Mari Onozuka

Cancer prevention by carotenoids

Various natural carotenoids have been proven to have anticarcinogenic activity. Epidemiological investigations have shown that cancer risk is inversely related to the consumption of green and yellow vegetables and fruits. As b-carotene is present in abundance in these vegetables and fruits, it has been investigated extensively as a possible cancer preventive agent. However, various carotenoids which coexist with b-carotene in vegetables and fruits also have anticarcinogenic activity, and some of these, such as a-carotene, lutein and lycopene, show a higher potency than b-carotene in suppressing experimental carcinogenesis. Thus, we have carried out more extensive studies on cancer preventive activities of natural carotenoids in foods. For example, we found that b-cryptoxanthin showed antitumor initiating activity, as well as antitumor promoting activity. It is of interest that not only carotenoids distributed in vegetables and fruits, but also animal carotenoids, such as astaxanthin, are promising as cancer preventive agents. In the present study, the cancer preventive potential of phytoene was also con®rmed. The establishment of NIH3T3 cells that produce phytoene by introducing the crtB gene provides evidence that resistance against transformation, imposed by transfection of activated H-ras oncogene, was acquired by phytoene production. Analysis of the action mechanism of these natural carotenoids is now in progress, and some interesting results have already been obtained; for example, various carotenoids were suggested to stimulate the expression of RB gene, an antioncogene.

Genetic regulation mediated by thiamin pyrophosphate-binding motif in Saccharomyces cerevisiae.

The expression of genes of Saccharomyces cerevisiae encoding the enzymes involved in the metabolism of thiamin (THI genes) is co‐ordinately repressed by exogenous thiamin and induced in the absence of thiamin. In this yeast THI regulatory system acts mainly at the transcriptional level, thiamin pyrophosphate (TDP) seems to serve as a corepressor, and genetic studies have identified three positive regulatory factors (Thi2p, Thi3p and Pdc2p). We found in a DNA microarray analysis that the expression of THI genes increased 10‐ to 90‐fold in response to thiamin deprivation, and likewise, the expression of THI2 and THI3 increased 17‐fold and threefold, respectively. After transfer from repressing to inducing medium, the promoter activity of both THI2 and THI3 increased in parallel with that of PHO3, one of THI genes. The stimulation of THI3 promoter activity was diminished by deletion of THI3, indicative of the autoregulation of THI3. The THI genes were not induced when THI2 was expressed from the yeast GAL1 promoter in a thi3Δ strain or when THI3 was expressed in a thi2Δ strain, suggesting that Thi2p and Thi3p participate simultaneously in the induction. When mutant Thi3p proteins lacking TDP‐binding activity were produced in the thi3Δ strain, THI genes were expressed even under thiamin‐replete conditions. This result supports the hypothesis that Thi3p senses the intracellular signal of the THI regulatory system to exert transcriptional control. Furthermore, Thi2p and Thi3p were demonstrated to bind each other and this interaction was partially diminished by exogenous thiamin, suggesting that Thi2p and Thi3p stimulate the expression as a complex whose function is disturbed by TDP bound to Thi3p. We discuss the possibility that the induction of THI genes is triggered by the activation of the complex attributed to decrease in intracellular TDP and the elevated complex in the autoregulatory fashion further upregulates THI genes. This is the first report of the involvement of the TDP‐binding motif in genetic regulation.

Molecular cloning and expression of a mouse thiamin pyrophosphokinase cDNA.

Thiamin pyrophosphokinase (EC 2.7.6.2) catalyzes the pyrophosphorylation of thiamin with adenosine 5′-triphosphate to form thiamin pyrophosphate. A mouse thiamin pyrophosphokinase cDNA clone (mTPK1) was isolated using a combination of mouse expressed sequence tag database analysis, a two-step polymerase chain reaction procedure, and functional complementation screening with aSaccharomyces cerevisiae thiamin pyrophosphokinase-deficient mutant (thi80). The predicted protein contained 243 amino acid residues with a calculated molecular weight of 27,068. When the intact mTPK1 open reading frame was expressed as a glutathione S-transferase fusion protein in Escherichia coli lacking thiamin pyrophosphokinase, marked enzyme activity was detected in the bacterial cells. The corresponding 2.5-kilobase pair mRNA was expressed in a tissue-dependent manner and was found at relatively high levels in the kidney and liver, indicating that the mode of expression of mTPK1 genes differs with cell type. The expression ofmTPK1 genes in cultured mouse neuroblastoma and normal liver cells was unaffected by the thiamin concentration in the medium (10 μm versus 3.0 nm). This is the first report on identification of the primary sequence for mammalian thiamin pyrophosphokinase.

Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant

Abstract The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis. The R188I mutant digested a conserved undecapeptide substrate with a K m of 33.8μM and k cat of 4753s−1. Compared with the value reported for the mature protease containing a C-terminal His-tag, the relative activity of the mutant was nearly 106. Novel peptide-aldehyde derivatives containing a side-chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde derivatives.

Isolation and characterization of a human thiamine pyrophosphokinase cDNA

A human thiamine pyrophosphokinase cDNA clone (hTPK1) was isolated and sequenced. When the intact hTPK1 open reading frame was expressed as a histidine-tag fusion protein in Escherichia coli, marked enzyme activity was detected in the bacterial cells. The hTPK1 mRNA was widely expressed in various human tissues at a very low level, and the mRNA content in cultured fibroblasts was unaffected by the thiamine concentration of the medium. The chromosome localization of the hTPK1 gene was assigned to 7q34.

Biosynthesis of hydroxymethylpyrimidine pyrophosphate in Saccharomyces cerevisiae

Two redundant genes, THI20 and THI21, of Saccharomyces cerevisiae encode a 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate (HMP-P) kinase required for thiamin biosynthesis. Using functional complementation analysis with an Escherichia coli mutant strain and a defined biochemical system containing partially purified proteins for the reconstitution of thiamin monophosphate synthesis, we demonstrate that both Thi20p and Thi21p proteins also have HMP kinase activity. Although each isoform independently can synthesize HMP pyrophosphate (HMP-PP) from HMP, there is a marked difference in efficiency between the two proteins. The thi20 deletion strain grows at the same rate as the parental strain in minimal medium without thiamin, but its ability to synthesize HMP-PP from HMP is significantly decreased. We discuss the possibility that HMP is not involved in the pathway of de novo thiamin synthesis in S. cerevisiae.

Thiamin-dependent transactivation activity of PDC2 in Saccharomyces cerevisiae.

MINT‐6797334, MINT‐6797355: THI3 (uniprotkb:Q07471) physically interacts (MI:0218) with PDC2 (uniprotkb:P32896) by two hybrid (MI:0018) MINT‐6797316: THI3 (uniprotkb:Q07471) physically interacts (MI:0218) with THI2 (uniprotkb:P38141) by two hybrid (MI:0018) MINT‐6797373: THI3 (uniprotkb:Q07471) binds (MI:0407) to PDC2 (uniprotkb:P32896) by pull down (MI:0096)

Facilitated recruitment of Pdc2p, a yeast transcriptional activator, in response to thiamin starvation

In Saccharomyces cerevisiae, genes involved in thiamin pyrophosphate (TPP) synthesis (THI genes) and the pyruvate decarboxylase structural gene PDC5 are transcriptionally induced in response to thiamin starvation. Three positive regulatory factors (Thi2p, Thi3p, and Pdc2p) are involved in the expression of THI genes, whereas only Pdc2p is required for the expression of PDC5. Thi2p and Pdc2p serve as transcriptional activators and each factor can interact with Thi3p. The target consensus DNA sequence of Thi2p has been deduced. When TPP is not bound to Thi3p, the interactions between the regulatory factors are increased and THI gene expression is upregulated. In this study, we demonstrated that Pdc2p interacts with the upstream region of THI genes and PDC5. The association of Pdc2p or Thi2p with THI gene promoters was enhanced by thiamin starvation, suggesting that Pdc2p and Thi2p assist each other in their recruitment to the THI promoters via interaction with Thi3p. It is highly likely that, under thiamin-deprived conditions, a ternary Thi2p/Thi3p/Pdc2p complex is formed and transactivates THI genes in yeast cells. On the other hand, the association of Pdc2p with PDC5 was unaffected by thiamin. We also identified a DNA element in the upstream region of PDC5, which can bind to Pdc2p and is required for the expression of PDC5.