Project description:Arsenic metalloid is a double-edge sword. On the one hand it is a very toxic and powerful carcinogen, and on the other it has been successfully used in the treatment of acute promyelocytic leukemia. In order to prevent the deleterious effects caused by arsenic compounds, almost all living organisms have developed mechanisms to eliminate it. In this study genome-wide response of S. cerevisiae to arsenic shows that this metal interferes with genes involved in the iron homeostasis including those encoding proteins that function in iron uptake, incorporation into Fe–S clusters, and more. In addition our data indicate that Yap1 transcriptionally controls the iron homeostasis regulator AFT2 as well as its direct target, MRS4. Most importantly in response to arsenate exposure Yap1 strongly regulates the expression of several genes involved in the Fe-S proteins biosynthesis, namely NBP35 and YFH1. Interestingly mRNA levels encoding Fet3, Ferro-O2-oxidoreductase required for high-affinity iron uptake, are drastically destabilized upon arsenic exposure. Such destabilization is due to the 5’ to 3’ exonuclease Xrn1 localized in the P Bodies. Moreover FET3 mRNA decay is not mediated by Cth2 and is independent on the formation of ROS responsible for the toxic effects of arsenic compounds. Strikingly, in presence of arsenate fet3 mutant shows resistance over the wild-type which leads us to suggest that Fet3 has a role in arsenic toxicity. Unexpectedly arsenic treatment seems to activate the non-reductive iron uptake in order to maintain the cellular iron homeostasis. Furthermore our genetic, biochemical, and physiological analysis demonstrate that aft1 mutant is sensitive to arsenic compounds and such phenotype is reversible upon addition of iron. We also show that arsenic exposure induces iron deficiency in aft1 mutant. In conclusion this work shows for the first time that arsenic, a chemotherapy drug used to treat a specific type of acute promyelocytic leukemia (APL), disrupts iron homeostasis and our results suggest that this disruption is independent on ROS generation. Finally we provide preliminary data confirming that such disruption also takes place in mammalian cells, an observation that can be very relevant in term of clinical applications.

Project description:Saxitoxin is a potent neurotoxin produced by several species of dinoflagellates and cyanobacteria. The molecular target of saxitoxin in higher eukaryotes is the voltage-gated sodium channel; however, its target in lower eukaryotic organisms remains unknown. The goal of this study was to obtain the transcriptional fingerprint of the model lower eukaryote Saccharomyces cerevisiae upon exposure to saxitoxin to identify potential genes suitable for biomarker development. Microarray analyses identified multiple genes associated with copper and iron homeostasis and sulfur metabolism as significantly differentially expressed upon exposure to saxitoxin; these results were verified with quantitative reverse-transcriptase PCR (qRT-PCR). Additionally, the qRT-PCR assays were used to generate expression profiles in a subset of the differentially regulated genes across multiple exposure times and concentrations, the results of which demonstrated that overall, genes tended to respond in a consistent manner to the toxin. In general, the genes encoding the metallothioneins CUP1 and CRS5 were induced following exposure to saxitoxin, while those encoding the ferric/cupric reductase FRE1 and the copper uptake transporter CTR1 were repressed. The gene encoding the multicopper ferroxidase FET3, part of the high-affinity iron uptake system, was also induced in all treatments, along with the STR3 gene, which codes for the cystathionine beta-lyase found in the methionine biosynthetic pathway. Experiment Overall Design: Cultures of S. cerevisiae S288C were grown to OD600=1.0 in YPD (1% yeast extract, 2% peptone, 2% dextrose). Four hundred microliters from individual 10 mL overnight cultures were transferred to sterile tubes and saxitoxin added to a final concentration of 16 ÂµM. Sample were mixed gently and incubated at 30Â° C for 45 min. Controls consisted of deionized water. Three biological replicates were performed under these conditions for microarray analysis. Cells were harvested via sequential centrifugation and placed immediately in liquid nitrogen.Total RNA was extracted from frozen cell pellets using a protocol that combined the hot acid phenol method with the RNEasy kit. Total RNA was processed by the Affymetrix Core Facility at the University of Tennessee (Knoxville, TN) following the protocols of the Affymetrix GeneChip Expression Analysis Technical Manual. MAS5.0 was used to provide present/absent calls; genes absent in all six samples were then removed from the data set. Intensity values for all transcripts were obtained from the scanned image files of the chips. The intensity values of the six arrays were background-corrected and normalized with the GC-RMA algorithm.

Project description:Arsenic metalloid is a double-edge sword. On the one hand it is a very toxic and powerful carcinogen, and on the other it has been successfully used in the treatment of acute promyelocytic leukemia. In order to prevent the deleterious effects caused by arsenic compounds, almost all living organisms have developed mechanisms to eliminate it. In this study genome-wide response of S. cerevisiae to arsenic shows that this metal interferes with genes involved in the iron homeostasis including those encoding proteins that function in iron uptake, incorporation into Fe–S clusters, and more. In addition our data indicate that Yap1 transcriptionally controls the iron homeostasis regulator AFT2 as well as its direct target, MRS4. Most importantly in response to arsenate exposure Yap1 strongly regulates the expression of several genes involved in the Fe-S proteins biosynthesis, namely NBP35 and YFH1. Interestingly mRNA levels encoding Fet3, Ferro-O2-oxidoreductase required for high-affinity iron uptake, are drastically destabilized upon arsenic exposure. Such destabilization is due to the 5’ to 3’ exonuclease Xrn1 localized in the P Bodies. Moreover FET3 mRNA decay is not mediated by Cth2 and is independent on the formation of ROS responsible for the toxic effects of arsenic compounds. Strikingly, in presence of arsenate fet3 mutant shows resistance over the wild-type which leads us to suggest that Fet3 has a role in arsenic toxicity. Unexpectedly arsenic treatment seems to activate the non-reductive iron uptake in order to maintain the cellular iron homeostasis. Furthermore our genetic, biochemical, and physiological analysis demonstrate that aft1 mutant is sensitive to arsenic compounds and such phenotype is reversible upon addition of iron. We also show that arsenic exposure induces iron deficiency in aft1 mutant. In conclusion this work shows for the first time that arsenic, a chemotherapy drug used to treat a specific type of acute promyelocytic leukemia (APL), disrupts iron homeostasis and our results suggest that this disruption is independent on ROS generation. Finally we provide preliminary data confirming that such disruption also takes place in mammalian cells, an observation that can be very relevant in term of clinical applications. yap1yap8 mutant cells independently transformed with pRS416 and YcpLac111, YcpLac111-YAP1, or pRS416-YAP8 were grown in triplicates in SC-URA-LEU containing 2mM of AsV until exponential growth phase, and RNA was extracted, labeled, and hybridized to Affymetrix Yeast Genome S98 arrays.

Dataset Information

Activation of two different resistance mechanisms in Saccharomyces cerevisiae upon exposure to octanoic and decanoic acids.

Publications

Activation of two different resistance mechanisms in Saccharomyces cerevisiae upon exposure to octanoic and decanoic acids.

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