RESUMO
Acyl-coenzyme A (acyl-CoA) species are cofactors for numerous enzymes that acylate thousands of proteins. Here, we describe an enzyme that uses S-nitroso-CoA (SNO-CoA) as its cofactor to S-nitrosylate multiple proteins (SNO-CoA-assisted nitrosylase, SCAN). Separate domains in SCAN mediate SNO-CoA and substrate binding, allowing SCAN to selectively catalyze SNO transfer from SNO-CoA to SCAN to multiple protein targets, including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1). Insulin-stimulated S-nitrosylation of INSR/IRS1 by SCAN reduces insulin signaling physiologically, whereas increased SCAN activity in obesity causes INSR/IRS1 hypernitrosylation and insulin resistance. SCAN-deficient mice are thus protected from diabetes. In human skeletal muscle and adipose tissue, SCAN expression increases with body mass index and correlates with INSR S-nitrosylation. S-nitrosylation by SCAN/SNO-CoA thus defines a new enzyme class, a unique mode of receptor tyrosine kinase regulation, and a revised paradigm for NO function in physiology and disease.
Assuntos
Insulina , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Transdução de Sinais , Animais , Humanos , Camundongos , Acil Coenzima A/metabolismo , Tecido Adiposo/metabolismo , Resistência à Insulina , Óxido Nítrico/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismoRESUMO
Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a â¼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.
Assuntos
DNA Glicosilases/química , DNA Helicases/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Endonucleases/química , Genoma , Proteínas Ferro-Enxofre/química , Animais , Bactérias/genética , Bactérias/metabolismo , DNA/metabolismo , DNA/ultraestrutura , Dano ao DNA , DNA Glicosilases/metabolismo , DNA Glicosilases/ultraestrutura , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/ultraestrutura , Espectroscopia de Ressonância de Spin Eletrônica , Endonucleases/metabolismo , Endonucleases/ultraestrutura , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/ultraestrutura , Oxirredução , Ligação Proteica , Transdução de Sinais , TermodinâmicaRESUMO
Severe changes in the environmental redox potential, and resulting alterations in the oxidation states of intracellular metabolites and enzymes, have historically been considered negative stressors, requiring responses that are strictly defensive. However, recent work in diverse organisms has revealed that more subtle changes in the intracellular redox state can act as signals, eliciting responses with benefits beyond defense and detoxification. Changes in redox state have been shown to influence or trigger chromosome segregation, sporulation, aerotaxis, and social behaviors, including luminescence as well as biofilm establishment and dispersal. Connections between redox state and complex behavior allow bacteria to link developmental choices with metabolic state and coordinate appropriate responses. Promising future directions for this area of study include metabolomic analysis of species- and condition-dependent changes in metabolite oxidation states and elucidation of the mechanisms whereby the redox state influences circadian regulation.
Assuntos
Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Esporos Bacterianos/metabolismo , Aliivibrio fischeri/genética , Aliivibrio fischeri/crescimento & desenvolvimento , Aliivibrio fischeri/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Caulobacter crescentus/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Glutationa/metabolismo , Proteínas de Membrana/genética , Oxirredução , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismoRESUMO
Oxidative stress is two sided: Whereas excessive oxidant challenge causes damage to biomolecules, maintenance of a physiological level of oxidant challenge, termed oxidative eustress, is essential for governing life processes through redox signaling. Recent interest has focused on the intricate ways by which redox signaling integrates these converse properties. Redox balance is maintained by prevention, interception, and repair, and concomitantly the regulatory potential of molecular thiol-driven master switches such as Nrf2/Keap1 or NF-κB/IκB is used for system-wide oxidative stress response. Nonradical species such as hydrogen peroxide (H2O2) or singlet molecular oxygen, rather than free-radical species, perform major second messenger functions. Chemokine-controlled NADPH oxidases and metabolically controlled mitochondrial sources of H2O2 as well as glutathione- and thioredoxin-related pathways, with powerful enzymatic back-up systems, are responsible for fine-tuning physiological redox signaling. This makes for a rich research field spanning from biochemistry and cell biology into nutritional sciences, environmental medicine, and molecular knowledge-based redox medicine.
Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo , Regulação da Expressão Gênica , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , NADPH Oxidases/genética , Fator 2 Relacionado a NF-E2/genética , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/genética , Oxirredução , Transdução de Sinais , Oxigênio Singlete/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
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
Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.
Assuntos
Peroxirredoxinas , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Cisteína/metabolismo , Dissulfetos , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Oxirredução , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis in vivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis.
Assuntos
Carcinoma Ductal Pancreático , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Pancreáticas , Hormônios Tireóideos/metabolismo , Carcinoma Ductal Pancreático/genética , Humanos , Metionina , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/metabolismo , Oxirredução , Neoplasias Pancreáticas/genética , Piruvato Quinase/metabolismo , Proteínas de Ligação a Hormônio da Tireoide , Neoplasias PancreáticasRESUMO
Redox reactions are intrinsically linked to energy metabolism. Therefore, redox processes are indispensable for organismal physiology and life itself. The term reactive oxygen species (ROS) describes a set of distinct molecular oxygen derivatives produced during normal aerobic metabolism. Multiple ROS-generating and ROS-eliminating systems actively maintain the intracellular redox state, which serves to mediate redox signaling and regulate cellular functions. ROS, in particular hydrogen peroxide (H2O2), are able to reversibly oxidize critical, redox-sensitive cysteine residues on target proteins. These oxidative post-translational modifications (PTMs) can control the biological activity of numerous enzymes and transcription factors (TFs), as well as their cellular localization or interactions with binding partners. In this review, we describe the diverse roles of redox regulation in the context of physiological cellular metabolism and provide insights into the pathophysiology of diseases when redox homeostasis is dysregulated.
Assuntos
Metabolismo Energético/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Cisteína/metabolismo , Homeostase , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Estresse Oxidativo , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Hydrogen peroxide (H2O2) sensing and signaling involves the reversible oxidation of particular thiols on particular proteins to modulate protein function in a dynamic manner. H2O2 can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous "hotspots" of H2O2 generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H2O2 sensor HyPer7 was fused to the C-terminus of all protein-coding open reading frames (ORFs). We also generated a control library in which a redox-insensitive mutant of HyPer7 (SypHer7) was fused to all ORFs. Both libraries were screened side-by-side to identify proteins located within H2O2-generating environments. Screening under a variety of different metabolic conditions revealed dynamic changes in H2O2 availability highly specific to individual proteins and protein complexes. These findings suggest that intracellular H2O2 generation is much more localized and functionally differentiated than previously recognized.
Assuntos
Técnicas Biossensoriais , Peróxido de Hidrogênio , Peróxido de Hidrogênio/metabolismo , Proteoma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , OxirreduçãoRESUMO
One of the major challenges that remain in the fields of aging and lifespan determination concerns the precise roles that reactive oxygen species (ROS) play in these processes. ROS, including superoxide and hydrogen peroxide, are constantly generated as byproducts of aerobic metabolism, as well as in response to endogenous and exogenous cues. While ROS accumulation and oxidative damage were long considered to constitute some of the main causes of age-associated decline, more recent studies reveal a signaling role in the aging process. In fact, accumulation of ROS, in a spatiotemporal manner, can trigger beneficial cellular responses that promote longevity and healthy aging. In this review, we discuss the importance of timing and compartmentalization of external and internal ROS perturbations in organismal lifespan and the role of redox regulated pathways.
Assuntos
Envelhecimento , Longevidade , Estresse Oxidativo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento/metabolismo , Humanos , AnimaisRESUMO
Chemogenetics refers to experimental systems that dynamically regulate the activity of a recombinant protein by providing or withholding the protein's specific biochemical stimulus. Chemogenetic tools permit precise dynamic control of specific signaling molecules to delineate the roles of those molecules in physiology and disease. Yeast d-amino acid oxidase (DAAO) enables chemogenetic manipulation of intracellular redox balance by generating hydrogen peroxide only in the presence of d-amino acids. Advances in biosensors have allowed the precise quantitation of these signaling molecules. The combination of chemogenetic approaches with biosensor methodologies has opened up new lines of investigation, allowing the analysis of intracellular redox pathways that modulate physiological and pathological cell responses. We anticipate that newly developed transgenic chemogenetic models will permit dynamic modulation of cellularredox balance in diverse cells and tissues and will facilitate the identification and validation of novel therapeutic targets involved in both physiological redox pathways and pathological oxidative stress.
Assuntos
Peróxido de Hidrogênio , Estresse Oxidativo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxirredução , Transdução de SinaisRESUMO
Cytochrome c (cyt c) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping-mass spectrometry-bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40-57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.
Assuntos
Citocromos c , Heme , Animais , Sequência de Aminoácidos , Anticorpos Monoclonais , Citocromos c/química , Heme/química , Hibridomas , Oxirredução , Melanoma Experimental , CamundongosRESUMO
Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. [Formula: see text], the reduced semiquinone form of the nonexchangeable quinone, is often considered capable of a side reaction with O2, forming superoxide, but this reaction has not yet been demonstrated experimentally. Here, using chlorophyll fluorescence in plant PSII membranes, we show that O2 does oxidize [Formula: see text] at physiological O2 concentrations with a t1/2 of 10 s. Superoxide is formed stoichiometrically, and the reaction kinetics are controlled by the accessibility of O2 to a binding site near [Formula: see text], with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, [Formula: see text] could only reduce O2 when bicarbonate was absent from its binding site on the nonheme iron (Fe2+) and the addition of bicarbonate or formate blocked the O2-dependant decay of [Formula: see text] These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from [Formula: see text] to O2 occurs when the O2 is bound to the empty bicarbonate site on Fe2+ A protective role for bicarbonate in PSII was recently reported, involving long-lived [Formula: see text] triggering bicarbonate dissociation from Fe2+ [Brinkert et al, Proc. Natl. Acad. Sci. U.S.A. 113, 12144-12149 (2016)]. The present findings extend this mechanism by showing that bicarbonate release allows O2 to bind to Fe2+ and to oxidize [Formula: see text] This could be beneficial by oxidizing [Formula: see text] and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain.
Assuntos
Bicarbonatos/metabolismo , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Chlamydomonas reinhardtii/metabolismo , Clorofila/metabolismo , Transporte de Elétrons/fisiologia , Formiatos/metabolismo , Oxirredução , Quinonas/metabolismo , Spinacia oleracea/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
Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.
Assuntos
Caspase 8 , Necroptose , Oxidantes , Fatores de Necrose Tumoral , Animais , Camundongos , Caspase 8/química , Caspase 8/metabolismo , Inflamação/metabolismo , Necroptose/efeitos dos fármacos , Oxidantes/metabolismo , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Fatores de Necrose Tumoral/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Peroxidase , Lactoperoxidase , Domínio CatalíticoRESUMO
Cancer cells experience increased levels of oxidant stress as a consequence of oncogene activation, nucleotide biosynthesis, and growth factor receptor signaling. Mitochondria contribute to this redox stress by generating reactive oxygen species (ROS) along the electron transport chain, which are released to the matrix and the intermembrane space (IMS). Assessing the contribution of mitochondrial ROS in cancer cells is technically difficult, as electron transport chain inhibitors can increase or decrease ROS generation, while they also block oxidative phosphorylation and ATP synthesis. Mitochondria-targeted antioxidant compounds can scavenge ROS in the matrix compartment but do not act on ROS released to the IMS. We assessed the importance of mitochondrial ROS for tumor cell proliferation, survival, and for tumor xenograft growth by stably expressing a hydrogen peroxide (H2O2) scavenger, peroxiredoxin-5, in the mitochondrial IMS (IMS-Prdx5) in 143B osteosarcoma and HCT116 colorectal cancer cell lines. IMS-Prdx5 attenuates hypoxia-induced ROS signaling as assessed independently in cytosol and IMS, HIF-1α stabilization and activity, and cellular proliferation under normoxic and hypoxic culture conditions. It also suppressed tumor growth in vivo. Stable expression of nondegradable HIF-1α only partially rescued proliferation in IMS-Prdx5-expressing cells, indicating that mitochondrial H2O2 signaling contributes to tumor cell proliferation and survival through HIF-dependent and HIF-independent mechanisms.
Assuntos
Peróxido de Hidrogênio , Neoplasias , Humanos , Proliferação de Células , Peróxido de Hidrogênio/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) are vital entry points for monosaccharides and amino acids into the Krebs cycle and thus integral for mitochondrial bioenergetics. Both complexes produce mitochondrial hydrogen peroxide (mH2O2) and are deactivated by electrophiles. Here, we provide an update on the role of PDH and KGDH in mitochondrial redox balance and their function in facilitating metabolic reprogramming for the propagation of oxidative eustress signals in hepatocytes and how defects in these pathways can cause liver diseases. PDH and KGDH are known to account for â¼45% of the total mH2O2 formed by mitochondria and display rates of production several-fold higher than the canonical source complex I. This mH2O2 can also be formed by reverse electron transfer (RET) in vivo, which has been linked to metabolic dysfunctions that occur in pathogenesis. However, the controlled emission of mH2O2 from PDH and KGDH has been proposed to be fundamental for oxidative eustress signal propagation in several cellular contexts. Modification of PDH and KGDH with protein S-glutathionylation (PSSG) and S-nitrosylation (PSNO) adducts serves as a feedback inhibitor for mH2O2 production in response to glutathione (GSH) pool oxidation. PSSG and PSNO adduct formation also reprogram the Krebs cycle to generate metabolites vital for interorganelle and intercellular signaling. Defects in the redox modification of PDH and KGDH cause the over generation of mH2O2, resulting in oxidative distress and metabolic dysfunction-associated fatty liver disease (MAFLD). In aggregate, PDH and KGDH are essential platforms for emitting and receiving oxidative eustress signals.
Assuntos
Hepatócitos , Peróxido de Hidrogênio , Complexo Cetoglutarato Desidrogenase , Mitocôndrias Hepáticas , Complexo Piruvato Desidrogenase , Peróxido de Hidrogênio/metabolismo , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/metabolismo , Oxirredução , Estresse Oxidativo , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/metabolismo , Humanos , Hepatócitos/enzimologia , Mitocôndrias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Animais , CamundongosRESUMO
Nitric oxide (NO) is widely recognized for its role in regulating renal function and blood pressure. However, the precise mechanisms by which NO affects renal epithelial cells remain understudied. Our previous research has shown that NO signaling in glomerular podocytes can be initiated by Angiotensin II (Ang II) but not by ATP. This study aims to elucidate the crucial interplay between the renin-angiotensin system (RAS) and NO production in podocytes. To conduct our research, we utilized cultured human podocytes and freshly isolated rat glomeruli. A variety of RAS peptides were employed, alongside confocal microscopy, to detect NO production and NO/Ca2+ crosstalk. Dynamic changes in the podocyte cytoskeleton, mediated by RAS-NO intracellular signaling, were observed using fluorescent labeling for F-actin and scanning probe microscopy. The experiments demonstrated that Ang II and Ang III generated high levels of NO through the activation of the Angiotensin II Type 2 Receptor (AT2R). We did not detect functional MAS receptor presence in podocytes, and the moderate NO response to Ang 1-7 was also mediated through AT2R. Furthermore, NO production impacted intracellular Ca2+ signaling and correlated with an increase in podocyte volume and growth. Scanning probe experiments revealed that AT2R activation and the corresponding NO generation are responsible for the protrusion of podocyte lamellipodia. Taken together, our data indicate that AT2R activation enhances NO production in podocytes and subsequently mediates changes in Ca2+ signaling and podocyte volume dynamics. These mechanisms may play a significant role in both physiological and pathophysiological interactions between the RAS and podocytes.
RESUMO
In eukaryotic cells, mitochondria perform cellular respiration through a series of redox reactions ultimately reducing molecular oxygen to water. The system responsible for this process is the respiratory chain or electron transport system (ETS) composed of complexes I-IV. Due to its function, the ETS is the main source of reactive oxygen species (ROS), generating them on both sides of the mitochondrial inner membrane, i.e. the intermembrane space (IMS) and the matrix. A correct balance between ROS generation and scavenging is important for keeping the cellular redox homeostasis and other important aspects of cellular physiology. However, ROS generated in the mitochondria are important signaling molecules regulating mitochondrial biogenesis and function. The IMS contains a large number of redox sensing proteins, containing specific Cys-rich domains, that are involved in ETS complex biogenesis. The large majority of these proteins function as cytochrome c oxidase (COX) assembly factors, mainly for the handling of copper ions necessary for the formation of the redox reactive catalytic centers. A particular case of ROS-regulated COX assembly factor is COA8, whose intramitochondrial levels are increased by oxidative stress, promoting COX assembly and/or protecting the enzyme from oxidative damage. In this review, we will discuss the current knowledge concerning the role played by ROS in regulating mitochondrial activity and biogenesis, focusing on the COX enzyme and with a special emphasis on the functional role exerted by the redox sensitive Cys residues contained in the COX assembly factors.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Mitocôndrias , Oxirredução , Espécies Reativas de Oxigênio , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Animais , Estresse OxidativoRESUMO
Plants fully depend on their immune systems to defend against pathogens. Upon pathogen attack, plants not only activate immune responses at the infection site but also trigger a defense mechanism known as systemic acquired resistance (SAR) in distal systemic tissues to prevent subsequent infections by a broad-spectrum of pathogens. SAR is induced by mobile signals produced at the infection site. Accumulating evidence suggests that reactive oxygen species (ROS) play a central role in SAR signaling. ROS burst at the infection site is one of the earliest cellular responses following pathogen infection and can spread to systemic tissues through membrane-associated NADPH oxidase-dependent relay production of ROS. It is well known that ROS ignite redox signaling and, when in excess, cause oxidative stress, damaging cellular components. In this review, we summarize current knowledge on redox regulation of several SAR signaling components. We discuss the ROS amplification loop in systemic tissues involving multiple SAR mobile signals. Moreover, we highlight the essential role of oxidative stress in generating SAR signals including azelaic acid and extracellular NAD(P) [eNAD(P)]. Finally, we propose that eNAD(P) is a damage-associated molecular pattern serving as a converging point of SAR mobile signals in systemic tissues.