Linkage of oxidative stress and mitochondrial dysfunctions to spontaneous culture degeneration in Aspergillus nidulans.
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SUBMITTER: Li L
PROVIDER: S-EPMC3916646 | biostudies-literature | 2014 Feb
REPOSITORIES: biostudies-literature
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Linkage of oxidative stress and mitochondrial dysfunctions to spontaneous culture degeneration in Aspergillus nidulans.
Li Lin L Hu Xiao X Xia Yongliang Y Xiao Guohua G Zheng Peng P Wang Chengshu C
Molecular & cellular proteomics : MCP 20131217 2
Filamentous fungi including mushrooms frequently and spontaneously degenerate during subsequent culture maintenance on artificial media, which shows the loss or reduction abilities of asexual sporulation, sexuality, fruiting, and production of secondary metabolites, thus leading to economic losses during mass production. To better understand the underlying mechanisms of fungal degeneration, the model fungus Aspergillus nidulans was employed in this study for comprehensive analyses. First, linkag ...[more]
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Project description:Filamentous fungi including mushrooms frequently and spontaneously degenerate during subsequent culture maintenance on artificial media, which shows the loss or reduction abilities of asexual sporulation, sexuality, fruiting and production of secondary metabolites, thus leading to economic losses during mass production. To better understand the underlying mechanisms of fungal degeneration, the model fungus Aspergillus nidulans was employed in this study for comprehensive analyses. First, linkage of oxidative stress to culture degeneration was evident in A. nidulans. Taken together with the verifications of cell biology and biochemical data, a comparative mitochondrial proteome analysis revealed that, unlike the healthy wild type, a spontaneous fluffy sector culture of A. nidulans demonstrated the characteristics of mitochondrial dysfunctions. Relative to the wild type, the features of cytochrome c release, calcium overload and up-regulation of apoptosis inducing factors evident in sector mitochondria suggested a linkage of fungal degeneration to cell apoptosis. However, the sector culture could still be maintained for generations without the signs of growth arrest. Up-regulation of the heat shock protein chaperones, anti-apoptotic factors and DNA repair proteins in the sector could account for the compromise in cell death. The results of this study not only shed new lights on the mechanisms of spontaneous degeneration of fungal cultures but will also provide alternative biomarkers to monitor fungal culture degeneration. Label-free quantitative proteomic analysis of the WT and Sec mt proteins was performed as described previously. Briefly, purified mitochondrial proteins (100 ug) were diluted in the lysis buffer to a concentration of 5 g/ul and carboxyamidomethylated in 50 mM iodoacetamide for 40 min at room temperature in the dark. The proteins were digested with an endoprotease Lys-C (Roche Applied Science, Indianapolis, IN) at a final substrate/enzyme ratio of 100:1 (w/w) at 37 C for 3 h. The Lys-C digests were further treated with modified sequencing grade trypsin (Roche Applied Science, Indianapolis, IN) at a final substrate/enzyme ratio of 50:1 (w/w) at 37 C for 20 h. After digestion, the peptide mixture was passed through an ultra-filter unit (Millipore, Billerica, MA) with a molecular weight cut-off of 10 kDa and acidified by formic acid (0.1% final concentration) for mass spectrum analysis. A linear ion trap-orbitrap liquid chromatography-tandem mass spectrometry system (LTQ-Orbitrap, Thermo Fisher Scientific, San Josse, CA) equipped with a nanospray ion source was used for full MS scan analysis followed by five MS/MS scans in the LTQ on the five most intense ions from the MS spectrum. Three parallel runs were performed consecutively for each sample. The software DeCyder MS (ver 2.0) (GE Healthcare, Pittsburgh, PA) was used to generate the peak lists from all the runs and the data were then automatically searched using the SEQUEST (ver. 2.7) (Thermo Fisher Scientific, San Josse, CA) against the A. nidulans genome archive (ver. S03-M05-R01, containing 10,644 protein entries) at AspGD database (www.aspgd.org) with the following parameters: peptide mass tolerance set to 10 ppm; fragment tolerance set to 0.8 Da, and two missed trypsin cleavages. Carbamidomethylation of cysteine was searched as a fixed modification, whereas N-acetyl protein and oxidation of methionine were searched as variable modifications. For protein identification, all peptide matches were filtered by a maximum false discovery rate (FDR) index less than 0.01, delta Cn larger than 0.1 and Xcorr scores of greater than 1.7, 2.0 and 3.0 for +1, +2, and +3 charged ions, respectively. The peptides were discarded for further analysis if mapped to more than two different proteins. A protein was considered identifiable with at least one unique peptide detected for more than twice in the three replicates of each sample. The MS data have been deposited in the PRIDE proteomics identification database under accession numbers ? List of the identified peptides and matched proteins is provided in Supplemental Table 1. Quantification of proteins was performed by estimating the normalized spectral index (SIN) for each protein, which combines the features of peptide count, spectral count, fragment-ion intensity and protein length as described by Griffin et al.. Reliability of technical repeats was analyzed by calculating the multivariate Pearson correlation coefficients using the software SigmaPlot (ver. 8.0). FDR estimation of differentially expressed proteins was conducted using a mixture model-based method. Heat mapping analysis was conducted using the program Matlab (R2009a). The significance of differentially expressed proteins between the samples was tested with the cutoff values of P less than or equal to 0.05 and FDR less than or equal to 0.05.
2019-11-25 | PXD000422 | Pride
Project description:BackgroundThe b-Zip transcription factor AtfA plays a key role in regulating stress responses in the filamentous fungus Aspergillus nidulans. To identify the core regulons of AtfA, we examined genome-wide expression changes caused by various stresses in the presence/absence of AtfA using A. nidulans microarrays. We also intended to address the intriguing question regarding the existence of core environmental stress response in this important model eukaryote.ResultsExamination of the genome wide expression changes caused by five different oxidative stress conditions in wild type and the atfA null mutant has identified a significant number of stereotypically regulated genes (Core Oxidative Stress Response genes). The deletion of atfA increased the oxidative stress sensitivity of A. nidulans and affected mRNA accumulation of several genes under both unstressed and stressed conditions. The numbers of genes under the AtfA control appear to be specific to a stress-type. We also found that both oxidative and salt stresses induced expression of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of additional clusters. Moreover, certain clusters were down-regulated by the stresses tested.ConclusionOur data suggest that the observed co-regulations were most likely consequences of the overlapping physiological effects of the stressors and not of the existence of a general environmental stress response. The function of AtfA in governing various stress responses is much smaller than anticipated and/or other regulators may play a redundant or overlapping role with AtfA. Both stress inducible and stress repressive regulations of secondary metabolism seem to be frequent features in A. nidulans.
| S-EPMC4482186 | biostudies-literature
Project description:Genome wide transcriptional changes induced by various types of oxidative stresses as well as salt stress were studied in a DatfA mutant and the appropriate control A. nidulans strains. Although a significant number of stereotypically regulated genes was identified (Core Oxidative Stress Response or COSR genes) when the global transcriptional effects of five different oxidative stress conditions were compared the number of co-regulated genes decreased to 13 when NaCl stress was included into the analyses. The appearance of only a few co-regulated genes and the great number of genes regulated merely by one certain type of stress do not support the existence of a S. cerevisiae-type Environmental Stress Response in A. nidulans. Deletion of atfA, a true functional ortholog of fission yeast’s “all-purpose” stress response transcription factor, increased the oxidative stress sensitivity of A. nidulans and affected the transcription of several genes under both unstressed and stressed conditions. The number of genes under AtfA control was quite stress-type dependent; e.g. deletion of atfA altered the transcription of a wide spectrum of genes under menadione sodium bisulfite stress but had only a minor effect on the transcriptome profiles when A. nidulans cultures were exposed to H2O2, tBOOH, NaCl and, especially, to diamide stress. These observations suggest that the function of AtfA in the regulation of various stress responses is much smaller than we thought before or other transcription factors can take over a number of AtfA’s functions when the atfA gene is deleted. It is noteworthy that both oxidative and salt stress induced the transcription of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of further clusters. Surprisingly, certain clusters were down-regulated by the stress conditions tested and the majority of them were not stress-responsive at all. Therefore, stress dependent regulation seems to be a frequent but far not a general feature of the regulation of secondary metabolism in A. nidulans.
2015-12-31 | GSE63019 | GEO
Project description:Genome wide transcriptional changes induced by various types of oxidative stresses as well as salt stress were studied in a DatfA mutant and the appropriate control A. nidulans strains. Although a significant number of stereotypically regulated genes was identified (Core Oxidative Stress Response or COSR genes) when the global transcriptional effects of five different oxidative stress conditions were compared the number of co-regulated genes decreased to 13 when NaCl stress was included into the analyses. The appearance of only a few co-regulated genes and the great number of genes regulated merely by one certain type of stress do not support the existence of a S. cerevisiae-type Environmental Stress Response in A. nidulans. Deletion of atfA, a true functional ortholog of fission yeastâs âall-purposeâ stress response transcription factor, increased the oxidative stress sensitivity of A. nidulans and affected the transcription of several genes under both unstressed and stressed conditions. The number of genes under AtfA control was quite stress-type dependent; e.g. deletion of atfA altered the transcription of a wide spectrum of genes under menadione sodium bisulfite stress but had only a minor effect on the transcriptome profiles when A. nidulans cultures were exposed to H2O2, tBOOH, NaCl and, especially, to diamide stress. These observations suggest that the function of AtfA in the regulation of various stress responses is much smaller than we thought before or other transcription factors can take over a number of AtfAâs functions when the atfA gene is deleted. It is noteworthy that both oxidative and salt stress induced the transcription of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of further clusters. Surprisingly, certain clusters were down-regulated by the stress conditions tested and the majority of them were not stress-responsive at all. Therefore, stress dependent regulation seems to be a frequent but far not a general feature of the regulation of secondary metabolism in A. nidulans. 14 samples, 7 with the control strain and 7 with an DatfA strain (each series contains samples from untreated as well as menadione, low concentration hidrogen-peroxide, high concentration hidrogen-peroxide, tert-butylhydroperoxide, diamide and NaCl treated cultures)
2015-12-31 | E-GEOD-63019 | biostudies-arrayexpress
Project description:Catalases are ubiquitous hydrogen peroxide-detoxifying enzymes that are central to the cellular antioxidant response. Of two catalase activities detected in the fungus Aspergillus nidulans, the catA gene encodes the spore-specific catalase A (CatA). Here we characterize a second catalase gene, identified after probing a genomic library with catA, and demonstrate that it encodes catalase B. This gene, designated catB, predicts a 721-amino-acid polypeptide (CatB) showing 78% identity to an Aspergillus fumigatus catalase and 61% identity to Aspergillus niger CatR. Notably, similar levels of identity are found when comparing CatB to Escherichia coli catalase HPII (43%), A. nidulans CatA (40%), and the predicted peptide of a presumed catA homolog from A. fumigatus (38%). In contrast, the last two peptides share a 79% identity. The catalase B activity was barely detectable in asexual spores (conidia), disappeared after germination, and started to accumulate 10 h after spore inoculation, throughout growth and conidiation. The catB mRNA was absent from conidia, and its accumulation correlated with catalase activity, suggesting that catB expression is regulated at the transcription level. In contrast, the high CatA activity found in spores was lost gradually during germination and growth. In addition to its developmental regulation, CatB was induced by H2O2, heat shock, paraquat, or uric acid catabolism but not by osmotic stress. This pattern of regulation and the protective role against H2O2 offered by CatA and CatB, at different stages of the A. nidulans life cycle, suggest that catalase gene redundancy performs the function of satisfying catalase demand at the two different stages of metabolic and genetic regulation represented by growing hyphae versus spores. Alternative H2O2 detoxification pathways in A. nidulans were indicated by the fact that catA/catB double mutants were able to grow in substrates whose catabolism generates H2O2.
| S-EPMC179108 | biostudies-other
Project description:Axonal degeneration is a prominent feature of many neurological disorders that are associated with mitochondrial dysfunction, including Parkinson's disease, motor neuron disease, and inherited peripheral neuropathies. Studies of the Wld(s) mutant mouse, which undergoes delayed Wallerian degeneration in response to axonal injury, suggest that axonal degeneration is an active process. Wld(s) mice also have slower axonal degeneration and disease progression in numerous models of neurodegenerative disease. The Wld(s) mutation results in the production of a chimeric protein that contains the full-length coding sequence of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), which alone is sufficient for axonal protection in vitro. To test the effects of increased Nmnat expression on axonal degeneration induced by mitochondrial dysfunction, we examined dorsal root ganglion (DRG) neurons treated with rotenone. Rotenone induced profound axonal degeneration in DRG neurons; however, this degeneration was delayed by expression of Nmnat. Nmnat-mediated protection resulted in decreased axonal accumulation and sensitivity to reactive oxygen species (ROS) but did not affect the change in the rate of rotenone-induced loss in neuronal ATP. Nmnat also prevented axonal degeneration caused by exposure to exogenous oxidants and reduced the level of axonal ROS after treatment with vincristine, further supporting the idea that Nmnat promotes axonal protection by mitigating the effects of ROS.
| S-EPMC2678678 | biostudies-other
Project description:Microorganisms have developed mechanisms to combat reactive nitrogen species (RNS); however, only a few of the fungal genes involved have been characterized. Here we screened RNS-resistant Aspergillus nidulans strains from fungal transformants obtained by introducing a genomic DNA library constructed in a multicopy vector. We found that the AN0121.3 gene (hemC) encodes a protein similar to the heme biosynthesis enzyme porphobilinogen deaminase (PBG-D) and facilitates RNS-tolerant fungal growth. The overproduction of PBG-D in A. nidulans promoted RNS tolerance, whereas PBG-D repression caused growth that was hypersensitive to RNS. PBG-D levels were comparable to those of cellular protoheme synthesis as well as flavohemoglobin (FHb; encoded by fhbA and fhbB) and nitrite reductase (NiR; encoded by niiA) activities. Both FHb and NiR are hemoproteins that consume nitric oxide and nitrite, respectively, and we found that they are required for maximal growth in the presence of RNS. The transcription of hemC was upregulated by RNS. These results demonstrated that PBG-D is a novel NO-tolerant protein that modulates the reduction of environmental NO and nitrite levels by FHb and NiR.
| S-EPMC3255638 | biostudies-literature
Project description:Microorganisms produce various secondary metabolites for growth and survival. During iron stress, they produce secondary metabolites termed siderophores. In the current investigation, antifungal activity of catecholate siderophore produced by Escherichia coli has been assessed against Aspergillus nidulans. Exogenous application of the bacterial siderophore to fungal cultures resulted in decreased colony size, increased filament length, and changes in hyphal branching pattern. Growth inhibition was accompanied with increased intracellular iron content. Scanning electron microscopy revealed dose-dependent alteration in fungal morphology. Fluorescent staining by propidium iodide revealed cell death in concert with growth inhibition with increasing siderophore concentration. Antioxidative enzyme activity was also compromised with significant increase in catalase activity and decrease in ascorbate peroxidase activity. Siderophore-treated cultures showed increased accumulation of reactive oxygen species as observed by fluorescence microscopy and enhanced membrane damage in terms of malondialdehyde content. Antifungal property might thus be attributed to xenosiderophore-mediated iron uptake leading to cell death. STRING analysis showed interaction of MirB (involved in transport of hydroxamate siderophore) and MirA (involved in transport of catecholate siderophore), confirming the possibility of uptake of iron-xenosiderophore complex through fungal transporters. MirA structure was modeled and validated with 95% residues occurring in the allowed region. In silico analysis revealed MirA-Enterobactin-Fe3+ complex formation. Thus, the present study reveals a promising antifungal agent in the form of catecholate siderophore and supports involvement of MirA fungal receptors in xenosiderophore uptake.
| S-EPMC8596375 | biostudies-literature
Project description:To better understand the molecular functions of the master stress-response regulator AtfA in Aspergillus nidulans, transcriptomic analyses of the atfA null mutant and the appropriate control strains exposed to menadione sodium bisulfite- (MSB-), t-butylhydroperoxide- and diamide-induced oxidative stresses were performed. Several elements of oxidative stress response were differentially expressed. Many of them, including the downregulation of the mitotic cell cycle, as the MSB stress-specific upregulation of FeS cluster assembly and the MSB stress-specific downregulation of nitrate reduction, tricarboxylic acid cycle, and ER to Golgi vesicle-mediated transport, showed AtfA dependence. To elucidate the potential global regulatory role of AtfA governing expression of a high number of genes with very versatile biological functions, we devised a model based on the comprehensive transcriptomic data. Our model suggests that an important function of AtfA is to modulate the transduction of stress signals. Although it may regulate directly only a limited number of genes, these include elements of the signaling network, for example, members of the two-component signal transduction systems. AtfA acts in a stress-specific manner, which may increase further the number and diversity of AtfA-dependent genes. Our model sheds light on the versatility of the physiological functions of AtfA and its orthologs in fungi.
| S-EPMC5523550 | biostudies-literature
Project description:Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration--defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.
| S-EPMC4503542 | biostudies-literature