Radiobiological Characterization of 64CuCl2 as a Simple Tool for Prostate Cancer Theranostics.

64CuCl2 has recently been proposed as a promising agent for prostate cancer (PCa) theranostics, based on preclinical studies in cellular and animal models, and on the increasing number of human studies documenting its use for PCa diagnosis. Nevertheless, the use of 64CuCl2 raises important radiobiological questions that have yet to be addressed. In this work, using a panel of PCa cell lines in comparison with a non-tumoral prostate cell line, we combined cytogenetic approaches with radiocytotoxicity assays to obtain significant insights into the cellular consequences of exposure to 64CuCl2. PCa cells were found to exhibit increased 64CuCl2 uptake, which could not be attributed to increased expression of the main copper cellular importer, hCtr1, as had been previously suggested. Early DNA damage and genomic instability were also higher in PCa cells, with the tumoral cell lines exhibiting deficient DNA-damage repair upon exposure to 64CuCl2. This was corroborated by the observation that 64CuCl2 was more cytotoxic in PCa cells than in non-tumoral cells. Overall, we showed for the first time that PCa cells had a higher sensitivity to 64CuCl2 than healthy cells, supporting the idea that this compound deserved to be further evaluated as a theranostic agent in PCa.

The Zygosaccharomyces bailii transcription factor Haa1 is required for acetic acid and copper stress responses suggesting subfunctionalization of the ancestral bifunctional protein Haa1/Cup2.

BACKGROUND: The food spoilage yeast species Zygosaccharomyces bailii exhibits an extraordinary capacity to tolerate weak acids, in particular acetic acid. In Saccharomyces cerevisiae, the transcription factor Haa1 (ScHaa1) is considered the main player in genomic expression reprogramming in response to acetic acid stress, but the role of its homologue in Z. bailii (ZbHaa1) is unknown. RESULTS: In this study it is demonstrated that ZbHaa1 is a ScHaa1 functional homologue by rescuing the acetic acid susceptibility phenotype of S. cerevisiae haa1Δ. The disruption of ZbHAA1 in Z. bailii IST302 and the expression of an extra ZbHAA1 copy confirmed ZbHAA1 as a determinant of acetic acid tolerance. ZbHaa1 was found to be required for acetic acid stress-induced transcriptional activation of Z. bailii genes homologous to ScHaa1-target genes. An evolutionary analysis of the Haa1 homologues identified in 28 Saccharomycetaceae species genome sequences, including Z bailii, was carried out using phylogenetic and gene neighbourhood approaches. Consistent with previous studies, this analysis revealed a group containing pre-whole genome duplication species Haa1/Cup2 single orthologues, including ZbHaa1, and two groups containing either Haa1 or Cup2 orthologues from post-whole genome duplication species. S. cerevisiae Cup2 (alias Ace1) is a transcription factor involved in response and tolerance to copper stress. Taken together, these observations led us to hypothesize and demonstrate that ZbHaa1 is also involved in copper-induced transcriptional regulation and copper tolerance. CONCLUSIONS: The transcription factor ZbHaa1 is required for adaptive response and tolerance to both acetic acid and copper stresses. The subfunctionalization of the single ancestral Haa1/Cup2 orthologue that originated Haa1 and Cup2 paralogues after whole genome duplication is proposed.

Search for genes responsible for the remarkably high acetic acid tolerance of a Zygosaccharomyces bailii-derived interspecies hybrid strain.

BACKGROUND: Zygosaccharomyces bailii is considered the most problematic acidic food spoilage yeast species due to its exceptional capacity to tolerate high concentrations of weak acids used as fungistatic preservatives at low pH. However, the mechanisms underlying its intrinsic remarkable tolerance to weak acids remain poorly understood. The identification of genes and mechanisms involved in Z. bailii acetic acid tolerance was on the focus of this study. For this, a genomic library from the highly acetic acid tolerant hybrid strain ISA1307, derived from Z. bailii and a closely related species and isolated from a sparkling wine production plant, was screened for acetic acid tolerance genes. This screen was based on the transformation of an acetic acid susceptible Saccharomyces cerevisiae mutant deleted for the gene encoding the acetic acid resistance determinant transcription factor Haa1. RESULTS: The expression of 31 different DNA inserts from ISA1307 strain genome was found to significantly increase the host cell tolerance to acetic acid. The in silico analysis of these inserts was facilitated by the recently available genome sequence of this strain. In total, 65 complete or truncated ORFs were identified as putative determinants of acetic acid tolerance and an S. cerevisiae gene homologous to most of them was found. These include genes involved in cellular transport and transport routes, protein fate, protein synthesis, amino acid metabolism and transcription. The role of strong candidates in Z. bailii and S. cerevisiae acetic acid tolerance was confirmed based on homologous and heterologous expression analyses. CONCLUSIONS: ISA1307 genes homologous to S. cerevisiae genes GYP8, WSC4, PMT1, KTR7, RKR1, TIF3, ILV3 and MSN4 are proposed as strong candidate determinants of acetic acid tolerance. The ORF ZBAI_02295 that contains a functional domain associated to the uncharacterised integral membrane proteins of unknown function of the DUP family is also suggested as a relevant tolerance determinant. The genes ZbMSN4 and ZbTIF3, encoding a putative stress response transcription factor and a putative translation initiation factor, were confirmed as determinants of acetic acid tolerance in both Z. bailii and S. cerevisiae. This study provides valuable indications on the cellular components, pathways and processes to be targeted in order to control food spoilage by the highly acetic acid tolerant Z. bailii and Z. bailii-derived strains. Additionally, this information is essential to guide the improvement of yeast cells robustness against acetic acid if the objective is their use as cell factories.

The YEASTRACT database: an upgraded information system for the analysis of gene and genomic transcription regulation in Saccharomyces cerevisiae.

The YEASTRACT (http://www.yeastract.com) information system is a tool for the analysis and prediction of transcription regulatory associations in Saccharomyces cerevisiae. Last updated in June 2013, this database contains over 200,000 regulatory associations between transcription factors (TFs) and target genes, including 326 DNA binding sites for 113 TFs. All regulatory associations stored in YEASTRACT were revisited and new information was added on the experimental conditions in which those associations take place and on whether the TF is acting on its target genes as activator or repressor. Based on this information, new queries were developed allowing the selection of specific environmental conditions, experimental evidence or positive/negative regulatory effect. This release further offers tools to rank the TFs controlling a gene or genome-wide response by their relative importance, based on (i) the percentage of target genes in the data set; (ii) the enrichment of the TF regulon in the data set when compared with the genome; or (iii) the score computed using the TFRank system, which selects and prioritizes the relevant TFs by walking through the yeast regulatory network. We expect that with the new data and services made available, the system will continue to be instrumental for yeast biologists and systems biology researchers.