The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence

This is the final version (corrected proof). The final published version is available from Public Library of Science via the DOI in this recordData Availability: Short reads were submitted to the NCBI’s Sequence Read Archive under accession number SRP154617 (https://www.ncbi.nlm.nih.gov/sra/?term=SRP154617). The ChIPseq data are available from NCBI SRA (sequence read archive) database under accession number PRJNA574873 (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA574873&o=acc_s%3Aa).Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES)Wellcome TrustUniversity of MacauNational Science Foundation (NSF)Vanderbilt UniversityHoward Hughes Medical Institut

Cytosolic thioredoxin peroxidase I is essential for the antioxidant defense of yeast with dysfunctional mitochondria

The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol-speciric antioxidant), was investigated in the oxidative stress response of Saccharomyces ceravisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H2O2 in Cells, which could be related to their elevated sensitivity to oxidative stress. In. fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H2O2. In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.509343043

Regulation of mitochondrial thioredoxin peroxidase I expression by two different pathways: One dependent on cAMP and the other on heme

Mitochondrial isoform of thioredoxin peroxidase (mTPx I) is an antioxidant protein recently described in Saccharomyces cerevisiae. Here we characterized pathways that lead to mTPx I induction in two situations: growth in media containing low glucose concentrations and treatment with peroxides. The induction of mTPx I by growth on low glucose concentrations was dependent on cAMP and on the transcription factors Msn2p/Msn4p as demonstrated by northern blot experiments using yeast strains with deletion of MSN2 and MSN4 genes and also using a strain permeable to cAMP. mTPx I expression was also induced by peroxides in a time- and dose-dependent manner and varied with the carbon source present in the media. Deletion of HAP1 or inhibition of heme synthesis abolished induction of mTPx I by H2O2 on cells which were grown in media containing glucose, indicating that Hap1p is involved in the regulation of this process. mTPx I was induced by H2O2 on glycerol/ethanol-containing media, but we could not associate any transcription factor with this phenomenon. Finally, mTPx I also induced by t-butyl hydroperoxide in a Hap1p-independent manner. In conclusion, mTPx I expression is under a complex regulatory network, which involves, at least, two signaling pathways: one sensing the carbon source (which is signalized by cAMP) and the other sensing the intracellular redox state (which is signalized by heme). (C) 2002 Elsevier Science Inc.32327828

Yeast oxidative stress response - Influences of cytosolic thioredoxin peroxidase I and of the mitochondrial functional state

We investigated the changes in the oxidative stress response of yeast cells suffering mitochondrial dysfunction that could impair their viability. First, we demonstrated that cells with this dysfunction rely exclusively on cytosolic thioredoxin peroxidase I (cTPxI) and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2-induced death. This cTPxI-dependent protection could be related to its dual functions, as peroxidase and as molecular chaperone, suggested by mixtures of low and high molecular weight oligomeric structures of cTPxI observed in cells challenged with H2O2. We found that cTPxI deficiency leads to increased basal sulfhydryl levels and transcriptional activation of most of the H2O2-responsive genes, interpreted as an attempt by the cells to improve their antioxidant defense. On the other hand, mitochondrial dysfunction, specifically the electron transport blockage, provoked a huge depletion of sulfhydryl groups after H2O2 treatment and reduced the H2O2-mediated activation of some genes otherwise observed, impairing cell defense and viability. The transcription factors Yap1 and Skn7 are crucial for the antioxidant response of cells under inhibited electron flow condition and probably act in the same pathway of cTPxI to protect cells affected by this disorder. Yap1 cellular distribution was not affected by cTpxI deficiency and by mitochondrial dysfunction, in spite of the observed expression alterations of several Yap1-target genes, indicating alternative mechanisms of Yap1 activation/deactivation. Therefore, we propose that cTPxI is specifically important in the protection of yeast with mitochondrial dysfunction due to its functional versatility as an antioxidant, chaperone and modulator of gene expression.273480581

Catalases and thioredoxin peroxidase protect Saccharomyces cerevisiae against Ca2+-induced mitochondrial membrane permeabilization and cell death

The involvement of reactive oxygen species in Ca2+-induced mitochondrial membrane permeabilization and cell viability was studied using yeast cells in which the thioredoxin peroxidase (TPx) gene was disrupted and/or catalase was inhibited by 3-amino-1,2,4-triazole (ATZ) treatment. Wild-type Saccharomyces cerevisiae cells were very resistant to Ca2+ and inorganic phosphate or t-butyl hydroperoxide-induced mitochondrial membrane permeabilization, but suffered an immediate decrease in mitochondrial membrane potential when treated with Ca2+ and the dithiol binding reagent phenylarsine oxide. In contrast, S, cerevisiae spheroblasts lacking the TPx gene and/or treated with ATZ suffered a decrease in mitochondrial membrane potential, generated higher amounts of hydrogen peroxide and had decreased viability under these conditions. In all cases, the decrease in mitochondrial membrane potential could be inhibited by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, dithiothreitol or ADP, but not by cyclosporin A. We conclude that TPx and catalase act together, maintaining cell viability and protecting S, cerevisiae mitochondria against Ca2+-promoted membrane permeabilization, which presents similar characteristics to mammalian permeability transition. (C) 2000 Federation of European Biochemical Societies.473217718

Selectively labeling the heterologous protein in Escherichia coli for NMR studies: A strategy to speed up NMR spectroscopy

Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process, In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin, Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The H-1/N-15 heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective N-15-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein, The method has high yields and a good H-1/N-15 HMQC spectrum can be obtained with 50 mi of Mo growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The N-15-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the H-1 spectrum shows several other resonances from other proteins of the cell lysate, The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others, (C) 2001 Academic Press.148114214