Biomedical subjects

Wataru Nomura

Publications and source records attributed to Wataru Nomura.

9 recordsLinked to original sources

PubMed2026

Acetic acid-induced translational repression involves eIF2B body formation and Ded1 sequestration into stress granules in yeast.

Elucidating the physiological impact of acetic acid stress and the corresponding yeast responses is essential for advancing fundamental biology and improving industrial alcoholic fermentation. Despite numerous genome-wide studies, information on the effects of acetic acid stress on yeast translational regulation remains limited. We found that a sublethal concentration of acetic acid (35 mM, 0.2% v/v) causes translational repression, accompanied by the formation of eIF2B bodies and the phosphorylation of eIF2α, both of which are involved in the regulation of translation initiation. Acetic acid also caused the sequestration of Ded1, a DEAD-box RNA helicase crucial for translation initiation, into stress granules. Removal of acetic acid restored translational activity and the proper localization of eIF2B and Ded1, indicating the reversibility of acetic acid-induced translational repression. Furthermore, when yeast cells were pretreated with 0.05% acetic acid, translational repression under subsequent 0.2% acetic acid stress was attenuated in wild-type cells but not in hrk1Δ cells. This indicates that Hrk1, a Pma1 activator, is required to sufficiently enhance tolerance to acetic acid-induced translational repression. These findings provide novel insights into the physiological effects of acetic acid stress on translational activity and translation-related factors in yeast cells.

Saccharomyces cerevisiae↗

PubMed2007

Efficient extraction of thioreodoxin from Saccharomyces cerevisiae by ethanol.

Thioredoxin, an antioxidant protein, is a promising molecule for development of functional foods because it protects the gastric mucosa and reduces the allergenicity of allergens. To establish a method for obtaining an ample amount of yeast thioredoxin, we found here that thioredoxin is released from Saccharomyces cerevisiae by treatment with 20% ethanol. We also found that Japanese sake contains a considerable amount of thioredoxin.

Ethanol↗

PubMed2007

Design and synthesis of artificial zinc finger proteins.

Of the DNA-binding motifs, the (Cys)2(His)2-type zinc finger motif has great potential for manipulation. The zinc finger motif offers an attractive framework for the design of novel DNA-binding proteins. Specially, a structure-based design strategy is valuable for the creation of new artificial zinc finger proteins that have novel DNA-binding properties, namely, long-DNA recognition, DNA bending, and AT-rich sequence recognition. Herein, new strategies for the design of multi-zinc finger proteins for the recognition of a target DNA sequence, a DNA-bending zinc finger protein, a (His)4-type zinc finger protein, and an AT-recognizing zinc finger protein are described based on recent experimental results.

Base Sequence↗

PubMed2006

Green tea polyphenols function as prooxidants to activate oxidative-stress-responsive transcription factors in yeasts.

Epigallocatechin gallate (EGCG) is the most abundant polyphenolic flavonoid in green tea. Catechin and its derivatives, including EGCG, are widely believed to function as antioxidants. Here we demonstrate that both EGCG and green tea extract (GTE) cause oxidative stress-related responses in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe under weak alkaline conditions in terms of the activation of oxidative-stress-responsive transcription factors. GTE as well as EGCG induced the nuclear localization of Yap1 in S. cerevisiae, which was repressed by the addition of catalase but not by the addition of superoxide dismutase. The same phenomena were observed for the nucleocytoplasmic localization of Msn2 in S. cerevisiae and Pap1, a Yap1 homologue, in S. pombe. The formation of intramolecular disulfide bonds has been proposed to be crucial for the H(2)O(2)-induced nuclear localization of Yap1, and we verified the importance of cysteine residues of Yap1 in response to EGCG and GTE. Additionally, we show that EGCG and GTE produce H(2)O(2) in a weak alkaline medium. Finally, we conclude that tea polyphenols are able to act as prooxidants to cause a response to oxidative stress in yeasts under certain conditions.

Camellia sinensis↗

PubMed2005

Effects of linking 15-zinc finger domains on DNA binding specificity and multiple DNA binding modes.

To assess the possibility of multi-connection of zinc finger domains for understanding of DNA binding mechanisms and gene regulation, the longest artificial zinc finger protein, Sp1ZF15, has been constructed. This zinc finger consists of 5 units of Sp1 zinc finger peptide connected by canonical short linker sequences (TGEKP). Recognition of the 50 base pairs of DNA and potential binding to shorter targets by Sp1ZF15 were determined. Sequence alterations of the GCG triplet to ATA at a target site clearly showed that Sp1ZF15 changes its DNA binding mode depending on the target sequences. Of special interest is the fact that Sp1ZF15 controls the number of finger domains active in DNA binding corresponding to the length and sequence of the target DNA. These results suggest that artificial transcription factors based upon these multi-zinc finger proteins have great potential for the regulation of a vast number of cellular processes.

Amino Acid Sequence↗

PubMed2005

Self-assembly of size- and position-controlled ultralong nanodot chains using near-field optical desorption.

We report the self-assembly of a size- and position-controlled ultralong nanodot chain using a novel effect of near-field optical desorption. A sub-100-nm dot chain with a deviation of 5 nm forms at a size based on plasmon resonance, depending on the photon energy; the resulting structure forms a high-transmission-efficiency nanoscale waveguide. Using this method with simple lithographically patterned substrates allows one to increase the throughput of the production of nanoscale structures dramatically at all scales.

Adsorption↗

PubMed2003

Effects of length and position of an extended linker on sequence-selective DNA recognition of zinc finger peptides.

Engineered zinc finger proteins revealed that a linker sequence connecting zinc finger units has a significant effect on the DNA binding property of the protein. The recognition for a noncontiguous DNA target beyond the current recognition code of zinc finger proteins has never been determined because of the limitation of a zinc finger framework. DNA recognition of zinc finger proteins is limited only to a contiguous subset of three base pairs. We propose the recognition for a noncontiguous DNA target by inserting amino acids into the canonical linker between zinc finger units. The sequence selectivity of the new zinc finger peptides was evaluated by gel mobility shift assays. DNase I footprinting analyses clearly showed different DNA binding of various linker-extended zinc finger peptides. The application of a SPR measurement also revealed a DNA sequence selectivity of peptides. Insertion of three amino acids is enough for recognition of a noncontiguous DNA target with sequence selectivity. An extended linker will be useful for expansion of the recognition code of zinc finger proteins and for development of a new role for linker sequences in DNA binding of zinc finger proteins.

Amino Acid Substitution↗

PubMed2003

Influence of TFIIIA-type linker at the N- or C-terminal of nine-zinc finger protein on DNA-binding site.

The central three-zinc finger connection of the native nine-zinc finger protein transcription factor IIIA (TFIIIA) is composed of unique linker sequences, -NIKICV-, -TQQLP-, -AG-, and -QDL-. New artificial nine-zinc finger proteins, Sp1ZF9TC and Sp1ZF9TN, which use the TFIIIA-type linker for their C- and N-terminal three-zinc finger connections have been created. To investigate the influence of TFIIIA-type linker sequences by their different locations in the proteins, gel mobility shift assays (GMSA), DNase I footprinting assays, methylation interference analyses, and hydroxyl radical footprinting assays were performed. The GMSA revealed similar DNA-binding affinities of these two proteins. The footprinting analyses indicated that the two zinc finger proteins recognize the same part of GCII or GCIII DNA. Moreover, the specific base contacts were observed in the same sites of the substrate DNA. In the present proteins, Sp1ZF9TC and Sp1ZF9TN, the four zinc fingers (fingers 1-4 or 5-9) situated in the site opposite to the TFIIIA-type linker position participate in their DNA bindings. The position of the TFIIIA-type linker is important in DNA recognition by multi-zinc finger proteins.

Amino Acid Sequence↗

PubMed2002

Interconversion between serine and aspartic acid in the alpha helix of the N-terminal zinc finger of Sp1: implication for general recognition code and for design of novel zinc finger peptide recognizing complementary strand.

In the typical base recognition mode of the C(2)H(2)-type zinc finger, the amino acid residues at alpha-helical positions -1, 3, and 6 make a contact with the base in one strand (the primary strand), and the residue at position 2 interacts with the base in a complementary strand (the secondary strand). The N-terminal zinc finger of the three-zinc-finger domain of Sp1 has inherently a unique five-base-pair binding mode in which the guanine bases are recognized in both strands. To clarify the effect of the amino acid at position 2 on DNA binding affinity and base specificity, we have created a library of the mutants by the interconversion between serine and aspartic acid in the N-terminal zinc finger of Sp1 and recombinant variants of finger order. Gel mobility shift and methylation interference assays showed that the combination of arginine and serine at positions -1 and 2, respectively, provides a newly strong guanine contact in the secondary strand and a higher binding affinity than that of wild-type Sp1. Of special interest are the facts that the mutant with lysine and aspartic acid at positions -1 and 2 in the alpha helix predominantly recognizes the bases in the secondary strand and that its DNA binding affinity is higher than that of the wild-type. The aspartic acid or serine at position 2 independently contributes to the DNA binding affinity and base specificity. The present results provide useful information for the design of a novel zinc finger protein with priority for the bases in the secondary strand.

Aspartic Acid↗