Reactive oxygen species accumulation is synchronised with growth inhibition of temperature-sensitive recAts polA Escherichia coli.
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ABSTRACT:
SUBMITTER: Kaidow A
PROVIDER: S-EPMC9200703 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redox-gated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feed-back mechanism to prevent ROS production.
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Project description:Purpose: Despite considerable efforts to improve treatment modalities for cholangiocarcinoma, a common form of malignant tumor, its long-term survival rate remains poor. Hydroxychloroquine (HCQ) is a 4-aminoquinoline derivative antimalarial drug that has antimalarial and autophagy inhibition effects and exhibits comprehensive therapeutic effects on various cancers. In this study, we aimed to explore the anticancer potential and the underlying molecular mechanism of HCQ in cholangiocarcinoma treatment in vitro and in vivo. Methods: Autophagy-related genes (ARGs) were obtained from the Human Autophagy Database and Molecular Signatures Database, and the expression profiles of ARGs were downloaded from the database of The Cancer Genome Atlas. Different expression gene sets were performed using R software. The Gene Ontology and KEGG enrichment analyses were performed to reveal significantly enriched signaling pathways and to identify differentially expressed genes in cholangiocarcinoma tissues. HuCCT-1 and CCLP-1 cells were exposed to different concentrations of HCQ. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. Cell apoptosis and cycle arrest were detected by the Live/Dead cell assay and flow cytometry (FCM). The inhibition of autophagy was observed using fluorescence microscopy. The reactive oxygen species levels were assessed by fluorescence microscopy and flow cytometry. The protein levels were determined by western blot. A cholangiocarcinoma cell line xenograft model was used to evaluate the antitumor activity of HCQ in vivo. Results: Compared with normal tissues, there were 141 ARGs with an aberrant expression in cholangiocarcinoma tissues which were mainly enriched in autophagy-related processes. Inhibition of autophagy by HCQ effectively suppressed cholangiocarcinoma in vitro and in vivo. HCQ inhibited cell proliferation and induced apoptosis and cycle arrest in vitro by increasing ROS accumulation, which was involved in autophagy inhibition. The ROS scavenger reduced l-glutathione distinctly weakened HCQ-induced cell apoptosis and viability inhibition in cholangiocarcinoma cells. In addition, HCQ inhibited growth of cholangiocarcinoma cell line xenograft tumors. Conclusion: HCQ could inhibit cell proliferation and induce apoptosis in cholangiocarcinoma by triggering ROS accumulation via autophagy inhibition, which makes HCQ a potential antitumor drug candidate for cholangiocarcinoma treatment.
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Project description:Persister cells are difficult to eliminate because they are tolerant to antibiotic stress. In the present study, using artificially induced Escherichia coli persister cells, we found that reactive oxygen species (ROS) have greater effects on persister cells than on exponential cells. Thus, we examined which types of ROS could effectively eliminate persister cells and determined the mechanisms underlying the effects of these ROS. Ultraviolet (UV) light irradiation can kill persister cells, and bacterial viability is markedly increased under UV shielding. UV induces the production of ROS, which kill bacteria by moving toward the shielded area. Electron spin resonance-based analysis confirmed that hydroxyl radicals are produced by UV irradiation, although singlet oxygen is not produced. These results clearly revealed that ROS sterilizes persister cells more effectively compared to the sterilization of exponential cells (** p < 0.01). These ROS do not injure the bacterial cell wall but rather invade the cell, followed by cell killing. Additionally, the sterilization effect on persister cells was increased by exposure to oxygen plasma during UV irradiation. However, vapor conditions decreased persister cell sterilization by reducing the levels of hydroxyl radicals. We also verified the effect of ROS against bacteria in biofilms that are more resistant than planktonic cells. Although UV alone could not completely sterilize the biofilm bacteria, UV with ROS achieved complete sterilization. Our results demonstrate that persister cells strongly resist the effects of antibiotics and starvation stress but are less able to withstand exposure to ROS. It was shown that ROS does not affect the cell membrane but penetrates it and acts internally to kill persister cells. In particular, it was clarified that the hydroxy radical is an effective sterilizer to kill persister cells.
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Project description:Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.
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Project description:BACKGROUND:Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). METHODS:Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. RESULTS:Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 ?g/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. CONCLUSION:The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 .
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