RESUMO
DNA methyltransferases (DNMTs) catalyze methylation at the C5 position of cytosine with S-adenosyl-L-methionine. Methylation regulates gene expression, serving a variety of physiological and pathophysiological roles. The chemical mechanisms regulating DNMT enzymatic activity, however, are not fully elucidated. Here, we show that protein S-nitrosylation of a cysteine residue in DNMT3B attenuates DNMT3B enzymatic activity and consequent aberrant upregulation of gene expression. These genes include Cyclin D2 (Ccnd2), which is required for neoplastic cell proliferation in some tumor types. In cell-based and in vivo cancer models, only DNMT3B enzymatic activity, and not DNMT1 or DNMT3A, affects Ccnd2 expression. Using structure-based virtual screening, we discovered chemical compounds that specifically inhibit S-nitrosylation without directly affecting DNMT3B enzymatic activity. The lead compound, designated DBIC, inhibits S-nitrosylation of DNMT3B at low concentrations (IC50 ≤ 100 nM). Treatment with DBIC prevents nitric oxide (NO)-induced conversion of human colonic adenoma to adenocarcinoma in vitro. Additionally, in vivo treatment with DBIC strongly attenuates tumor development in a mouse model of carcinogenesis triggered by inflammation-induced generation of NO. Our results demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by NO, and DBIC protects against tumor formation by preventing aberrant S-nitrosylation of DNMT3B.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Epigênese Genética , Animais , Humanos , Camundongos , Transformação Celular Neoplásica/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , DNA Metiltransferase 3BRESUMO
Emerging evidence indicates the importance of S-nitrosation in regulating protein function and activity. This chemical reaction has been termed protein S-nitrosylation to emphasize its biological importance as a posttranslational modification, in some ways reminiscent of phosphorylation. The reaction at cysteine thiols is distinct from other chemical reactions of nitric oxide (NO) that activate soluble guanylate cyclase via nitrosylation of heme or formation of peroxynitrite via reaction with superoxide anion to produce tyrosine nitration. Here, we review the importance of pathological, aberrant transnitrosylation reactions, i.e., transfer of the NO group from one protein to another, and its consequent effect on the pathogenesis of neurological disorders, to date on Alzheimer's disease (AD), but also expected to affect Parkinson's disease (PD)/Lewy body dementia (LBD), HIV-associated neurocognitive disorder (HAND), and other neurodegenerative and neurodevelopmental disorders.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/metabolismo , Proteínas/metabolismo , Nitrosação , Óxido Nítrico/metabolismo , Sinapses/patologiaRESUMO
Redox modifications are described that can be harnessed for the treatment of neurodegenerative disorders, including Alzheimer's disease (AD). The approach has shown potential therapeutic efficacy in AD in both transgenic mouse and hiPSC cerebral organoids models. In this review, two such redox targets are highlighted. First, protein S-nitrosylation of the NMDA-type of glutamate receptor is described as a potential therapeutic target. Second, an S-alkylation reaction of critical, redox-active cysteine thiol(s) on the protein KEAP1 to activate the anti-oxidant/anti-inflammatory transcription factor NRF2 is proposed. In both approaches, we utilize compounds described as pathologically activated therapeutics (or "PAT" drugs), which can only be activated by the disease process that they then combat. Thus, PAT drugs remain relatively innocuous and therefore clinically-tolerated in normal tissue in the absence of disease, thereby avoiding severe side effects both systemically and in the brain.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Animais , Antioxidantes/uso terapêutico , Biologia , Cisteína/metabolismo , Cisteína/uso terapêutico , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , N-Metilaspartato/metabolismo , N-Metilaspartato/uso terapêutico , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/uso terapêutico , OxirreduçãoRESUMO
Protein S-nitrosylation is known to regulate enzymatic function. Here, we report that nitric oxide (NO)-related species can contribute to Alzheimer's disease (AD) by S-nitrosylating the lysosomal protease cathepsin B (forming SNO-CTSB), thereby inhibiting CTSB activity. This posttranslational modification inhibited autophagic flux, increased autolysosomal vesicles, and led to accumulation of protein aggregates. CA-074Me, a CTSB chemical inhibitor, also inhibited autophagic flux and resulted in accumulation of protein aggregates similar to the effect of SNO-CTSB. Inhibition of CTSB activity also induced caspase-dependent neuronal apoptosis in mouse cerebrocortical cultures. To examine which cysteine residue(s) in CTSB are S-nitrosylated, we mutated candidate cysteines and found that three cysteines were susceptible to S-nitrosylation. Finally, we observed an increase in SNO-CTSB in both 5XFAD transgenic mouse and flash-frozen postmortem human AD brains. These results suggest that S-nitrosylation of CTSB inhibits enzymatic activity, blocks autophagic flux, and thus contributes to AD pathogenesis.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Animais , Humanos , Camundongos , Catepsina B , Agregados Proteicos , Doenças Neurodegenerativas/genética , Proteínas/metabolismo , Doença de Alzheimer/metabolismo , Cisteína , Óxido NítricoRESUMO
Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1ß production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.
Assuntos
Trifosfato de Adenosina/metabolismo , DNA Mitocondrial/biossíntese , Inflamassomos/efeitos dos fármacos , Metformina/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia/prevenção & controle , Animais , COVID-19/metabolismo , COVID-19/prevenção & controle , Citocinas/genética , Citocinas/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Metformina/uso terapêutico , Camundongos , Núcleosídeo-Fosfato Quinase/metabolismo , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/prevenção & controle , SARS-CoV-2/patogenicidadeRESUMO
Significance: Physiological concentrations of nitric oxide (NOâ¢) and related reactive nitrogen species (RNS) mediate multiple signaling pathways in the nervous system. During inflammaging (chronic low-grade inflammation associated with aging) and in neurodegenerative diseases, excessive RNS contribute to synaptic and neuronal loss. "NO signaling" in both health and disease is largely mediated through protein S-nitrosylation (SNO), a redox-based posttranslational modification with "NO" (possibly in the form of nitrosonium cation [NO+]) reacting with cysteine thiol (or, more properly, thiolate anion [R-S-]). Recent Advances: Emerging evidence suggests that S-nitrosylation occurs predominantly via transnitros(yl)ation. Mechanistically, the reaction involves thiolate anion, as a nucleophile, performing a reversible nucleophilic attack on a nitroso nitrogen to form an SNO-protein adduct. Prior studies identified transnitrosylation reactions between glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-nuclear proteins, thioredoxin-caspase-3, and X-linked inhibitor of apoptosis (XIAP)-caspase-3. Recently, we discovered that enzymes previously thought to act in completely disparate biochemical pathways can transnitrosylate one another during inflammaging in an unexpected manner to mediate neurodegeneration. Accordingly, we reported a concerted tricomponent transnitrosylation network from Uch-L1-to-Cdk5-to-Drp1 that mediates synaptic damage in Alzheimer's disease. Critical Issues: Transnitrosylation represents a critical chemical mechanism for transduction of redox-mediated events to distinct subsets of proteins. Although thousands of thiol-containing proteins undergo S-nitrosylation, how transnitrosylation regulates a myriad of neuronal attributes is just now being uncovered. In this review, we highlight recent progress in the study of the chemical biology of transnitrosylation between proteins as a mechanism of disease. Future Directions: We discuss future areas of study of protein transnitrosylation that link our understanding of aging, inflammation, and neurodegenerative diseases. Antioxid. Redox Signal. 35, 531-550.
Assuntos
Doenças Neurodegenerativas , Humanos , Doenças Neurodegenerativas/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional , Espécies Reativas de Nitrogênio/metabolismo , Transdução de SinaisRESUMO
In the brain, both HIV-1 and methamphetamine (meth) use result in increases in oxidative and nitrosative stress. This redox stress is thought to contribute to the pathogenesis of HIV-associated neurocognitive disorder (HAND) and further worsening cognitive activity in the setting of drug abuse. One consequence of such redox stress is aberrant protein S-nitrosylation, derived from nitric oxide, which may disrupt normal protein activity. Here, we report an improved, mass spectrometry-based technique to assess S-nitrosylated protein in human postmortem brains using selective enrichment of S-nitrosocysteine residues with an organomercury resin. The data show increasing S-nitrosylation of tricarboxylic acid (TCA) enzymes in the setting of HAND and HAND/meth use compared with HIV+ control brains without CNS pathology. The consequence is systematic inhibition of multiple TCA cycle enzymes, resulting in energy collapse that can contribute to the neuronal and synaptic damage observed in HAND and meth use.
Assuntos
Ciclo do Ácido Cítrico/efeitos dos fármacos , Disfunção Cognitiva/metabolismo , Infecções por HIV/metabolismo , Metanfetamina/efeitos adversos , Processamento de Proteína Pós-Traducional , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Autopsia , Bancos de Espécimes Biológicos , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Ciclo do Ácido Cítrico/genética , Disfunção Cognitiva/complicações , Disfunção Cognitiva/patologia , Disfunção Cognitiva/virologia , Cisteína/análogos & derivados , Cisteína/metabolismo , Infecções por HIV/complicações , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Óxido Nítrico/metabolismo , S-Nitrosotióis/metabolismo , Transtornos Relacionados ao Uso de Substâncias/complicações , Transtornos Relacionados ao Uso de Substâncias/patologia , Transtornos Relacionados ao Uso de Substâncias/virologia , Sinapses/efeitos dos fármacos , Sinapses/patologiaRESUMO
Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of "sporadic" cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/metabolismo , S-Nitrosotióis/metabolismo , Esclerose Lateral Amiotrófica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Cisteína/metabolismo , Proteínas de Ligação a DNA/química , Demência Frontotemporal/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Óxido Nítrico/metabolismo , Agregação Patológica de Proteínas , Processamento Pós-Transcricional do RNA , Espécies Reativas de Nitrogênio/metabolismo , S-Nitrosotióis/química , Estresse FisiológicoRESUMO
Here we describe mechanistically distinct enzymes (a kinase, a guanosine triphosphatase, and a ubiquitin protein hydrolase) that function in disparate biochemical pathways and can also act in concert to mediate a series of redox reactions. Each enzyme manifests a second, noncanonical function-transnitrosylation-that triggers a pathological biochemical cascade in mouse models and in humans with Alzheimer's disease (AD). The resulting series of transnitrosylation reactions contributes to synapse loss, the major pathological correlate to cognitive decline in AD. We conclude that enzymes with distinct primary reaction mechanisms can form a completely separate network for aberrant transnitrosylation. This network operates in the postreproductive period, so natural selection against such abnormal activity may be decreased.
Assuntos
Doença de Alzheimer/enzimologia , Quinase 5 Dependente de Ciclina/metabolismo , Dinaminas/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Sinapses/enzimologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Cisteína/genética , Cisteína/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Nitroarginina/farmacologia , Oxirredução , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sinapses/patologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Significance: Most brains affected by neurodegenerative diseases manifest mitochondrial dysfunction as well as elevated production of reactive oxygen species and reactive nitrogen species (RNS), contributing to synapse loss and neuronal injury. Recent Advances: Excessive production of RNS triggers nitric oxide (NO)-mediated post-translational modifications of proteins, such as S-nitrosylation of cysteine residues and nitration of tyrosine residues. Proteins thus affected impair mitochondrial metabolism, mitochondrial dynamics, and mitophagy in the nervous system. Critical Issues: Identification and better characterization of underlying molecular mechanisms for NO-mediated mitochondrial dysfunction will provide important insights into the pathogenesis of neurodegenerative disorders. In this review, we highlight recent discoveries concerning S-nitrosylation of the tricarboxylic acid cycle enzymes, mitochondrial fission GTPase dynamin-related protein 1, and mitophagy-related proteins Parkin and phosphatase and tensin homolog-induced putative kinase protein 1. We delineate signaling cascades affected by pathologically S-nitrosylated proteins that diminish mitochondrial function in neurodegenerative diseases. Future Directions: Further elucidation of the pathological events resulting from aberrant S-nitrosothiol or nitrotyrosine formation may lead to new therapeutic approaches to ameliorate neurodegenerative disorders.
Assuntos
Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Óxido Nítrico/metabolismo , Animais , Humanos , Processamento de Proteína Pós-Traducional , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by heterozygous mutations in the TSC1 or TSC2 gene. TSC is often associated with neurological, cognitive, and behavioral deficits. TSC patients also express co-morbidity with anxiety and mood disorders. The mechanism of pathogenesis in TSC is not entirely clear, but TSC-related neurological symptoms are accompanied by excessive glutamatergic activity and altered synaptic spine structures. To address whether extrasynaptic (e)NMDA-type glutamate receptor (NMDAR) antagonists, as opposed to antagonists that block physiological phasic synaptic activity, can ameliorate the synaptic and behavioral features of this disease, we utilized the Tsc2+/- mouse model of TSC to measure biochemical, electrophysiological, histological, and behavioral parameters in the mice. We found that antagonists that preferentially block tonic activity as found at eNMDARs, particularly the newer drug NitroSynapsin, provide biological and statistically significant improvement in Tsc2+/- phenotypes. Accompanying this improvement was correction of activity in the p38 MAPK-TSC-Rheb-mTORC1-S6K1 pathway. Deficits in hippocampal long-term potentiation (LTP), histological loss of synapses, and behavioral fear conditioning in Tsc2+/- mice were all improved after treatment with NitroSynapsin. Taken together, these results suggest that amelioration of excessive excitation, by limiting aberrant eNMDAR activity, may represent a novel treatment approach for TSC.
Assuntos
Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Hipocampo/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Esclerose Tuberosa/tratamento farmacológico , Animais , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
Cerebrovascular changes occur in Alzheimer's disease (AD). Using in vivo phage display, we searched for molecular markers of the neurovascular unit, including endothelial cells and astrocytes, in mouse models of AD. We identified a cyclic peptide, CDAGRKQKC (DAG), that accumulates in the hippocampus of hAPP-J20 mice at different ages. Intravenously injected DAG peptide homes to neurovascular unit endothelial cells and to reactive astrocytes in mouse models of AD. We identified connective tissue growth factor (CTGF), a matricellular protein that is highly expressed in the brain of individuals with AD and in mouse models, as the target of the DAG peptide. We also showed that exogenously delivered DAG homes to the brain in mouse models of glioblastoma, traumatic brain injury, and Parkinson's disease. DAG may potentially be used as a tool to enhance delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos Cíclicos/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Modelos Animais de Doenças , Hipocampo/irrigação sanguínea , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Ligação ProteicaRESUMO
The prevalence of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), is currently a major public health concern due to the lack of efficient disease-modifying therapeutic options. Recent evidence suggests that mitochondrial dysfunction and nitrosative/oxidative stress are key common mediators of pathogenesis. In this review, we highlight molecular mechanisms linking NO-dependent post-translational modifications, such as cysteine S-nitrosylation and tyrosine nitration, to abnormal mitochondrial metabolism. We further discuss the hypothesis that pathological levels of NO compromise brain energy metabolism via aberrant S-nitrosylation of key enzymes in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, contributing to neurodegenerative conditions. A better understanding of these pathophysiological events may provide a potential pathway for designing novel therapeutics to ameliorate neurodegenerative disorders.
Assuntos
Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Ciclo do Ácido Cítrico/fisiologia , Transporte de Elétrons/fisiologia , Metabolismo Energético/fisiologia , Humanos , OxirreduçãoRESUMO
Purpose: Photoreceptor degeneration in the retina is a major cause of blindness in humans. Elucidating mechanisms of degenerative and neuroprotective pathways in photoreceptors should afford identification and development of therapeutic strategies. Methods: We used mouse genetic models and improved methods for retinal explant cultures. Retinas were enucleated from Mef2d+/+ and Mef2d-/- mice, stained for MEF2 proteins and outer nuclear layer thickness, and assayed for apoptotic cells. Chromatin immunoprecipitation (ChIP) assays revealed MEF2 binding, and RT-qPCR showed levels of transcription factors. We used AAV2 and electroporation to express genes in retinal explants and electroretinograms to assess photoreceptor functionality. Results: We identify a prosurvival MEF2D-PGC1α pathway that plays a neuroprotective role in photoreceptors. We demonstrate that Mef2d-/- mouse retinas manifest decreased expression of PGC1α and increased photoreceptor cell loss, resulting in the absence of light responses. Molecular repletion of PGC1α protects Mef2d-/- photoreceptors and preserves light responsivity. Conclusions: These results suggest that the MEF2-PGC1α cascade may represent a new therapeutic target for drugs designed to protect photoreceptors from developmental- and age-dependent loss.
Assuntos
Regulação da Expressão Gênica/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Células Fotorreceptoras de Vertebrados/fisiologia , Degeneração Retiniana/prevenção & controle , Envelhecimento , Animais , Apoptose , Sobrevivência Celular/fisiologia , Dependovirus/genética , Modelos Animais de Doenças , Eletroporação , Eletrorretinografia , Feminino , Terapia Genética , Marcação In Situ das Extremidades Cortadas , Fatores de Transcrição MEF2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/genética , Degeneração Retiniana/patologiaRESUMO
Recent studies have pointed to protein S-nitrosylation as a critical regulator of cellular redox homeostasis. For example, S-nitrosylation of peroxiredoxin-2 (Prx2), a peroxidase widely expressed in mammalian neurons, inhibits both enzymatic activity and protective function against oxidative stress. Here, using in vitro and in vivo approaches, we identify a role and reaction mechanism of the reductase sulfiredoxin (Srxn1) as an enzyme that denitrosylates (thus removing -SNO) from Prx2 in an ATP-dependent manner. Accordingly, by decreasing S-nitrosylated Prx2 (SNO-Prx2), overexpression of Srxn1 protects dopaminergic neural cells and human-induced pluripotent stem cell (hiPSC)-derived neurons from NO-induced hypersensitivity to oxidative stress. The pathophysiological relevance of this observation is suggested by our finding that SNO-Prx2 is dramatically increased in murine and human Parkinson's disease (PD) brains. Our findings therefore suggest that Srxn1 may represent a therapeutic target for neurodegenerative disorders such as PD that involve nitrosative/oxidative stress.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Doença de Parkinson/metabolismo , Peroxirredoxinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Neurônios Dopaminérgicos/citologia , Humanos , Hidrólise , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Óxido Nítrico/química , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Peroxirredoxinas/química , FosforilaçãoRESUMO
S-Nitrosylation is well established as an important post-translational regulator in protein function and signaling. However, relatively little is known about its structural and dynamical consequences. We have investigated the effects of S-nitrosylation on the rhodanese domain of the Escherichia coli integral membrane protein YgaP by NMR, X-ray crystallography, and mass spectrometry. The results show that the active cysteine in the rhodanese domain of YgaP is subjected to two competing modifications: S-nitrosylation and S-sulfhydration, which are naturally occurring in vivo. It has been observed that in addition to inhibition of the sulfur transfer activity, S-nitrosylation of the active site residue Cys63 causes an increase in slow motion and a displacement of helix 5 due to a weakening of the interaction between the active site and the helix dipole. These findings provide an example of how nitrosative stress can exert action at the atomic level.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Processamento de Proteína Pós-Traducional , Tiossulfato Sulfurtransferase/química , Tiossulfato Sulfurtransferase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Cisteína/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Conformação ProteicaRESUMO
Reactive nitrogen species, such as nitric oxide (NO), exert their biological activity in large part through post-translational modification of cysteine residues, forming S-nitrosothiols. This chemical reaction proceeds via a process that we and our colleagues have termed protein S-nitrosylation. Under conditions of normal NO production, S-nitrosylation regulates the activity of many normal proteins. However, in degenerative conditions characterized by nitrosative stress, increased levels of NO lead to aberrant S-nitrosylation that contributes to the pathology of the disease. Thus, S-nitrosylation has been implicated in a wide range of cellular mechanisms, including mitochondrial function, proteostasis, transcriptional regulation, synaptic activity, and cell survival. In recent years, the research area of protein S-nitrosylation has become prominent due to improvements in the detection systems as well as the demonstration that protein S-nitrosylation plays a critical role in the pathogenesis of neurodegenerative and other neurological disorders. To further promote our understanding of how protein S-nitrosylation affects cellular systems, guidelines for the design and conduct of research on S-nitrosylated (or SNO-)proteins would be highly desirable, especially for those newly entering the field. In this review article, we provide a strategic overview of designing experimental approaches to study protein S-nitrosylation. We specifically focus on methods that can provide critical data to demonstrate that an S-nitrosylated protein plays a (patho-)physiologically-relevant role in a biological process. Hence, the implementation of the approaches described herein will contribute to further advancement of the study of S-nitrosylated proteins, not only in neuroscience but also in other research fields.
Assuntos
Óxido Nítrico/metabolismo , Proteínas/metabolismo , Projetos de Pesquisa , Animais , Cisteína/metabolismo , Humanos , Doadores de Óxido Nítrico/farmacologia , Nitrosação , Processamento de Proteína Pós-Traducional , S-Nitrosotióis/metabolismoRESUMO
At physiological levels, nitric oxide (NO) contributes to the maintenance of normal neuronal activity and survival, thus serving as an important regulatory mechanism in the central nervous system. By contrast, accumulating evidence suggests that exposure to environmental toxins or the normal aging process can trigger excessive production of reactive oxygen/nitrogen species (such as NO), contributing to the etiology of several neurodegenerative diseases. We highlight here protein S-nitrosylation, resulting from covalent attachment of an NO group to a cysteine thiol of the target protein, as a ubiquitous effector of NO signaling in both health and disease. We review our current understanding of this redox-dependent post-translational modification under neurodegenerative conditions, and evaluate how targeting dysregulated protein S-nitrosylation can lead to novel therapeutics.
Assuntos
Doenças Neurodegenerativas/metabolismo , Óxido Nítrico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Nitrosação , Processamento de Proteína Pós-Traducional , Espécies Reativas de Nitrogênio/metabolismo , S-Nitrosotióis/metabolismo , Sistemas do Segundo MensageiroRESUMO
Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson's disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.
Assuntos
Estresse do Retículo Endoplasmático/genética , Endorribonucleases/metabolismo , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/metabolismo , Substituição de Aminoácidos , Animais , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Cisteína/química , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Endorribonucleases/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Serina/química , Serina/metabolismo , Transdução de Sinais , eIF-2 Quinase/genéticaRESUMO
Effective treatment of diabetic neuropathy (DN) remains unsolved. We serendipitously observed dramatic relief of pain in several patients with painful DN receiving granulocyte-colony stimulating factor (G-CSF). The aim of this study was to determine if G-CSF could treat DN in an animal model and to ascertain its mechanism of action. In a rodent model of DN, G-CSF dramatically recovered nerve function, retarded histological nerve changes and increased the expression of neurotrophic factors within nerve. A sex-mismatched bone marrow transplantation (BMT) study revealed that G-CSF treatment increased the abundance of bone marrow (BM)-derived cells in nerves damaged by DN. However, we did not observe evidence of transdifferentiation or cell fusion of BM-derived cells. The beneficial effects of G-CSF were dependent on the integrity of BM. In conclusion, G-CSF produced a therapeutic effect in a rodent model of DN, which was attributed, at least in part, to the actions of BM-derived cells.