Pinostrobin suppresses the Ca2+-signal-dependent growth arrest in yeast by inhibiting the Swe1-mediated G2 cell-cycle regulation.

Pinostrobin, a flavonoid compound known for its diverse pharmacological actions, including anti-leukemic and anti-inflammatory activities, has been repeatedly isolated by various screenings, but its action mechanism is still obscure. Previously, pinostrobin was rediscovered in our laboratory using a yeast-based assay procedure devised specifically for the inhibitory effect on the activated Ca2+ signaling that leads the cells to severe growth retardation in the G2 phase. Here, we attempted to identify target of pinostrobin employing the genetic techniques available in the yeast. Using various genetically engineered yeast strains in which the Ca2+-signaling cascade can be activated by the controlled expression of the various signaling molecules of the cascade, its target was narrowed down to Swe1, the cell-cycle regulatory protein kinase. The Swe1 kinase is situated at the downstream of the Ca2+-signaling cascade and downregulates the Cdc28/Clb complex by phosphorylating the Cdc28 moiety of the complex in the G2 phase. We further demonstrated that pinostrobin inhibits the protein kinase activity of Swe1 in vivo as estimated by the decreased level of Cdc28 phosphorylation at Tyr-19. Since the yeast SWE1 gene is an ortholog for the human WEE1 gene, our finding implied a potentiality of pinostrobin as the G2 checkpoint abrogator in cancer chemotherapy.

Stimulating S-adenosyl-l-methionine synthesis extends lifespan via activation of AMPK.

Dietary restriction (DR), such as calorie restriction (CR) or methionine (Met) restriction, extends the lifespan of diverse model organisms. Although studies have identified several metabolites that contribute to the beneficial effects of DR, the molecular mechanism underlying the key metabolites responsible for DR regimens is not fully understood. Here we show that stimulating S-adenosyl-l-methionine (AdoMet) synthesis extended the lifespan of the budding yeast Saccharomyces cerevisiae The AdoMet synthesis-mediated beneficial metabolic effects, which resulted from consuming both Met and ATP, mimicked CR. Indeed, stimulating AdoMet synthesis activated the universal energy-sensing regulator Snf1, which is the S. cerevisiae ortholog of AMP-activated protein kinase (AMPK), resulting in lifespan extension. Furthermore, our findings revealed that S-adenosyl-l-homocysteine contributed to longevity with a higher accumulation of AdoMet only under the severe CR (0.05% glucose) conditions. Thus, our data uncovered molecular links between Met metabolites and lifespan, suggesting a unique function of AdoMet as a reservoir of Met and ATP for cell survival.

Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells.

Small-molecule inhibitors of Ca2+-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca2+-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl2 in the growth medium of Ca2+-sensitive Δzds1 strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca2+ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δmpk1 (lacking the Mpk1 MAP kinase pathway) but not Δcnb1 (lacking the calcineurin pathway) strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant.

Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin.

Calcineurin, which is a Ca(2+)/calmodulin-dependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcineurin is activated, inhibiting cellular growth as a result, we screened for a mutant(s) whose temperature sensitivity would be suppressed by FK506 from the budding yeast non-essential gene deletion library. We found that the temperature sensitivity of cells in which the conserved Verprolin VRP1 gene had been deleted, which gene is required for actin organization and endocytosis, was suppressed by either FK506 or by cnb1 deletion. Indeed, the calcineurin activity increased significantly in the ∆vrp1 cells. Finally, we demonstrated that the ∆vrp1 strain to be useful as an indicator in a positive screening for bioactive compounds inhibiting calcineurin.

Kujigamberol, a new dinorlabdane diterpenoid isolated from 85million years old Kuji amber using a biotechnological assay.

A new compound, 15,20-dinor-5,7,9-labdatriene-18-ol (1), named kujigamberol, was isolated from amber, fossilized tree resin from the Kuji area in Japan, has been dated as being 85 million years old (late Cretaceous). Kujigamberol was identified using the hypersensitive mutant yeast (zds1∆ erg3∆ pdr1∆ pdr3∆) with respect to Ca(2+)-signal transduction. The structure was elucidated on the basis of spectroscopic analysis including 1D NMR, 2D NMR and HR-EI-MS. It was different from known diterpenoids with a similar activity isolated from Baltic amber (agathic acid 15-monomethyl ester (2), dehydroabietic acid (3) and pimaric acid (4)). Kujigamberol showed glycogen synthase kinase-3ß (GSK-3ß) inhibition activity involving the growth restored activity against the mutant yeast and was cytotoxic to HL60 cells (IC(50)=19.6 µM).

Implication of Ca2+ in the regulation of replicative life span of budding yeast.

In eukaryotic cells, Ca(2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca(2+)/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca(2+) sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca(2+) signaling and life span in yeast. Here we show that Ca(2+) affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca(2+) levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca(2+) homeostasis/Ca(2+) signaling are required to regulate longevity in budding yeast.

Identification of ricinoleic acid as an inhibitor of Ca2+ signal-mediated cell-cycle regulation in budding yeast.

Free fatty acids exhibit diverse biological effects such as the regulation of immune responses in humans and animals. To investigate the biological effect of fatty acids in the model eukaryotic organism yeast, we examined the activity of various fatty acids in a yeast-based drug-screening system designed to detect the small-molecule compounds that inhibit Ca(2+)-signal-mediated cell-cycle regulation. Among the fatty acids examined, ricinoleic acid markedly alleviated the deleterious physiological effects induced by the compelled activation of Ca(2+) signaling by external CaCl(2), such as the polarized bud growth and the growth arrest in the G(2) phase. In accordance with the physiological consequences induced by ricinoleic acid, it diminished the Ca(2+)-induced phosphorylation of Cdc28p at Tyr-19, concomitant with the decrease in the Ca(2+)-stimulated expression levels of Cln2p and Swe1p.

Pinostrobin from Boesenbergia pandurata is an inhibitor of Ca2+-signal-mediated cell-cycle regulation in the yeast Saccharomyces cerevisiae.

Upon searching plant extracts for inhibitors of the Ca(2+) signaling pathway using the zds1Delta-yeast proliferation based assay, a crude rhizome extract of Boesenbergia pandurata was found to be strongly positive, and from this extract pinostrobin, alpinetin, and pinocembrin chalcone were isolated as active components. Further biochemical experiments confirmed that pinostrobin possesses inhibitory activity on the Ca(2+) signals involved in the control of G2/M phase cell cycle progression in Saccharomyces cerevisiae.

Combinatorial gene overexpression and recessive mutant gene introduction in sake yeast.

Industrial yeast strains are generally diploid and are often defective in sporulation. Such strains are hence thought to be less tractable for manipulation by genetic engineering. To facilitate more reliable genetic manipulation of the diploid yeast Japanese sake, we constructed variants of this strain that were homozygous for a URA3 deletion, homozygous for either MATa or MATalpha, and homozygous for either the his3 or the lys4 mutation. A ura3-null genotype enabled gene targeting to be undertaken more easily. The TDH3 promoter was inserted upstream of six yeast genes that have been implicated in flavor control to drive their constitutive overexpression. The homozygous MAT alleles, combined with the non-complementary auxotrophic mutations in the targeted transformants, allowed for tetraploid selection through mating. This resulted in the combinatorial construction of tetraploid strains that overexpress two different genes simultaneously. In addition, a recessive mutant gene, sah1-1, that is known to overproduce S-adenosylmethionine, was introduced into the diploid sake strain by the replacement of one wild-type allele and subsequent disruption of the other. The resulting sah1-1/sah1Delta::URA3 strain produced higher amounts of S-adenosylmethionine than the wild type. The novel sake yeast diploid strains we generated in this study can thus undergo simple PCR-mediated gene manipulation and mating in a manner analogous to established laboratory strains. Moreover, none of these sake strains had extraneous sequences, and they are thus suitable for use in commercial applications.

New eremophilane sesquiterpenoid compounds, eremoxylarins A and B directly inhibit calcineurin in a manner independent of immunophilin.

In the course of our screening program for a new Ca2+-signal transduction inhibitor using the hypersensitive mutant strain of Saccharomyces cerevisiae (zds1Delta erg3Delta pdr1Delta pdr3Delta), new eremophilane sesquiterpenoid compounds eremoxylarins A and B were found to restore the growth inhibition caused by the hyperactivated Ca2+-signal. These compounds showed lethal activity against the mpk1Delta strain, specifically, compared to the cnb1Delta strain, and ion-sensitive activity against the wild-type strain in the presence of LiCl, indicating that their molecular target might be the calcineurin pathway. They inhibited calcineurin directly without immunophilins at IC50=2.7 and 1.4 microM with competitive inhibition in vitro. The eremophilane sesquiterpenoid structure in eremoxylarins could be a good leading compound for immunosuppressants and anti-allergy drugs.

Identification of Saccharomyces cerevisiae Tub1 alpha-tubulin as a potential target for NKH-7, a cytotoxic 1-naphthol derivative compound.

In a screening for small-molecule compounds that alleviate the deleterious effects of external CaCl(2) on zds1 Delta strain yeast, we found 2-((1-(hydroxymethyl) cyclohexyl) methyl) naphthalen-1-ol (NKH-7) to be an active compound. NKH-7 also inhibited cell growth at higher concentrations. To identify its target in growth inhibition, we isolated NKH-7-resistant mutants and selected those mutants that exhibited dominant or semi-dominant resistance specifically to NKH-7. By gene cloning, a TUB1 mutant gene encoding alpha-tubulin with a Ser248Pro mutation was identified. Deletion of the TUB3 gene, a minor gene encoding alpha-tubulin, led to supersensitivity to NKH-7. Cellular tubulin-containing arrays as visualized by green fluorescent protein (GFP)-labeled alpha-tubulin diminished rapidly on exposure to the inhibitor. The mutation was situated proximal to the alpha-beta interface of alpha-tubulin in microtubule protofilaments, suggesting the possibility that NKH-7 affects the hydrolysis of GTP bound to beta-tubulin. A functional connection perhaps exists between the tubulin inhibition and Ca(2+)-dependent cell-cycle regulation.

Identification of Tup1 and Cyc8 mutations defective in the responses to osmotic stress.

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general transcriptional corepressor. This repression is mediated by recruitment of the Tup1-Cyc8 complex to target promoters through sequence-specific DNA-binding proteins such as Sko1, which mediates the HOG pathway-dependent regulation. We identified tup1 and cyc8 mutant alleles as the suppressor of osmo-sensitivity of the hog1Delta strain. In these mutants, although the expression of the genes under the control of DNA-binding proteins other than Sko1 was apparently normal, the Sko1-regulated genes GRE2 and AHP1 were derepressed under non-stress conditions, suggesting that the Tup1 and Cyc8 mutant proteins were specifically defective in the repression of the Sko1-dependent genes. Chromatin immunoprecipitation analyses of the GRE2 promoter in the mutants demonstrated that the Sko1-Tup1-Cyc8 complex was localized to the promoter, together with Gcn5/SAGA, suggesting that the erroneous recruitment of SAGA to the promoter led to the derepression.

Inhibition of Ca2+-signal-dependent growth regulation by radicicol in budding yeast.

Compelled activation of Ca(2+) signaling by exposure of zds1Delta strain Saccharomyces cerevisiae cells to external CaCl(2) leads to characteristic physiological consequences such as growth inhibition in the G(2) phase and polarized bud growth. Screening of microbial metabolites for activity alleviating the deleterious physiological effects of external CaCl(2) identified the Hsp90 inhibitor radicicol as an inhibitor of Ca(2+)-signal-dependent cell-cycle regulation in yeast. Radicicol alleviated analogous physiological effects due to the expression of a constitutively active form of calcineurin or overexpression of Swe1, the negative regulatory kinase of the Cdc28-Clb complex. Western blot analysis indicated that radicicol inhibited Ca(2+)-induced accumulation of Swe1 and Cln2.

The cytoplasmic region of alpha-1,6-mannosyltransferase Mnn9p is crucial for retrograde transport from the Golgi apparatus to the endoplasmic reticulum in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Och1p and Mnn9p mannosyltransferases are localized in the cis-Golgi. Attempts to live image Och1p and Mnn9p tagged with green fluorescent protein or red fluorescent protein, respectively, using a high-performance confocal laser scanning microscope system resulted in simultaneous visualization of the native proteins in a living cell. Our observations revealed that Och1p and Mnn9p are not always colocalized to the same cisternae. The difference in the dynamics of these mannosyltransferases may reflect differences in the mechanisms for their retention in the cis-Golgi, since it has been reported that Mnn9p cycles between the endoplasmic reticulum and the cis-Golgi whereas Och1p does not (Z. Todorow, A. Spang, E. Carmack, J. Yates, and R. Schekman, Proc. Natl. Acad. Sci. USA 97:13643-13648, 2000). We investigated the localization of chimeric proteins of Mnn9p and Och1p in sec12 and erd1 mutant cells. A chimeric protein, M16/O16, which consists of the N-terminal cytoplasmic region of Mnn9p and the transmembrane and luminal region of Och1p, behaved like Mnn9p, suggesting that the N-terminal cytoplasmic region is important for the intracellular dynamics of Mnn9p. This observation is supported by results from subcellular-fractionation experiments. Mutational analysis revealed that two arginine residues in the N-terminal region of Mnn9p are important for the chimeric protein to cycle between the endoplasmic reticulum and the Golgi apparatus.

YCM1008A, a novel Ca2+-signaling inhibitor, produced by Fusarium sp. YCM1008.

In the course of screening for drugs that suppress the Ca(2+)-mediated growth inhibition in a yeast mutant, we found that the metabolite of Fusarium sp. strain YCM1008 inhibited Ca(2+)-signaling. A novel pyrano-pyridone, YCM1008A was isolated from the fermentation broth using HLB column chromatography followed by HPLC, and the structure was elucidated by spectral analysis. YCM1008A suppressed Ca(2+)-induced growth inhibition of the Saccharomyces cerevisiae (Deltazds1Deltasyr1) mutant.

Physiological roles of calcineurin in Saccharomyces cerevisiae with special emphasis on its roles in G2/M cell-cycle regulation.

Calcineurin, a highly conserved Ca(2+)/CaM-dependent protein phosphatase, plays key regulatory roles in diverse biological processes from yeast to humans. Genetic and molecular analyses of the yeast model system have proved successful in dissecting complex regulatory pathways mediated by calcineurin. Saccharomyces cerevisiae calcineurin is not essential for growth under laboratory conditions, but becomes essential for survival under certain stress conditions, and is required for stress-induced expression of the genes for ion transporters and cell-wall synthesis. Yeast calcineurin, in collaboration with a Mpk1 MAP kinase cascade, is also important in G(2) cell-cycle regulation due to its action in a checkpoint-like mechanism. Genetic and molecular analysis of the Ca(2+)-dependent cell-cycle regulation has revealed an elaborate mechanism for the calcineurin-dependent regulation of the G(2)/M transition, in which calcineurin multilaterally activates Swe1, a negative regulator of the Cdc28/Clb complex, at the transcriptional, posttranslational, and degradation levels.

Identification of Saccharomyces cerevisiae ribosomal protein L3 as a target of curvularol, a G1-specific inhibitor of mammalian cells.

The cellular target of curvularol, a G1-specific cell-cycle inhibitor of mammalian cells, was identified by a genetic approach in Saccharomyces cerevisiae. Since the wild-type W303 strain was highly resistant to curvularol, a drug hypersensitive parental strain was constructed in which various genes implicated in general drug resistance had been disrupted. Curvularol resistant mutants were isolated, and strains that exhibited a semi-dominant, curvularol-specific resistance phenotype were selected. All five strains examined were classified into a single genetic complementation group designated YCR1. A mutant gene responsible for curvularol resistance was identified as an allele of the RPL3 gene encoding the ribosomal protein L3. Sequence analysis of the mutant genes revealed that Trp255Cys and Trp255Leu substitutions of Rpl3p are responsible for curvularol resistance. Rpl3p mutants in which Trp255 residue was replaced by other amino acids were constructed. All of these replacements led to varying degrees of increased resistance to curvularol and growth defects.

Involvement of calcineurin-dependent degradation of Yap1p in Ca2+-induced G2 cell-cycle regulation in Saccharomyces cerevisiae.

The Ca2+-activated pathways in Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1p, a negative regulatory kinase that inhibits the Cdc28p/Clb complex. We isolated the YAP1 gene as a multicopy suppressor of calcium sensitivity owing to the loss of ZDS1, a negative regulator of SWE1 and CLN2 gene expression. YAP1 deletion on a zds1delta background exacerbated the Ca2+-related phenotype. Yap1p was degraded in a calcineurin-dependent manner when cells were exposed to calcium. In yap1delta cells, the expression level of the RPN4 gene encoding a transcription factor for the subunits of the ubiquitin-proteasome system was diminished. The deletion of YAP1 gene or RPN4 gene led to the accumulation of Swe1p and Cln2p. Yap1p was a substrate of calcineurin in vivo and in vitro. The calcineurin-mediated Yap1p degradation seems to be a long adaptive response that assures a G2 delay in response to a stress that causes the activation of the calcium signalling pathways.