Biomedical subjects

Takeshi Todo

Publications and source records attributed to Takeshi Todo.

28 records · Page 2Linked to original sources

PubMed2004

Identification of cryptochrome DASH from vertebrates.

A new type of cryptochrome, CRY-DASH, has been recently identified. The CRY-DASH proteins constitute the fifth subfamily of the photolyase/cryptochrome family. CRY-DASHs have been identified from Synechocystis sp. PCC 6803, Vibrio cholerae, and Arabidopsis thaliana. The Synechocystis CRY-DASH was the first cryptochrome identified from bacteria, and its biochemical features and tertiary structure have been extensively investigated. To determine how broadly the subfamily is distributed within living organisms, we searched for new CRY-DASH candidates within several databases. We found five sequences as new CRY-DASH candidates, which are derived from four marine bacteria and Neurospora crassa. We also found many CRY-DASH candidates from the EST databases, which included sequences from fish and amphibians. We cloned and sequenced the cDNAs of the zebrafish and Xenopus laevis candidates, based on the EST sequences. The proteins encoded by the two genes were purified and characterized. Both proteins contained folate and flavin cofactors, and have a weak DNA photolyase activity. A phylogenetic analysis revealed that the seven candidates actually belong to the new type of cryptochrome subfamily. This is the first report of the CRY-DASH members from vertebrates and fungi.

Amino Acid Sequence↗

PubMed2004

Serine phosphorylation of mCRY1 and mCRY2 by mitogen-activated protein kinase.

The circadian oscillator is composed of a transcription/translation-based autoregulatory feedback loop in which Cryptochromes and Periods function as negative regulators for their own gene expression. Although post-translational modifications such as phosphorylation of these regulators appear crucial for circadian time-keeping mechanism, less is known about responsible protein kinases and their contribution to the function of the regulators. We found that mitogen-activated protein kinase (MAPK) associates with and phosphorylates mouse Cryptochromes (mCRY1 and mCRY2). Mass spectrometry analysis identified Ser265 and Ser557 of mCRY2 to be in vitro phospho-acceptor residues. Mutations of both the Ser residues to Ala completely abolished MAPK-mediated mCRY2 phosphorylation, suggesting that the two residues are the principal phosphorylation sites in mCRY2. Similarly, MAPK phosphorylates mCRY1 at Ser247, a site corresponding to Ser265 of mCRY2. An effect of the Ser phosphorylation was investigated by mutating Ser247 of mCRY1 and Ser265 of mCRY2 to Asp, which resulted in attenuation of each mCRYs' ability to inhibit BMAL1: CLOCK-mediated transcription, whereas a similar mutation at Ser557 of mCRY2 induced no measurable change in its activity. These results illustrate a model of MAPK-mediated negative regulation of mCRY function by phosphorylation at the specific Ser residue.

Amino Acid Sequence↗

PubMed2003

Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes.

CLOCK is a positive component of a transcription/translation-based negative feedback loop of the central circadian oscillator in the suprachiasmatic nucleus in mammals. To examine CLOCK-regulated circadian transcription in peripheral tissues, we performed microarray analyses using liver RNA isolated from Clock mutant mice. We also compared expression profiles with those of Cryptochromes (Cry1 and Cry2) double knockout mice. We identified more than 100 genes that fluctuated from day to night and of which expression levels were decreased in Clock mutant mice. In Cry-deficient mice, the expression levels of most CLOCK-regulated genes were elevated to the upper range of normal oscillation. Most of the screened genes had a CLOCK/BMAL1 binding site (E box) in the 5'-flanking region. We found that CLOCK was absolutely concerned with the circadian transcription of one type of liver genes (such as DBP, TEF, and Usp2) and partially with another (such as mPer1, mPer2, mDec1, Nocturnin, P450 oxidoreductase, and FKBP51) because the latter were damped but remained rhythmic in the mutant mice. Our results showed that CLOCK and CRY proteins are involved in the transcriptional regulation of many circadian output genes in the mouse liver. In addition to being a core component of the negative feedback loop that drives the circadian oscillator, CLOCK also appears to be involved in various physiological functions such as cell cycle, lipid metabolism, immune functions, and proteolysis in peripheral tissues.

Amino Acid Motifs↗

PubMed2003

Functional and structural analyses of cryptochrome. Vertebrate CRY regions responsible for interaction with the CLOCK:BMAL1 heterodimer and its nuclear localization.

Mouse mCRY1 and zebrafish zCRY1a and zCRY3 belong to the DNA photolyase/Cryptochrome family. mCRY1 and zCRY1a repress CLOCK:BMAL1-mediated transcription, whereas zCRY3 does not. Reciprocal chimeras between zCRY1a and zCRY3 were generated to determine the zCRY1a regions responsible for nuclear translocation, interaction with the CLOCK:BMAL1 heterodimer, and repression of CLOCK:BMAL1-mediated transcription. Three regions, RD-2a-(126-196), RD-1-(197-263), and RD-2b-(264-293), were identified. Proteins in this family consist of an N-terminal alpha/beta domain and a C-terminal helical domain connected by an interdomain loop. RD-2a is within this loop, RD-1 is at the N-terminal 50 amino acids, and RD-2b at the following 31 amino acid residues of the helical domain. Either RD-2a or RD-1 is required for interaction with the CLOCK: BMAL1 heterodimer, and either RD-1 or RD-2b is required for the nuclear translocation of CRY. Both of these functions are prerequisites for the transcriptional repressor activity. The functional nuclear localizing signal in the RD-2b region also was identified. The sequence is well conserved among repressor-type CRYs, including mCRY1. Mutations in the nuclear localizing signal of mCRY1 reduce the extent of its nuclear localization. These findings show that both nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY.

Amino Acid Sequence↗

PubMed2003

New role of zCRY and zPER2 as regulators of sub-cellular distributions of zCLOCK and zBMAL proteins.

The core oscillator that generates circadian rhythm in eukaryotes consists of transcription/translation-based autoregulatory feedback loops by which clock gene products negatively regulate their own expression. Control of the accumulation and nuclear entry of the negative regulators PER and CRY is believed to be a key step in these loops. We clarified the mutual interaction between zebrafish clock-related proteins and their sub-cellular localizations in NIH3T3 cells. Six CRYs exist in zebrafish, of which zCRY1a strongly represses zCLOCK1: zBMAL3-mediated transcription, but zCRY3 does not. We show that zCRY1a interacts with zCLOCK1 and zBMAL3, facilitating nuclear accumulation, whereas zCRY3 associates with neither one and does not influence their sub-cellular distributions. We cloned zPer2 cDNA and showed that the protein product encoded by the cDNA acts as a moderate transcriptional repressor. In our sub-cellular localization studies we also found that zPER2 interacts with the zCLOCK1:zBMAL3 heterodimer, causing its cytoplasmic retention. zCRY1a and zPER2 apparently have opposite effects on the sub-cellular distribution of zCLOCK:zBMAL heterodimer. We speculate that the opposite regulation of the sub-cellular distribution of this is associated with the different transcriptional repression abilities of zCRY1a and zPER2. zCRY1a acts as a potent transcriptional inhibitor by interacting directly with the zCLOCK:zBMAL heterodimer in the nucleus, whereas zPER2 maintains the zCLOCK:zBMAL heterodimer in the cytoplasm, resulting in transactivation repression.

3T3 Cells↗

PubMed2003

Identification of a new cryptochrome class. Structure, function, and evolution.

Cryptochrome flavoproteins, which share sequence homology with light-dependent DNA repair photolyases, function as photoreceptors in plants and circadian clock components in animals. Here, we coupled sequencing of an Arabidopsis cryptochrome gene with phylogenetic, structural, and functional analyses to identify a new cryptochrome class (cryptochrome DASH) in bacteria and plants, suggesting that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. The cryptochrome crystallographic structure, reported here for Synechocystis cryptochrome DASH, reveals commonalities with photolyases in DNA binding and redox-dependent function, despite distinct active-site and interaction surface features. Whole genome transcriptional profiling together with experimental confirmation of DNA binding indicated that Synechocystis cryptochrome DASH functions as a transcriptional repressor.

Amino Acid Sequence↗

PubMed2002

Photoactivation of the flavin cofactor in Xenopus laevis (6 - 4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance.

The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6-4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6-4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair.

Animals↗

PubMed2002

Zebrafish CRY represses transcription mediated by CLOCK-BMAL heterodimer without inhibiting its binding to DNA.

BACKGROUND: CLOCK and BMAL1 proteins, members of the basic helix-loop-helix PAS (PER-ARNT-SIM) superfamily of transcription factors which bind to the E-box DNA motif, are required for the high-level expression of the circadian clock genes period (per) and cryptochrome (cry). CRY inhibits transcriptional activity of the CLOCK-BMAL1 heterodimer, generating a negative-feedback loop that is the core element of the circadian oscillator. RESULTS: We show that zebrafish CRY (zCRY1a) neither disrupts the association between zfCLOCK and zfBMAL nor inhibits binding of the zfCLOCK-zfBMAL heterodimer to an E-box-bearing DNA fragment. Instead it binds to the heterodimer to form a stable zCRY1a-zfCLOCK-zfBMAL-E-box complex. Another zebrafish CRY protein, zCRY4, does not have transcriptional inhibitor activity, whereas zCRY1a has strong activity. zCRY4 does not associate with zfCLOCK and zfBMAL. We also show that the presence of a chemical reductant in the reaction mixture is crucial for efficient binding of the CLOCK-BMAL heterodimer to E-box bearing DNA, which is indicative of the reduction/oxidation (redox)-sensitive character of the heterodimer. CONCLUSIONS: Our findings suggest that CRY represses CLOCK-BMAL-mediated transcription by interacting directly with the zfCLOCK-zfBMAL-E-box complex.

ARNTL Transcription Factors↗

PubMed2002

Somatic mutation in larvae of the silkworm, Bombyx mori, induced by heavy ion irradiation to diapause eggs.

In order to investigate whether eggs of the black-striped strain (P(S)) of the silkworm, Bombyx mori, represent an appropriate model for estimating the biological effect of cosmic radiation, radiosensitivity of the eggs against X-rays and heavy ion particles was examined as ground-based experiments. The exposure of diapause eggs to X-rays or heavy ion particles resulted in somatic mutations appearing as a white spot on the black integument during larval stage. Irradiation of non-diapause eggs with X-rays demonstrated a significant difference in frequency of the mutation between fractionated and single administration doses, but no difference was observed in diapause eggs. Incidence of the mutation as induced by carbon ion beams for 15-day old eggs was higher for eggs that had been kept at 15 degrees C than those kept at 25 degrees C. Neon beam irradiation of diapause eggs displayed dose- and linear energy transfer (LET)-dependent effects, causing a maximal rate of the mutation at 150 keV/microm. These results confirm that B. mori eggs represent valid models for estimating the biological effects of cosmic radiation.

Animals↗

PubMedDate not supplied

Pyrimidine (6-4) pyrimidone photoproduct mapping after sublethal UVC doses: nucleotide resolution using terminal transferase-dependent PCR.

UVC irradiation of genomic DNA induces two main types of potentially mutagenic base modifications: cyclobutane pyrimidine dimers (CPDs) and the less frequent (15-30% of CPD levels) pyrimidine (6-4) pyrimidone photoproducts (6-4PP). Ligation-mediated PCR (LMPCR), a genomic sequencing technique, allows CPD mapping at nucleotide resolution following irradiation with sublethal doses of UVB or UVC for most cell types. In contrast, a dose of 80 J/m(2) of UVC that is lethal for the majority of cell types is necessary to map 6-4PP by the LMPCR technique. This compromises the use of LMPCR to study the repair of 6-4PP. To date, no other techniques have been developed to study 6-4PP repair at nucleotide resolution. We have therefore adapted a recently developed technique for the mapping of 6-4PP: terminal transferase-dependent PCR (TDPCR). TDPCR is in many ways similar to LMPCR. This technique is more sensitive and allows the mapping of 6-4PP at UVC doses as low as 10 J/m(2) in genomic DNA and in living cells.

Binding Sites↗