RESUMO
Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.
Assuntos
RNA Polimerases Dirigidas por DNA , Plastídeos , Cloroplastos/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Nicotiana/genética , Fotossíntese , Plastídeos/enzimologiaRESUMO
Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.
Assuntos
Cloraminas/metabolismo , Peróxido de Hidrogênio/metabolismo , Ácido Hipocloroso/metabolismo , Neutrófilos/imunologia , Superóxidos/metabolismo , Tiocianatos/metabolismo , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cloraminas/imunologia , Expressão Gênica , Humanos , Peróxido de Hidrogênio/imunologia , Ácido Hipocloroso/imunologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Oxirredução , Peroxidase/genética , Peroxidase/imunologia , Transdução de Sinais , Superóxidos/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Tiocianatos/imunologia , Zimosan/farmacologiaRESUMO
Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.
Assuntos
Chaperonas Moleculares , Dobramento de Proteína , Chaperonas Moleculares/metabolismoRESUMO
The free radical nitric oxide (·NO) is a key mediator in different physiological processes such as vasodilation, neurotransmission, inflammation, and cellular immune responses, and thus preserving its bioavailability is essential. In several disease conditions, superoxide radical (O2·-) production increases and leads to the rapid "inactivation" of ·NO by a diffusion-controlled radical termination reaction that yields a potent and short-lived oxidant, peroxynitrite. This reaction not only limits ·NO bioavailability for physiological signal transduction but also can divert and switch the biochemistry of ·NO toward nitrooxidative processes. Indeed, since the early 1990s peroxynitrite (and its secondary derived species) has been linked to the establishment and progression of different acute and chronic human diseases and also to the normal aging process. Here, we revisit an earlier and classical review on the role of peroxynitrite in human physiology and pathology (Pacher P, Beckman J, Liaudet L. Physiol Rev 87: 315-424, 2007) and further integrate, update, and interpret the accumulated evidence over 30 years of research. Innovative tools and approaches for the detection, quantitation, and sub- or extracellular mapping of peroxynitrite and its secondary products (e.g., protein 3-nitrotyrosine) have allowed us to unambiguously connect the complex biochemistry of peroxynitrite with numerous biological outcomes at the physiological and pathological levels. Furthermore, our current knowledge of the ·NO/O2·- and peroxynitrite interplay at the cell, tissue, and organ levels is assisting in the discovery of therapeutic interventions for a variety of human diseases.
Assuntos
Ácido Peroxinitroso , Superóxidos , Biologia , Humanos , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismoRESUMO
Water resources are indispensable basic resources and important environmental carriers; the presence of organic contaminants in wastewater poses considerable risks to the health of both humans and ecosystems. Although the Fenton-like reactions using H2O2 as the oxidant to destroy organic pollutants are attractive, there are still challenges in improving reaction activity under neutral or even alkaline conditions. Herein, we designed a H2O2 activation pathway with O2â¢- as the main active species and elucidated that the spin interaction between Fe sites and coordinated O atoms effectively promotes the generation of the key intermediate Fe-*OOH. Furthermore, we successfully captured and analyzed the Fe-*OOH intermediate by in situ Raman spectroscopy. When applying FBOB to a continuous-flow reactor, CIP removal efficiency remained at around 90% within 600 min of continuous operation, achieving excellent efficiency, stability, and pH tolerance in removing pollutants.
RESUMO
Many genes with distinct molecular functions have been linked to genetically heterogeneous amyotrophic lateral sclerosis (ALS), including SuperOxide Dismutase 1 (SOD1) and Valosin-Containing Protein (VCP). SOD1 converts superoxide to oxygen and hydrogen peroxide. VCP acts as a chaperon to regulate protein degradation and synthesis and various other cellular responses. Although the functions of these two genes differ, in the current report we show that overexpression of wild-type VCP in mice enhances lifespan and maintains the size of neuromuscular junctions (NMJs) of both male and female SOD1G93A mice, a well-known ALS mouse model. Although VCP exerts multiple functions, its regulation of ER formation and consequent protein synthesis has been shown to play the most important role in controlling dendritic spine formation and social and memory behaviors. Given that SOD1 mutation results in protein accumulation and aggregation, it may direct VCP to the protein degradation pathway, thereby impairing protein synthesis. Since we previously showed that the protein synthesis defects caused by Vcp deficiency can be improved by leucine supplementation, to confirm the role of the VCP-protein synthesis pathway in SOD1-linked ALS, we applied leucine supplementation to SOD1G93A mice and, similar to Vcp overexpression, we found that it extends SOD1G93A mouse lifespan. In addition, the phenotypes of reduced muscle strength and fewer NMJs of SOD1G93A mice are also improved by leucine supplementation. These results support the existence of crosstalk between SOD1 and VCP and suggest a critical role for protein synthesis in ASL. Our study also implies a potential therapeutic treatment for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Modelos Animais de Doenças , Leucina , Longevidade , Camundongos Transgênicos , Junção Neuromuscular , Fenótipo , Superóxido Dismutase-1 , Proteína com Valosina , Animais , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Camundongos , Junção Neuromuscular/metabolismo , Feminino , Masculino , Longevidade/genética , Leucina/farmacologia , Leucina/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismoRESUMO
Nutrients are not only organic compounds fueling bioenergetics and biosynthesis, but also key chemical signals controlling growth and metabolism. Nutrients enormously impact the production of reactive oxygen species (ROS), which play essential roles in normal physiology and diseases. How nutrient signaling is integrated with redox regulation is an interesting, but not fully understood, question. Herein, we report that superoxide dismutase 1 (SOD1) is a conserved component of the mechanistic target of rapamycin complex 1 (mTORC1) nutrient signaling. mTORC1 regulates SOD1 activity through reversible phosphorylation at S39 in yeast and T40 in humans in response to nutrients, which moderates ROS level and prevents oxidative DNA damage. We further show that SOD1 activation enhances cancer cell survival and tumor formation in the ischemic tumor microenvironment and protects against the chemotherapeutic agent cisplatin. Collectively, these findings identify a conserved mechanism by which eukaryotes dynamically regulate redox homeostasis in response to changing nutrient conditions.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nutrientes/metabolismo , Fosforilação/fisiologia , Superóxido Dismutase-1/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/fisiologia , Metabolismo Energético/fisiologia , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2â¢-) during substrate oxidation. The O2â¢- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2â¢- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.
Assuntos
Aminoácido Oxirredutases , Domínio Catalítico , Oxigênio , Pseudomonas aeruginosa , Superóxidos , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Oxigênio/metabolismo , Oxigênio/química , Superóxidos/metabolismo , Superóxidos/química , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/genética , Prótons , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cinética , Oxirredução , Mutação , Substituição de Aminoácidos , Arginina/química , Arginina/metabolismoRESUMO
With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) that is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with Km=2.7 µM and Vmax=794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in lungs of control mice with even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage.
RESUMO
A wide variety of stresses have been proposed to exert killing effects upon bacteria by stimulating the intracellular formation of reactive oxygen species (ROS). A key part of the supporting evidence has often been the ability of antioxidant compounds to protect the cells. In this study, some of the most-used antioxidants-thiourea, glutathione, N-acetylcysteine, and ascorbate-have been examined. Their ability to quench superoxide and hydrogen peroxide was verified in vitro, but the rate constants were orders of magnitude too slow for them to have an impact upon superoxide and peroxide concentrations in vivo, where these species are already scavenged by highly active enzymes. Indeed, the antioxidants were unable to protect the growth and ROS-sensitive enzymes of E. coli strains experiencing authentic oxidative stress. Similar logic posits that antioxidants cannot substantially quench hydroxyl radicals inside cells, which contain abundant biomolecules that react with them at diffusion-limited rates. Indeed, antioxidants were able to protect cells from DNA damage only if they were applied at concentrations that slow metabolism and growth. This protective effect was apparent even under anoxic conditions, when ROS could not possibly be involved, and it was replicated when growth was similarly slowed by other means. Experimenters should discard the use of antioxidants as a way of detecting intracellular oxidative stress and should revisit conclusions that have been based upon such experiments. The notable exception is that these compounds can effectively degrade hydrogen peroxide from environmental sources before it enters cells.
Assuntos
Antioxidantes , Escherichia coli , Peróxido de Hidrogênio , Estresse Oxidativo , Espécies Reativas de Oxigênio , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Superóxidos/metabolismo , Glutationa/metabolismo , Dano ao DNA , Ácido Ascórbico/farmacologia , Ácido Ascórbico/metabolismo , Tioureia/farmacologia , Tioureia/análogos & derivados , Acetilcisteína/farmacologia , Acetilcisteína/metabolismoRESUMO
The clinical manifestations of sporadic amyotrophic lateral sclerosis (ALS) vary widely. However, the current classification of ALS is mainly based on clinical presentations, while the roles of electrophysiological and biomedical biomarkers remain limited. Herein, we investigated a group of patients with sporadic ALS and an ALS mouse model with superoxide dismutase 1 (SOD1)/G93A transgenes using nerve excitability tests (NET) to investigate axonal membrane properties and chemical precipitation, followed by enzyme-linked immunosorbent assay analysis to measure plasma misfolded protein levels. Six of 19 patients (31.6%) with sporadic ALS had elevated plasma misfolded SOD1 protein levels. In sporadic ALS patients, only those with elevated misfolded SOD1 protein levels showed an increased inward rectification in the current-threshold (I/V) curve and an increased threshold reduction in the hyperpolarizing threshold electrotonus (TE) in the NET study. Two familial ALS patients with SOD1 mutations also exhibited similar electrophysiological patterns of NET. For patients with sporadic ALS showing significantly increased inward rectification in the I/V curve, we noted an elevation in plasma misfolded SOD1 level, but not in total SOD1, misfolded C9orf72, or misfolded phosphorylated TDP43 levels. Computer simulations demonstrated that the aforementioned axonal excitability changes are likely associated with an increase in hyperpolarization-activated cyclic nucleotide-gated (HCN) current. In SOD1/G93A mice, NET also showed an increased inward rectification in the I/V curve, which could be reversed by a single injection of the HCN channel blocker, ZD7288. Daily treatment of SOD1/G93A mice with ZD7288 partially prevented the early motor function decline and spinal motor neuron death. In summary, sporadic ALS patients with elevated plasma misfolded SOD1 exhibited similar patterns of motor axonal excitability changes as familial ALS patients and ALS mice with mutant SOD1 genes, suggesting the existence of SOD1-associated sporadic ALS. The observed NET pattern of increased inward rectification in the I/V curve was attributable to an elevation in the HCN current in SOD1-associated ALS.
RESUMO
The potent immunomodulatory function of mesenchymal stem/stromal cells (MSCs) elicited by proinflammatory cytokines IFN-γ and TNF-α (IT) is critical to resolve inflammation and promote tissue repair. However, little is known about how the immunomodulatory capability of MSCs is related to their differentiation competency in the inflammatory microenvironment. In this study, we demonstrate that the adipocyte differentiation and immunomodulatory function of human adipose tissue-derived MSCs (MSC(AD)s) are mutually exclusive. Mitochondrial reactive oxygen species (mtROS), which promote adipocyte differentiation, were decreased in MSC(AD)s due to IT-induced upregulation of superoxide dismutase 2 (SOD2). Furthermore, knockdown of SOD2 led to enhanced adipogenic differentiation but reduced immunosuppression capability of MSC(AD)s. Interestingly, the adipogenic differentiation was associated with increased mitochondrial biogenesis and upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A/PGC-1α) expression. IT inhibited PGC-1α expression and decreased mitochondrial mass but promoted glycolysis in an SOD2-dependent manner. MSC(AD)s lacking SOD2 were compromised in their therapeutic efficacy in DSS-induced colitis in mice. Taken together, these findings indicate that the adipogenic differentiation and immunomodulation of MSC(AD)s may compete for resources in fulfilling the respective biosynthetic needs. Blocking of adipogenic differentiation by mitochondrial antioxidant may represent a novel strategy to enhance the immunosuppressive activity of MSCs in the inflammatory microenvironment.
Assuntos
Células-Tronco Mesenquimais , Superóxido Dismutase , Camundongos , Humanos , Animais , Diferenciação Celular , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Adipócitos , Células-Tronco Mesenquimais/metabolismoRESUMO
Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2â¢-) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, GAPDH, which in turn reroutes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2â¢- to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2â¢- toxicity, which only requires <1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.
Assuntos
NADP/metabolismo , Oxigênio/metabolismo , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase/metabolismo , Animais , Antioxidantes/metabolismo , Glicólise , Peróxido de Hidrogênio/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Superóxidos/metabolismoRESUMO
Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function, and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations, they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the YS deletion mutants results from distinct enzymatic vulnerabilities.IMPORTANCEBacteria require multiple trace metal ions for survival. Metal homeostasis relies on the tightly regulated expression of metal uptake, storage, and efflux proteins. Metal intoxication occurs when metal homeostasis is perturbed and often results from enzyme mis-metalation. In Bacillus subtilis, Mn-dependent superoxide dismutase (MnSOD) is the most abundant Mn-containing protein and is important for oxidative stress resistance. Here, we report novel roles for MnSOD and a co-regulated membrane protein, YqgC, in Mn homeostasis. Loss of both MnSOD and YqgC (but not the individual proteins) prevents the efficient expression of Mn efflux proteins and leads to a large-scale perturbation of the metabolome due to inhibition of Mg-dependent enzymes, including key chorismate-utilizing MST (menaquinone, siderophore, and tryptophan) family enzymes.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Magnésio , Manganês , Óperon , Superóxido Dismutase , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/enzimologia , Manganês/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Magnésio/metabolismoRESUMO
Clostridioides difficile causes a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, the mechanistic details of C. difficile intestinal colonization remain undefined. C. difficile is highly sensitive to oxygen and requires anaerobic conditions for in vitro growth. However, the mammalian gut is not devoid of oxygen, and C. difficile tolerates moderate oxidative stress in vivo. The C. difficile genome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protecting C. difficile against oxygen exposure. Insertional inactivation of sor rendered C. difficile more sensitive to superoxide, indicating that SOR contributes to antioxidant defense. Heterologous C. difficile sor expression in Escherichia coli conferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, a C. difficile SOR mutant exhibited global proteome changes under oxygen stress when compared to the parent strain. Collectively, our data establish the enzymatic activity of C. difficile SOR, confirm its role in protection against oxidative stress, and demonstrate SOR's broader impacts on the C. difficile vegetative cell proteome.IMPORTANCEClostridioides difficile is an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobe in vitro, C. difficile has been shown to tolerate low levels of oxygen in the mammalian host. Among other well-characterized antioxidant proteins, the C. difficile genome encodes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of SOR's enzymatic activity, including confirmation of its role in protecting C. difficile against oxidative stress. This furthers our understanding of C. difficile pathogenesis and presents a potential new avenue for targeted therapies.
Assuntos
Clostridioides difficile , Estresse Oxidativo , Oxigênio , Superóxidos , Clostridioides difficile/genética , Clostridioides difficile/enzimologia , Clostridioides difficile/metabolismo , Oxigênio/metabolismo , Superóxidos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Oxirredutases/metabolismo , Oxirredutases/genética , Regulação Bacteriana da Expressão GênicaRESUMO
Hepatitis B Virus (HBV) infection significantly elevates the risk of hepatocellular carcinoma (HCC), with the HBV X protein (HBx) playing a crucial role in cancer progression. Sorafenib, the primary therapy for advanced HCC, shows limited effectiveness in HBV-infected patients due to HBx-related resistance. Numerous studies have explored combination therapies to overcome this resistance. Sodium diethyldithiocarbamate (DDC), known for its anticancer effects and its inhibition of superoxide dismutase 1 (SOD1), is hypothesized to counteract sorafenib (SF) resistance in HBV-positive HCCs. Our research demonstrates that combining DDC with SF significantly reduces HBx and SOD1 expressions in HBV-positive HCC cells and human tissues. This combination therapy disrupts the PI3K/Akt/mTOR signalling pathway and promotes apoptosis by increasing reactive oxygen species (ROS) levels. These cellular changes lead to reduced tumour viability and enhanced sensitivity to SF, as evidenced by the synergistic suppression of tumour growth in xenograft models. Additionally, DDC-mediated suppression of SOD1 further enhances SF sensitivity in HBV-positive HCC cells and xenografted animals, thereby inhibiting cancer progression more effectively. These findings suggest that the DDC-SF combination could serve as a promising strategy for overcoming SF resistance in HBV-related HCC, potentially optimizing therapy outcomes.
Assuntos
Carcinoma Hepatocelular , Vírus da Hepatite B , Neoplasias Hepáticas , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Espécies Reativas de Oxigênio , Transdução de Sinais , Sorafenibe , Superóxido Dismutase-1 , Serina-Treonina Quinases TOR , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/virologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/genética , Animais , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Camundongos , Vírus da Hepatite B/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Apoptose/efeitos dos fármacos , Hepatite B/complicações , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Ditiocarb/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Camundongos Nus , Proliferação de Células/efeitos dos fármacos , Transativadores , Proteínas Virais Reguladoras e AcessóriasRESUMO
Insulin resistance is a significant contributor to the development of type 2 diabetes (T2D) and is associated with obesity, physical inactivity, and low maximal oxygen uptake. While intense and prolonged exercise may have negative effects, physical activity can have a positive influence on cellular metabolism and the immune system. Moderate exercise has been shown to reduce oxidative stress and improve antioxidant status, whereas intense exercise can increase oxidative stress in the short term. The impact of exercise on pro-inflammatory cytokine production is complex and varies depending on intensity and duration. Exercise can also counteract the harmful effects of ageing and inflamm-ageing. This review aims to examine the molecular pathways altered by exercise in non-obese individuals at higher risk of developing T2D, including glucose utilization, lipid metabolism, mitochondrial function, inflammation and oxidative stress, with the potential to improve insulin sensitivity. The focus is on understanding the potential benefits of exercise for improving insulin sensitivity and providing insights for future targeted interventions before onset of disease.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/metabolismo , Antioxidantes/metabolismo , Estresse Oxidativo , Exercício Físico , Insulina/metabolismoRESUMO
Cu/Zn-superoxide dismutase (CuZnSOD) is an enzyme that binds a copper and zinc ion and also forms an intramolecular disulfide bond. Together with the copper ion as the active site, the disulfide bond is completely conserved among these proteins; indeed, the disulfide bond plays critical roles in maintaining the catalytically competent conformation of CuZnSOD. Here, we found that a CuZnSOD protein in Paenibacillus lautus (PaSOD) has no Cys residue but exhibits a significant level of enzyme activity. The crystal structure of PaSOD revealed hydrophobic and hydrogen-bonding interactions in substitution for the disulfide bond of the other CuZnSOD proteins. Also notably, we determined that PaSOD forms a homodimer through an additional domain with a novel fold at the N terminus. While the advantages of lacking Cys residues and adopting a novel dimer configuration remain obscure, PaSOD does not require a disulfide-introducing/correcting system for maturation and could also avoid misfolding caused by aberrant thiol oxidations under an oxidative environment.
Assuntos
Proteínas de Bactérias , Dissulfetos , Superóxido Dismutase-1 , Cobre , Cisteína , Dissulfetos/química , Superóxido Dismutase-1/química , Zinco , Proteínas de Bactérias/química , Paenibacillus , Dobramento de ProteínaRESUMO
Deinococcus radiodurans is known for its remarkable ability to withstand harsh stressful conditions. The outermost layer of its cell envelope is a proteinaceous coat, the S-layer, essential for resistance to and interactions with the environment. The S-layer Deinoxanthin-binding complex (SDBC), one of the main units of the characteristic multilayered cell envelope of this bacterium, protects against environmental stressors and allows exchanges with the environment. So far, specific regions of this complex, the collar and the stalk, remained unassigned. Here, these regions are resolved by cryo-EM and locally refined. The resulting 3D map shows that the collar region of this multiprotein complex is a trimer of the protein DR_0644, a Cu-only superoxide dismutase (SOD) identified here to be efficient in quenching reactive oxygen species. The same data also showed that the stalk region consists of a coiled coil that extends into the cell envelope for â¼280 Å, reaching the inner membrane. Finally, the orientation and localization of the complex are defined by in situ cryo-electron crystallography. The structural organization of the SDBC couples fundamental UV antenna properties with the presence of a Cu-only SOD, showing here coexisting photoprotective and chemoprotective functions. These features suggests how the SDBC and similar protein complexes, might have played a primary role as evolutive templates for the origin of photoautotrophic processes by combining primary protective needs with more independent energetic strategies.
Assuntos
Deinococcus , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Deinococcus/química , Deinococcus/citologia , Deinococcus/metabolismo , Estresse Oxidativo , Superóxido Dismutase/metabolismoRESUMO
Herbicides are small molecules that act by inhibiting specific molecular target sites within primary plant metabolic pathways resulting in catastrophic and lethal consequences. The stress induced by herbicides generates reactive oxygen species (ROS), but little is known about the nexus between each herbicide mode of action (MoA) and their respective ability to induce ROS formation. Indeed, some herbicides cause dramatic surges in ROS levels as part of their primary MoA, whereas other herbicides may generate some ROS as a secondary effect of the stress they imposed on plants. In this review, we discuss the types of ROS and their respective reactivity and describe their involvement for each known MoA based on the new Herbicide Resistance Action Committee classification.