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1.
Cell ; 172(3): 409-422.e21, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29290465

RESUMO

Selenoproteins are rare proteins among all kingdoms of life containing the 21st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided.


Assuntos
Apoptose , Glutationa Peroxidase/metabolismo , Convulsões/metabolismo , Selênio/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Feminino , Glutationa Peroxidase/genética , Células HEK293 , Humanos , Peróxido de Hidrogênio/toxicidade , Interneurônios/metabolismo , Peroxidação de Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Convulsões/etiologia
2.
Annu Rev Biochem ; 86: 715-748, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28441057

RESUMO

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/metabolismo
3.
Brain ; 143(4): 1127-1142, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32293668

RESUMO

Chronic disability in multiple sclerosis is linked to neuroaxonal degeneration. 4-aminopyridine (4-AP) is used and licensed as a symptomatic treatment to ameliorate ambulatory disability in multiple sclerosis. The presumed mode of action is via blockade of axonal voltage gated potassium channels, thereby enhancing conduction in demyelinated axons. In this study, we provide evidence that in addition to those symptomatic effects, 4-AP can prevent neuroaxonal loss in the CNS. Using in vivo optical coherence tomography imaging, visual function testing and histologic assessment, we observed a reduction in retinal neurodegeneration with 4-AP in models of experimental optic neuritis and optic nerve crush. These effects were not related to an anti-inflammatory mode of action or a direct impact on retinal ganglion cells. Rather, histology and in vitro experiments indicated 4-AP stabilization of myelin and oligodendrocyte precursor cells associated with increased nuclear translocation of the nuclear factor of activated T cells. In experimental optic neuritis, 4-AP potentiated the effects of immunomodulatory treatment with fingolimod. As extended release 4-AP is already licensed for symptomatic multiple sclerosis treatment, we performed a retrospective, multicentre optical coherence tomography study to longitudinally compare retinal neurodegeneration between 52 patients on continuous 4-AP therapy and 51 matched controls. In line with the experimental data, during concurrent 4-AP therapy, degeneration of the macular retinal nerve fibre layer was reduced over 2 years. These results indicate disease-modifying effects of 4-AP beyond symptomatic therapy and provide support for the design of a prospective clinical study using visual function and retinal structure as outcome parameters.


Assuntos
4-Aminopiridina/farmacologia , Esclerose Múltipla/patologia , Fármacos Neuroprotetores/farmacologia , Neurite Óptica/patologia , Degeneração Retiniana/patologia , Adulto , Idoso , Animais , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células-Tronco Neurais/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar
4.
Acta Neuropathol ; 137(2): 239-257, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30426203

RESUMO

Brain accumulation and aggregation of amyloid-ß (Aß) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aß peptides (mainly Aß1-40 and Aß1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aß peptides are truncated at the N-terminus, with Aß4-x peptides being particularly abundant. Aß4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aß peptide sequence, which facilitates Aß4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aß4-40 but unchanged levels of Aß1-x peptides. In the 5xFAD mouse model of amyloidosis, Aß4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4-/- knockout background, Aß4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aß species, but Aß4-40 peptides were absent in cultures derived from ADAMTS4-/- mice indicating that the enzyme was essential for Aß4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aß4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aß peptides.


Assuntos
Proteína ADAMTS4/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Oligodendroglia/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Oligodendroglia/patologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/patologia
5.
J Neuroinflammation ; 15(1): 71, 2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29514678

RESUMO

BACKGROUND: In multiple sclerosis (MS), neurodegeneration is the main reason for chronic disability. Alpha-lipoic acid (LA) is a naturally occurring antioxidant which has recently been demonstrated to reduce the rate of brain atrophy in progressive MS. However, it remains uncertain if it is also beneficial in the early, more inflammatory-driven phases. As clinical studies are costly and time consuming, optic neuritis (ON) is often used for investigating neuroprotective or regenerative therapeutics. We aimed to investigate the prospect for success of a clinical ON trial using an experimental autoimmune encephalomyelitis-optic neuritis (EAE-ON) model with visual system readouts adaptable to a clinical ON trial. METHODS: Using an in vitro cell culture model for endogenous oxidative stress, we compared the neuroprotective capacity of racemic LA with the R/S-enantiomers and its reduced form. In vivo, we analyzed retinal neurodegeneration using optical coherence tomography (OCT) and the visual function by optokinetic response (OKR) in MOG35-55-induced EAE-ON in C57BL/6J mice. Ganglion cell counts, inflammation, and demyelination were assessed by immunohistological staining of retinae and optic nerves. RESULTS: All forms of LA provided equal neuroprotective capacities in vitro. In EAE-ON, prophylactic LA therapy attenuated the clinical EAE score and prevented the thinning of the inner retinal layer while therapeutic treatment was not protective on visual outcomes. CONCLUSIONS: A prophylactic LA treatment is necessary to protect from visual loss and retinal thinning in EAE-ON, suggesting that a clinical ON trial starting therapy after the onset of symptoms may not be successful.


Assuntos
Encefalomielite Autoimune Experimental/patologia , Degeneração Neural/prevenção & controle , Retina/patologia , Ácido Tióctico/uso terapêutico , Transtornos da Visão/prevenção & controle , Complexo Vitamínico B/uso terapêutico , Animais , Complexo CD3/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/complicações , Feminino , Glutationa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/metabolismo , Degeneração Neural/etiologia , Nistagmo Optocinético/fisiologia , Carbonilação Proteica/fisiologia , Tomografia de Coerência Óptica , Transtornos da Visão/etiologia
6.
Proc Natl Acad Sci U S A ; 112(44): 13735-40, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26483494

RESUMO

The iron-sulfur cluster (ISC) is an ancient and essential cofactor of many proteins involved in electron transfer and metabolic reactions. In Arabidopsis, three pathways exist for the maturation of iron-sulfur proteins in the cytosol, plastids, and mitochondria. We functionally characterized the role of mitochondrial glutaredoxin S15 (GRXS15) in biogenesis of ISC containing aconitase through a combination of genetic, physiological, and biochemical approaches. Two Arabidopsis T-DNA insertion mutants were identified as null mutants with early embryonic lethal phenotypes that could be rescued by GRXS15. Furthermore, we showed that recombinant GRXS15 is able to coordinate and transfer an ISC and that this coordination depends on reduced glutathione (GSH). We found the Arabidopsis GRXS15 able to complement growth defects based on disturbed ISC protein assembly of a yeast Δgrx5 mutant. Modeling of GRXS15 onto the crystal structures of related nonplant proteins highlighted amino acid residues that after mutation diminished GSH and subsequently ISC coordination, as well as the ability to rescue the yeast mutant. When used for plant complementation, one of these mutant variants, GRXS15K83/A, led to severe developmental delay and a pronounced decrease in aconitase activity by approximately 65%. These results indicate that mitochondrial GRXS15 is an essential protein in Arabidopsis, required for full activity of iron-sulfur proteins.


Assuntos
Arabidopsis/metabolismo , Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Arabidopsis/crescimento & desenvolvimento , Teste de Complementação Genética
7.
Glia ; 65(9): 1521-1534, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28618115

RESUMO

Demyelinated brain lesions, a hallmark of autoimmune neuroinflammatory diseases like multiple sclerosis, result from oligodendroglial cell damage. Activated microglia are considered a major source of nitric oxide and subsequent peroxynitrite-mediated damage of myelin. Here, we provide biochemical and biophysical evidence that the oxidoreductase glutaredoxin 2 inhibits peroxynitrite formation by transforming nitric oxide into dinitrosyl-diglutathionyl-iron-complexes. Glutaredoxin 2 levels influence both survival rates of primary oligodendrocyte progenitor cells and preservation of myelin structure in cerebellar organotypic slice cultures challenged with activated microglia or nitric oxide donors. Of note, glutaredoxin 2-mediated protection is not linked to its enzymatic activity as oxidoreductase, but to the disassembly of its uniquely coordinated iron-sulfur cluster using glutathione as non-protein ligand. The protective effect of glutaredoxin 2 is connected to decreased protein carbonylation and nitration. In line, brain lesions of mice suffering from experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, show decreased glutaredoxin 2 expression and increased nitrotyrosine formation indicating that this type of protection is missing in the inflamed central nervous system. Our findings link inorganic biochemistry to neuroinflammation and identify glutaredoxin 2 as a protective factor against neuroinflammation-mediated myelin damage. Thus, improved availability of glutathione-coordinated iron-sulfur clusters emerges as a potential therapeutic approach in inflammatory demyelination.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Glutarredoxinas/metabolismo , Microglia/metabolismo , Óxido Nítrico/metabolismo , Oligodendroglia/metabolismo , Animais , Cerebelo/metabolismo , Cerebelo/patologia , Encefalomielite Autoimune Experimental/patologia , Escherichia coli , Feminino , Glutarredoxinas/genética , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos Endogâmicos C57BL , Microglia/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neuroproteção/fisiologia , Oligodendroglia/patologia , Ácido Peroxinitroso/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schistosoma japonicum , Técnicas de Cultura de Tecidos
8.
Biochim Biophys Acta ; 1850(8): 1575-87, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25450486

RESUMO

BACKGROUND: The cytoskeleton, unlike the bony vertebrate skeleton or the exoskeleton of invertebrates, is a highly dynamic meshwork of protein filaments that spans through the cytosol of eukaryotic cells. Especially actin filaments and microtubuli do not only provide structure and points of attachments, but they also shape cells, they are the basis for intracellular transport and distribution, all types of cell movement, and--through specific junctions and points of adhesion--join cells together to form tissues, organs, and organisms. SCOPE OF REVIEW: The fine tuned regulation of cytoskeletal dynamics is thus indispensible for cell differentiation and all developmental processes. Here, we discussed redox signalling mechanisms that control this dynamic remodeling. Foremost, we emphasised recent discoveries that demonstrated reversible thiol and methionyl switches in the regulation of actin dynamics. MAJOR CONCLUSIONS: Thiol and methionyl switches play an essential role in the regulation of cytoskeletal dynamics. GENERAL SIGNIFICANCE: The dynamic remodeling of the cytoskeleton is controlled by various redox switches. These mechanisms are indispensible during development and organogenesis and might contribute to numerous pathological conditions. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.


Assuntos
Desdiferenciação Celular/fisiologia , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Animais , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Oxirredução
9.
Biochim Biophys Acta ; 1850(8): 1543-54, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25662818

RESUMO

BACKGROUND: Accumulated data indicate that self-renewal, multipotency, and differentiation of neural stem cells are under an intrinsic control mediated by alterations in the redox homeostasis. These dynamic redox changes not only reflect and support the ongoing metabolic and energetic processes, but also serve to coordinate redox-signaling cascades. Controlling particular redox couples seems to have a relevant impact on cell fate decision during development, adult neurogenesis and regeneration. SCOPE OF REVIEW: Our own research provided initial evidence for the importance of NAD+-dependent enzymes in neural stem cell fate decision. In this review, we summarize recent knowledge on the active role of reactive oxygen species, redox couples and redox-signaling mechanisms on plasticity and function of neural stem and progenitor cells focusing on NAD(P)+/NAD(P)H-mediated processes. MAJOR CONCLUSIONS: The compartmentalized subcellular sources and availability of oxidizing/reducing molecules in particular microenvironment define the specificity of redox regulation in modulating the delicate balance between stemness and differentiation of neural progenitors. The generalization of "reactive oxygen species" as well as the ambiguity of their origin might explain the diametrically-opposed findings in the field of redox-dependent cell fate reflected by the literature. GENERAL SIGNIFICANCE: Increasing knowledge of temporary and spatially defined redox regulation is of high relevance for the development of novel approaches in the field of cell-based regeneration of nervous tissue in various pathological states. This article is part of a special issue entitled Redox regulation of differentiation and de-differentiation.


Assuntos
Diferenciação Celular , Proliferação de Células , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Humanos , Modelos Biológicos , NADP/metabolismo , Células-Tronco Neurais/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Adulto Jovem
10.
Plant Physiol ; 167(4): 1643-58, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25699589

RESUMO

Glutaredoxins (GRXs) catalyze the reduction of protein disulfide bonds using glutathione as a reductant. Certain GRXs are able to transfer iron-sulfur clusters to other proteins. To investigate the function of Arabidopsis (Arabidopsis thaliana) GRXS17, we applied a strategy combining biochemical, genetic, and physiological approaches. GRXS17 was localized in the nucleus and cytosol, and its expression was elevated in the shoot meristems and reproductive tissues. Recombinant GRXS17 bound Fe2S2 clusters, a property likely contributing to its ability to complement the defects of a Baker's yeast (Saccharomyces cerevisiae) strain lacking the mitochondrial GRX5. However, a grxs17 knockout Arabidopsis mutant exhibited only a minor decrease in the activities of iron-sulfur enzymes, suggesting that its primary function is as a disulfide oxidoreductase. The grxS17 plants were sensitive to high temperatures and long-day photoperiods, resulting in elongated leaves, compromised shoot apical meristem, and delayed bolting. Both environmental conditions applied simultaneously led to a growth arrest. Using affinity chromatography and split-Yellow Fluorescent Protein methods, a nuclear transcriptional regulator, the Nuclear Factor Y Subunit C11/Negative Cofactor 2α (NF-YC11/NC2α), was identified as a GRXS17 interacting partner. A mutant deficient in NF-YC11/NC2α exhibited similar phenotypes to grxs17 in response to photoperiod. Therefore, we propose that GRXS17 interacts with NF-YC11/NC2α to relay a redox signal generated by the photoperiod to maintain meristem function.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fator de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica de Plantas , Glutarredoxinas/metabolismo , Meristema/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Fator de Ligação a CCAAT/genética , Genes Reporter , Glutarredoxinas/genética , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Meristema/crescimento & desenvolvimento , Meristema/fisiologia , Meristema/efeitos da radiação , Modelos Biológicos , Mutação , Oxirredução , Fenótipo , Fotoperíodo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Brotos de Planta/efeitos da radiação , Plantas Geneticamente Modificadas , Proteínas Recombinantes , Transdução de Sinais
11.
Proc Natl Acad Sci U S A ; 110(50): 20057-62, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-24277839

RESUMO

Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione-disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S-glutathionylation of the NAD(+)-dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S-glutathionylation to vertebrate development and successful embryonic angiogenesis.


Assuntos
Sistema Cardiovascular/embriologia , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Sirtuína 1/metabolismo , Animais , Western Blotting , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Glutarredoxinas/genética , Células HeLa , Humanos , Espectrometria de Massas , Microscopia Confocal , Oxirredução , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Imagem com Lapso de Tempo , Peixe-Zebra
12.
Biol Chem ; 396(5): 523-37, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25581756

RESUMO

Cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.12) is present in all organisms and catalyzes the oxidation of triose phosphate during glycolysis. GAPDH is one of the most prominent cellular targets of oxidative modifications when reactive oxygen and nitrogen species are formed during metabolism and under stress conditions. GAPDH harbors a strictly conserved catalytic cysteine, which is susceptible to a variety of thiol modifications, including S-sulfenylation, S-glutathionylation, S-nitrosylation, and S-sulfhydration. Upon reversible oxidative thiol modification of GAPDH, glycolysis is inhibited leading to a diversion of metabolic flux through the pentose-phosphate cycle to increase NADPH production. Furthermore, oxidized GAPDH may adopt new functions in different cellular compartments including the nucleus, as well as in new microcompartments associated with the cytoskeleton, mitochondria and plasma membrane. This review focuses on the recently discovered mechanism underlying the eminent reactivity between GAPDH and hydrogen peroxide and the subsequent redox-dependent moonlighting functions discriminating between the induction either of adaptive responses and adjustment of metabolism or of cell death in yeast, plants, and mammals. In light of the summarized results, cytosolic GAPDH might function as a sensor for redox signals and an information hub to transduce these signals for appropriate responses.


Assuntos
Citosol/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Oxirredução
13.
Proc Natl Acad Sci U S A ; 108(51): 20532-7, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22139372

RESUMO

Cellular functions and survival are dependent on a tightly controlled redox potential. Currently, an increasing amount of data supports the concept of local changes in the redox environment and specific redox signaling events controlling cell function. Specific protein thiol groups are the major targets of redox signaling and regulation. Thioredoxins and glutaredoxins catalyze reversible thiol-disulfide exchange reactions and are primary regulators of the protein thiol redox state. Here, we demonstrate that embryonic brain development depends on the enzymatic activity of glutaredoxin 2. Zebrafish with silenced expression of glutaredoxin 2 lost virtually all types of neurons by apoptotic cell death and the ability to develop an axonal scaffold. As demonstrated in zebrafish and in a human cellular model for neuronal differentiation, glutaredoxin 2 controls axonal outgrowth via thiol redox regulation of collapsin response mediator protein 2, a central component of the semaphorin pathway. This study provides an example of a specific thiol redox regulation essential for vertebrate embryonic development.


Assuntos
Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Glutarredoxinas/química , Peixe-Zebra/embriologia , Animais , Apoptose , Axônios/fisiologia , Linhagem Celular Tumoral , Biologia do Desenvolvimento , Glutarredoxinas/genética , Humanos , Neuritos/metabolismo , Oxirredução , Proteínas Recombinantes/química , Transdução de Sinais , Vertebrados
14.
Biochim Biophys Acta Gen Subj ; 1868(6): 130603, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38521470

RESUMO

BACKGROUND: Redox control seems to be indispensable for proper embryonic development. The ratio between glutathione (GSH) and its oxidized disulfide (GSSG) is the most abundant cellular redox circuit. METHODS: We used zebrafish harboring the glutaredoxin 1-redox sensitive green fluorescent protein (Grx1-roGFP) probe either in mitochondria or cytosol to test the hypothesis that the GSH:GSSG ratio is strictly regulated through zebrafish embryogenesis to sustain the different developmental processes of the embryo. RESULTS: Following the GSSG:GSH ratio as a proxy for the GSH-dependent reduction potential (EhGSH) revealed increasing mitochondrial and cytosolic EhGSH during cleavage and gastrulation. During organogenesis, cytosolic EhGSH decreased, while that of mitochondria remained high. The similarity between EhGSH in brain and muscle suggests a central regulation. Modulation of GSH metabolism had only modest effects on the GSSG:GSH ratios of newly hatched larvae. However, inhibition of GSH reductase directly after fertilization led to dead embryos already 10 h later. Exposure to the emerging environmental pollutant Perfluorooctane Sulfonate (PFOS) disturbed the apparent regulated EhGSH as well. CONCLUSIONS: Mitochondrial and cytosolic GSSG:GSH ratios are almost identical in different organs during zebrafish development indicating that the EhGSH might follow H2O2 levels and rather indirectly affect specific enzymatic activities needed for proper embryogenesis. GENERAL SIGNIFICANCE: Our data confirm that vertebrate embryogenesis depends on strictly regulated redox homeostasis. Disturbance of the GSSG:GSH circuit, e.g. induced by environmental pollution, leads to malformation and death.


Assuntos
Citosol , Glutationa , Mitocôndrias , Oxirredução , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Glutationa/metabolismo , Mitocôndrias/metabolismo , Citosol/metabolismo , Desenvolvimento Embrionário , Dissulfeto de Glutationa/metabolismo , Embrião não Mamífero/metabolismo
15.
Front Pharmacol ; 15: 1468920, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39386028

RESUMO

Glioblastoma (GBM) is the most commonly occurring and most aggressive primary brain tumor. Transcriptomics-based tumor subtype classification has established the mesenchymal lineage of GBM (MES-GBM) as cancers with particular aggressive behavior and high levels of therapy resistance. Previously it was show that Trihexyphenidyl (THP), a market approved M1 muscarinic receptor-targeting oral drug can suppress proliferation and survival of GBM stem cells from the classical transcriptomic subtype. In a series of in vitro experiments, this study confirms the therapeutic potential of THP, by effectively suppressing the growth, proliferation and survival of MES-GBM cells with limited effects on non-tumor cells. Transcriptomic profiling of treated cancer cells identified genes and associated metabolic signaling pathways as possible underlying molecular mechanisms responsible for THP-induced effects. In vivo trials of THP in immunocompromised mice carry orthotopic MES-GBMs showed moderate response to the drug. This study further highlights the potential of THP repurposing as an anti-cancer treatment regimen but mode of action and d optimal treatment procedures for in vivo regimens need to be investigated further.

16.
Nat Commun ; 15(1): 411, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38195625

RESUMO

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.


Assuntos
COVID-19 , Cisteína , Humanos , SARS-CoV-2 , Desenho de Fármacos , Oxirredução
17.
Redox Biol ; 75: 103211, 2024 09.
Artigo em Inglês | MEDLINE | ID: mdl-38908072

RESUMO

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.


Assuntos
Ferroptose , Humanos , Animais , Ferro/metabolismo , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Peroxidação de Lipídeos , Oxirredução , Suscetibilidade a Doenças
18.
Biochem Biophys Res Commun ; 436(3): 491-6, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23756812

RESUMO

Glutaredoxins that contain a Cys-X-X-Cys active site motif are glutathione-dependent thiol-disulfide oxidoreductases. Vertebrate glutaredoxin 2 is characterized by two extra cysteines that form an intra-molecular disulfide bridge. Zebrafish glutaredoxin 2 contains four additional cysteines that are conserved within the infraclass of bony fish (teleosts). Here, we present a biochemical and biophysical characterization of zebrafish glutaredoxin 2, focusing on iron-sulfur-cluster coordination. The coordination of [2Fe2S](2+)-clusters in monomers of this protein was revealed by both absorption and Mössbauer spectroscopy as well as size exclusion chromatography. All other holo-glutaredoxins represent [FeS]-cluster bridged dimers using two molecules of non-covalently bound glutathione and the N-terminal active site cysteines as ligands. These cysteine residues were not required for [FeS]-cluster coordination in zebrafish glutaredoxin 2. A crystal structure of the teleost protein revealed high structural similarity to its human homologue. The two vertebrate-specific cysteines as well as two of the teleost-specific cysteines are positioned within a radius of 7Å near the C-terminus suggesting a potential role in [FeS]-cluster coordination. Indeed, mutated proteins lacking these teleost-specific cysteines lost the ability to bind the cofactor. Hence, the apparent mode of [FeS]-cluster coordination in zebrafish glutaredoxin 2 could be different from all yet described [FeS]-glutaredoxins.


Assuntos
Glutarredoxinas/química , Proteínas Ferro-Enxofre/química , Proteínas de Peixe-Zebra/química , Peixe-Zebra/metabolismo , Motivos de Aminoácidos , Animais , Domínio Catalítico , Cisteína/química , Ativação Enzimática , Humanos , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Homologia de Sequência de Aminoácidos
19.
Cells ; 12(17)2023 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-37681928

RESUMO

With a global increase in chronic kidney disease patients, alternatives to dialysis and organ transplantation are needed. Stem cell-based therapies could be one possibility to treat chronic kidney disease. Here, we used multipotent urine-derived renal progenitor cells (UdRPCs) to study nephrogenesis. UdRPCs treated with the JNK inhibitor-AEG3482 displayed decreased proliferation and downregulated transcription of cell cycle-associated genes as well as the kidney progenitor markers-SIX2, SALL1 and VCAM1. In addition, levels of activated SMAD2/3, which is associated with the maintenance of self-renewal in UdRPCs, were decreased. JNK inhibition resulted in less efficient oxidative phosphorylation and more lipid peroxidation via ferroptosis, an iron-dependent non-apoptotic cell death pathway linked to various forms of kidney disease. Our study is the first to describe the importance of JNK signalling as a link between maintenance of self-renewal and protection against ferroptosis in SIX2-positive renal progenitor cells.


Assuntos
Ferroptose , Sistema de Sinalização das MAP Quinases , Insuficiência Renal Crônica , Humanos , Rim , Diálise Renal , Células-Tronco
20.
Redox Biol ; 49: 102221, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34952462

RESUMO

Redox regulation of specific cysteines via oxidoreductases of the thioredoxin family is increasingly being recognized as an important signaling pathway. Here, we demonstrate that the cytosolic isoform of the vertebrate-specific oxidoreductase Glutaredoxin 2 (Grx2c) regulates the redox state of the transcription factor SP-1 and thereby its binding affinity to both the promoter and an enhancer region of the CSPG4 gene encoding chondroitin sulfate proteoglycan nerve/glial antigen 2 (NG2). This leads to an increased number of NG2 glia during in vitro oligodendroglial differentiation and promotes migration of these wound healing cells. On the other hand, we found that the same mechanism also leads to increased invasion of glioma tumor cells. Using in vitro (human cell lines), ex vivo (mouse primary cells), and in vivo models (zebrafish), as well as glioblastoma patient tissue samples we provide experimental data highlighting the Yin and Yang of redox signaling in the central nervous system and the enzymatic Taoism of Grx2c.


Assuntos
Glioma , Glutarredoxinas , Animais , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Glioma/genética , Glioma/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Neuroglia/metabolismo , Filosofias Religiosas , Cicatrização/genética , Peixe-Zebra/metabolismo
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