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Molecular Microbiology Oct 2023Saccharomyces cerevisiae Pso2/SNM1 is essential for DNA interstrand crosslink (ICL) repair; however, its mechanism of action remains incompletely understood. While...
Saccharomyces cerevisiae Pso2/SNM1 is essential for DNA interstrand crosslink (ICL) repair; however, its mechanism of action remains incompletely understood. While recent work has revealed that Pso2/Snm1 is dual-localized in the nucleus and mitochondria, it remains unclear whether cell-intrinsic and -extrinsic factors regulate its subcellular localization and function. Herein, we show that Pso2 undergoes ubiquitination and phosphorylation, but not SUMOylation, in unstressed cells. Unexpectedly, we found that methyl methanesulfonate (MMS), rather than ICL-forming agents, induced robust SUMOylation of Pso2 on two conserved residues, K97 and K575, and that SUMOylation markedly increased its abundance in the mitochondria. Reciprocally, SUMOylation had no discernible impact on Pso2 translocation to the nucleus, despite the presence of steady-state levels of SUMOylated Pso2 across the cell cycle. Furthermore, substitution of the invariant residues K97 and K575 by arginine in the Pso2 SUMO consensus motifs severely impaired SUMOylation and abolished its translocation to the mitochondria of MMS-treated wild type cells, but not in unstressed cells. We demonstrate that whilst Siz1 and Siz2 SUMO E3 ligases catalyze Pso2 SUMOylation, the former plays a dominant role. Notably, we found that the phenotypic characteristics of the SUMOylation-defective mutant Pso2 closely mirrored those observed in the Pso2Δ petite mutant. Additionally, leveraging next-generation sequencing analysis, we demonstrate that Pso2 mitigates MMS-induced damage to mitochondrial DNA (mtDNA). Viewed together, our work offers previously unknown insights into the link between genotoxic stress-induced SUMOylation of Pso2 and its preferential targeting to the mitochondria, as well as its role in attenuating MMS-induced mtDNA damage.
Topics: Humans; Saccharomyces cerevisiae; Methyl Methanesulfonate; DNA, Mitochondrial; Sumoylation; Saccharomyces cerevisiae Proteins; Endodeoxyribonucleases; DNA Damage; Mitochondria; Translocation, Genetic; Ubiquitin-Protein Ligases
PubMed: 37649278
DOI: 10.1111/mmi.15145 -
Scientific Reports Aug 2023Trichosporon asahii is an opportunistic pathogenic fungus that causes severe and sometimes fatal infections in immunocompromised patients. Hog1, a mitogen-activated...
Trichosporon asahii is an opportunistic pathogenic fungus that causes severe and sometimes fatal infections in immunocompromised patients. Hog1, a mitogen-activated protein kinase, regulates the stress resistance of some pathogenic fungi, however its role in T. asahii has not been investigated. Here, we demonstrated that the hog1 gene-deficient T. asahii mutant is sensitive to high temperature, cell membrane stress, oxidative stress, and antifungal drugs. Growth of the hog1 gene-deficient T. asahii mutant was delayed at 40 °C. The hog1 gene-deficient T. asahii mutant also exhibited sensitivity to sodium dodecyl sulfate, hydrogen peroxide, menadione, methyl methanesulfonate, UV exposure, and antifungal drugs such as amphotericin B under a glucose-rich condition. Under a glucose-restricted condition, the hog1 gene-deficient mutant exhibited sensitivity to NaCl and KCl. The virulence of the hog1 gene-deficient mutant against silkworms was attenuated. Moreover, the viability of the hog1 gene-deficient mutant decreased in the silkworm hemolymph. These phenotypes were restored by re-introducing the hog1 gene into the gene-deficient mutant. Our findings suggest that Hog1 plays a critical role in regulating cellular stress responses in T. asahii.
Topics: Animals; Antifungal Agents; Basidiomycota; Fungi; Bombyx; Glucose
PubMed: 37598230
DOI: 10.1038/s41598-023-40825-y -
Analytical Chemistry Aug 2023Genotoxic impurities (GTIs) occurred in drugs, and food and environment pose a threat to human health. Accurate and sensitive evaluation of GTIs is of significance. Ames...
Genotoxic impurities (GTIs) occurred in drugs, and food and environment pose a threat to human health. Accurate and sensitive evaluation of GTIs is of significance. Ames assay is the existing gold standard method. However, the pathogenic bacteria model lacks metabolic enzymes and requires mass GTIs, leading to insufficient safety, accuracy, and sensitivity. Whole-cell microbial sensors (WCMSs) can use normal strains to simulate the metabolic environment, achieving safe, sensitive, and high-throughput detection and evaluation for GTIs. Here, based on whether GTIs causing DNA alkylation required metabolic enzymes or not, two DNA repair-responsive engineered WCMS systems were constructed including -WCMS and yeast-WCMS. A DNA repair-responsive promoter as a sensing element was coupled with an enhanced green fluorescent protein as a reporter to construct plasmids for introduction into WCMS. The promoter was screened out in the -WCMS, while the promoter was selected for the yeast-WCMS. Different and yeast strains were modified by gene knockout and mutation to eliminate the interference and enhance the GTI retention in cells and further improved the sensitivity. Finally, GTI consumption of WCMS for the evaluation of methyl methanesulfonate (MMS) and nitrosamines was decreased to 0.46-8.53 μg and 0.068 ng-2.65 μg, respectively, decreasing 2-3 orders of magnitude compared to traditional methods. This study provided a novel approach to measure GTIs with different DNA damage pathways at a molecular level and facilitated the high-throughput screening and sensitive evaluation of GTIs.
Topics: Humans; High-Throughput Screening Assays; Saccharomyces cerevisiae; Escherichia coli; DNA Repair; DNA Damage
PubMed: 37589895
DOI: 10.1021/acs.analchem.3c02245 -
Journal of Analytical Methods in... 2023Methyl 7,7'-dimethoxy-5'-(morpholinomethyl)-[4,4'-bibenzo[d][1,3] dioxole]-5-carboxylate methanesulfonate (IMM) is an innovative drug for the treatment of nonalcoholic...
Methyl 7,7'-dimethoxy-5'-(morpholinomethyl)-[4,4'-bibenzo[d][1,3] dioxole]-5-carboxylate methanesulfonate (IMM) is an innovative drug for the treatment of nonalcoholic fatty liver disease (NAFLD) owing to its high efficacy and low toxicity. In this study, five minor impurities (I, II, III, IV, and V) were identified and analyzed using spectroscopic evidence, chemical synthetic methods, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The impurities included hydrolysates and oxidation by-products extracted from both the drug in its final formulation and during synthesis. Toxicity prediction revealed potential carcinogenicity of impurity V containing an N-oxygen fragment. A reliable and selective HPLC method for the quantitative analysis of impurities I-IV and a sensitive HPLC-MS/MS method for potential genotoxic impurity V were developed and optimized. The methods were validated based on the International Council for Harmonization guidelines. Satisfactory linearity was obtained for the analytes over the range of 0.1-2.0 g/mL for impurities I-IV and 0.3-30.0 ng/mL for impurity V, and in all cases, the fitting correlation coefficients exceeded 0.999. The obtained limits of detection values were 0.05 ng/mL and 0.005 g/mL for impurity V and impurities I-IV, respectively. The precision and repeatability of the methods were less than 1.08% and 8.72% for each impurity. The recovery percentages of all impurities were in the range of 91.18%-111.27%, with the relative standard deviation of less than 3.69%. The greenness assessment of the HPLC method and the HPLC-MS/MS method were evaluated by using AGREE software with a score value of 0.72 and 0.68, respectively. The recommended procedures that were accurate, specific, and ecofriendly were applied to the existing active pharmaceutical ingredients of IMM, and they generated satisfactory results.
PubMed: 37575214
DOI: 10.1155/2023/3116223 -
Cells Jul 2023The comet assay in Drosophila has been used in the last few years to study DNA damage responses (DDR) in different repair-mutant strains and to compare them to analyze...
The comet assay in Drosophila has been used in the last few years to study DNA damage responses (DDR) in different repair-mutant strains and to compare them to analyze DNA repair. We have used this approach to study interactions between DNA repair pathways in vivo. Additionally, we have implemented an ex vivo comet assay, in which nucleoids from treated and untreated cells were incubated ex vivo with cell-free protein extracts from individuals with distinct repair capacities. Four strains were used: wild-type OregonK (), nucleotide excision repair mutant , dmPolQ protein mutant , and the double mutant . Methyl methanesulfonate (MMS) was used as a genotoxic agent. Both approaches were performed with neuroblasts from third-instar larvae; they detected the effects of the NER and dmPolQ pathways on the DDR to MMS and that they act additively in this response. Additionally, the ex vivo approach quantified that , , and the double mutant strains presented, respectively, 21.5%, 52.9%, and 14.8% of strain activity over MMS-induced damage. Considering the homology between mammals and Drosophila in repair pathways, the detected additive effect might be extrapolated even to humans, demonstrating that Drosophila might be an excellent model to study interactions between repair pathways.
Topics: Humans; Animals; Comet Assay; Drosophila; Drosophila melanogaster; DNA Repair; DNA Damage; Methyl Methanesulfonate; Mammals
PubMed: 37566058
DOI: 10.3390/cells12151979 -
Genetics Aug 2023In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream...
In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences. Upon genotoxic stress, cells activate the DNA damage response to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1-to-S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA sequencing reveals that Opi1 plays a central role in modulating essential biological processes during methyl methanesulfonate (MMS)-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factor Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.
Topics: Basic Helix-Loop-Helix Transcription Factors; DNA Damage; Gene Expression Regulation, Fungal; Inositol; Phospholipids; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomycetales; Transcription Factors
PubMed: 37440469
DOI: 10.1093/genetics/iyad130 -
Plant Direct Jul 2023Phytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma...
Phytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. -methyladenosine (mA) is the most common internal modification of the eukaryotic Messenger RNA (mRNA). As one of the species susceptible to phytoplasma infection, the pathogenesis and mechanism of Paulownia has been extensively studied by scholars, but the mA transcriptome map of () has not been reported. Therefore, this study aimed to explore the effect of phytoplasma infection on mA modification of and obtained the whole transcriptome mA map in by mA-seq. The mA-seq results of Paulownia witches' broom (PaWB) disease and healthy samples indicate that PaWB infection increased the degree of mA modification of . The correlation analysis between the RNA-seq and mA-seq data detected that a total of 315 differentially methylated genes were predicted to be significantly differentially expressed at the transcriptome level. Moreover, the functions of PaWB-related genes were predicted by functional enrichment analysis, and two genes related to maintenance of the basic mechanism of stem cells in shoot apical meristem were discovered. One of the genes encodes the receptor protein kinase CLV2 (Paulownia_LG2G000076), and the other gene encodes the homeobox transcription factor STM (Paulownia_LG15G000976). In addition, genes F-box (Paulownia_LG17G000760) and MSH5 (Paulownia_LG8G001160) had exon skipping and mutually exclusive exon types of alternative splicing in PaWB-infected seedling treated with methyl methanesulfonate, and mA modification was found in mA-seq results. Moreover, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) verified that the alternative splicing of these two genes was associated with mA modification. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA mA modification in the process of PaWB. In future studies, we plan to verify genes directly related to PaWB and methylation-related enzymes in Paulownia to elucidate the pathogenic mechanism of PaWB caused by phytoplasma invasion.
PubMed: 37426893
DOI: 10.1002/pld3.508 -
Genes & Genetic Systems Sep 2023Homologous recombination (HR) is a highly accurate mechanism for repairing DNA double-strand breaks (DSBs) that arise from various genotoxic insults and blocked...
Homologous recombination (HR) is a highly accurate mechanism for repairing DNA double-strand breaks (DSBs) that arise from various genotoxic insults and blocked replication forks. Defects in HR and unscheduled HR can interfere with other cellular processes such as DNA replication and chromosome segregation, leading to genome instability and cell death. Therefore, the HR process has to be tightly controlled. Protein N-terminal acetylation is one of the most common modifications in eukaryotic organisms. Studies in budding yeast implicate a role for NatB acetyltransferase in HR repair, but precisely how this modification regulates HR repair and genome integrity is unknown. In this study, we show that cells lacking NatB, a dimeric complex composed of Nat3 and Mdm2, are sensitive to the DNA alkylating agent methyl methanesulfonate (MMS), and that overexpression of Rad51 suppresses the MMS sensitivity of nat3Δ cells. Nat3-deficient cells have increased levels of Rad52-yellow fluorescent protein foci and fail to repair DSBs after release from MMS exposure. We also found that Nat3 is required for HR-dependent gene conversion and gene targeting. Importantly, we observed that nat3Δ mutation partially suppressed MMS sensitivity in srs2Δ cells and the synthetic sickness of srs2Δ sgs1Δ cells. Altogether, our results indicate that NatB functions upstream of Srs2 to activate the Rad51-dependent HR pathway for DSB repair.
Topics: Acetyltransferases; DNA Repair; DNA-Binding Proteins; Homologous Recombination; Methyl Methanesulfonate; N-Terminal Acetyltransferase B; N-Terminal Acetyltransferases; Rad51 Recombinase; Rad52 DNA Repair and Recombination Protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37331807
DOI: 10.1266/ggs.23-00013 -
Free Radical Biology & Medicine Aug 2023Mitochondria-targeted coenzyme Q10 (Mito-ubiquinone, Mito-quinone mesylate, or MitoQ) was shown to be an effective antimetastatic drug in patients with triple-negative...
Redox-crippled MitoQ potently inhibits breast cancer and glioma cell proliferation: A negative control for verifying the antioxidant mechanism of MitoQ in cancer and other oxidative pathologies.
Mitochondria-targeted coenzyme Q10 (Mito-ubiquinone, Mito-quinone mesylate, or MitoQ) was shown to be an effective antimetastatic drug in patients with triple-negative breast cancer. MitoQ, sold as a nutritional supplement, prevents breast cancer recurrence. It potently inhibited tumor growth and tumor cell proliferation in preclinical xenograft models and in vitro breast cancer cells. The proposed mechanism of action involves the inhibition of reactive oxygen species by MitoQ via a redox-cycling mechanism between the oxidized form, MitoQ, and the fully reduced form, MitoQH2 (also called Mito-ubiquinol). To fully corroborate this antioxidant mechanism, we substituted the hydroquinone group (-OH) with the methoxy group (-OCH). Unlike MitoQ, the modified form, dimethoxy MitoQ (DM-MitoQ), lacks redox-cycling between the quinone and hydroquinone forms. DM-MitoQ was not converted to MitoQ in MDA-MB-231 cells. We tested the antiproliferative effects of both MitoQ and DM-MitoQ in human breast cancer (MDA-MB-231), brain-homing cancer (MDA-MB-231BR), and glioma (U87MG) cells. Surprisingly, DM-MitoQ was slightly more potent than MitoQ (IC = 0.26 μM versus 0.38 μM) at inhibiting proliferation of these cells. Both MitoQ and DM-MitoQ potently inhibited mitochondrial complex I-dependent oxygen consumption (IC = 0.52 μM and 0.17 μM, respectively). This study also suggests that DM-MitoQ, which is a more hydrophobic analog of MitoQ (logP: 10.1 and 8.7) devoid of antioxidant function and reactive oxygen species scavenging ability, can inhibit cancer cell proliferation. We conclude that inhibition of mitochondrial oxidative phosphorylation by MitoQ is responsible for inhibition of breast cancer and glioma proliferation and metastasis. Blunting the antioxidant effect using the redox-crippled DM-MitoQ can serve as a useful negative control in corroborating the involvement of free radical-mediated processes (e.g., ferroptosis, protein oxidation/nitration) using MitoQ in other oxidative pathologies.
Topics: Humans; Female; Antioxidants; Hydroquinones; Reactive Oxygen Species; Breast Neoplasms; Ubiquinone; Oxidation-Reduction; Cell Proliferation; Oxidative Stress; Glioma; Organophosphorus Compounds
PubMed: 37321281
DOI: 10.1016/j.freeradbiomed.2023.06.009 -
Redox Biology Aug 2023Using S-phase synchronized RPE1-hTERT cells exposed to the DNA damaging agent, methyl methanesulfonate, we show the existence of a redox state associated with...
Using S-phase synchronized RPE1-hTERT cells exposed to the DNA damaging agent, methyl methanesulfonate, we show the existence of a redox state associated with replication stress-induced senescence termed senescence-associated redox state (SA-redox state). SA-redox state is characterized by its reactivity with superoxide-sensing fluorescent probes such as dihydroethidine, lucigenin and mitosox and peroxynitrite or hydroxyl radical sensing probe hydroxyphenyl fluorescein (HPF) but not the hydrogen peroxide (HO) reactive fluorescent probe CM-HDCFDA. Measurement of GSH and GSSH also reveals that SA-redox state mitigates the level of total GSH rather than oxidizes GSH to GSSG. Moreover, supporting the role of superoxide (O) in the SA-redox state, we show that incubation of senescent RPE1-hTERT cells with the O scavenger, Tiron, decreases the reactivity of SA-redox state with the oxidants' reactive probes lucigenin and HPF while the HO antioxidant N-acetyl cysteine has no effect. SA-redox state does not participate in the loss of proliferative capacity, G2/M cell cycle arrest or the increase in SA-β-Gal activity. However, SA-redox state is associated with the activation of NF-κB, dictates the profile of the Senescence Associated Secretory Phenotype, increases TFEB protein level, promotes geroconversion evidenced by increased phosphorylation of S6K and S6 proteins, and influences senescent cells response to senolysis. Furthermore, we provide evidence for crosstalk between SA redox state, p53 and p21. While p53 mitigates the establishment of SA-redox state, p21 is critical for the sustained reinforcement of the SA-redox state involved in geroconversion and resistance to senolysis.
Topics: Superoxides; Hydrogen Peroxide; Cellular Senescence; Tumor Suppressor Protein p53; Oxidation-Reduction
PubMed: 37285741
DOI: 10.1016/j.redox.2023.102757