribonucleopeptides for small ligands.

Here we report a simple method to convert synthetic receptors to fluorescent sensors. An RNA-peptide complex (ribonucleopeptide) with a known three-dimensional structure is used as a framework of the receptor. Artificial ribonucleopeptide sensors were created with a combination of in vitro selection method and successive modification of the peptide with a fluorophore. A ribonucleopeptide complex of the fluorophore labeled peptide showed a remarkable fluorescence emission change upon binding cognate ligands.

Amines↗

PubMed2003

Optimization of an ATP-binding ribonucleopeptide receptor.

We describe here a new strategy for the selection and evolution of functional RNA-peptide complexes. An ATP-binding ribonucleopeptide was obtained by means of a structure-based design of ribonucleopeptide domain and by in vitro selection of the RNA subunit of the ribonucleopeptide. Approaches to optimize the ligand-binding selectivity of ribonucleopeptide will be discussed.

Adenosine Triphosphate↗

PubMed2003

Quantitative trait loci analysis for growth and carcass traits in a Meishan x Duroc F2 resource population.

We constructed a pig F2 resource population by crossing a Meishan sow and a Duroc boar to locate economically important trait loci. The F2 generation was composed of 865 animals (450 males and 415 females) from four F1 males and 24 F1 females and was genotyped for 180 informative microsatellite markers spanning 2,263.6 cM of the whole pig genome. Results of the genome scan showed evidence for significant quantitative trait loci (<1% genomewise error rate) affecting weight at 30 d and average daily gain on Sus scrofa chromosome (SSC) 6, carcass yield on SSC 7, backfat thickness on SSC 7 and SSC X, vertebra number on SSC 1 and SSC 7, loin muscle area on SSC 1 and SSC 7, moisture on SSC 13, intramuscular fat content on SSC 7, and testicular weight on SSC 3 and SSC X. Moreover, 5% genomewise significant QTL were found for birth weight on SSC 7, average daily gain on SSC 4, carcass length on SSC 6, SSC 7, and SSC X and lightness (L value) on SSC 3. We identified 38 QTL for 28 traits at the 5% genomewise level. Of the 38 QTL, 24 QTL for 17 traits were significant at the 1% genomewise level. Analysis of marker genotypes supported the breed of origin results and provided further evidence that a suggestive QTL for circumference of cannon bone also was segregating within the Meishan parent. We identified genomic regions related with growth and meat quality traits. Fine mapping will be required for their application in introgression programs and gene cloning.

Adipose Tissue↗

PubMed2002

Chemical approaches untangling sequence-specific DNA binding by proteins.

Structure-based design of novel DNA-binding proteins provides an ultimate test of our understanding of protein-DNA interactions. A combination of synthetic, organic, biochemical and molecular biological approaches has been developed to study the principle of molecular recognition associated with the protein-DNA interactions. The strategies enabled a specific formation of noncovalent peptide dimers and determination of the preferential DNA-binding sequence of short peptides.

Amino Acid Sequence↗

PubMed2002

In vitro selection of ATP-binding receptors using a ribonucleopeptide complex.

A recently described three-dimensional structure of the ribosome provides a sense of remarkable diversity of RNA-protein complexes. We have designed a new class of scaffold for artificial receptors, in which a short peptide and RNA with a randomized nucleotide region form a stable and specific complex. The randomized nucleotide region was placed next to the HIV-1 Rev response element to enable the formation of "ribonucleopeptide" pools in the presence of the Rev peptide. In vitro selection of RNA oligonucleotides from the randomized pool afforded a ribonucleopeptide receptor specific for ATP. The ATP-binding ribonucleopeptide did not share the known consensus nucleotide sequence for ATP aptamers and completely lost its ATP-binding ability in the absence of the Rev peptide. The ATP-binding activity of the ribonucleopeptide was increased by a substitution of the N-terminal amino acid of the Rev peptide. These results demonstrate directly that the peptide is incorporated in the functional structure of RNA and suggest that amino acids outside the RNA-binding region of the peptide modulate the ATP-binding of ribonucleopeptide. Our approach would provide an alternative strategy for the design of "tailor-made" ribonucleopeptide receptors and enzymes.

Adenosine Triphosphate↗

PubMed2002

Structure-based design of a leucine zipper protein with new DNA contacting region.

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.

Amino Acid Sequence↗

PubMed2002

A general strategy to determine a target DNA sequence of a short peptide: application to a d-peptide.

Short peptides could potentially provide a novel element to read-out DNA sequences from the major groove. However, it is difficult to determine sequence-preference of de novo designed monomeric short peptides. Because DNS-binding affinity and specificity of short peptides are usually much lower than those of native DNA-binding proteins, determining the sequence-preference of short peptides by conventional methods utilized to deduce the target sequence of proteins often produces an unclear outcome. We report here a general strategy to defining the sequence-preference of a DNA-binding short peptide by using the heterodimers. A GCN4 basic region peptide tethers a low-affinity DNA-binding peptide adjacent to a GCN4 binding sequence through the cyclodextrin-adamantane association, thereby increasing local concentration of the low-affinity peptide on degenerated DNA sequences. An increase of the local concentration allows one to select a preferential sequence for the low-affinity DNA binding peptide. The method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of d-amino acids. The method is potentially applicable not only to artificial peptides, but also to other synthethic ligands.

Amino Acid Sequence↗

PubMed2002

A new strategy to determine the target DNA sequence of a short peptide.

The design of molecules that target desired DNA sequences has been one of the major challenges in the field of molecular recognition. We report here a general strategy for defining the sequence-preference of DNA-binding short peptide by using its heterodimer. Our method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of D-amino acids.

Base Sequence↗