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1.
J Am Heart Assoc ; 11(5): e024008, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35191317

RESUMO

Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S-nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S-nitrosoglutathione reductase (GSNOR-/-), a denitrosylase regulating protein S-nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S-nitrosylated amino acid residues in GSNOR-/- mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S-nitrosoproteome, and identifies a unique set of S-nitrosylated proteins in GSNOR-/- mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S-nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.


Assuntos
Álcool Desidrogenase , Óxido Nítrico , Placenta , Pré-Eclâmpsia , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Feminino , Camundongos , Óxido Nítrico/metabolismo , Placenta/enzimologia , Placenta/metabolismo , Pré-Eclâmpsia/enzimologia , Pré-Eclâmpsia/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismo
2.
Nature ; 600(7887): 158-163, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34819667

RESUMO

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription1-4. This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins5-7. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5-/-) and CSB (Csbm/m; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5-/-Csbm/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.


Assuntos
Síndrome de Cockayne , Dano ao DNA , Formaldeído/efeitos adversos , Estresse Fisiológico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Caquexia/complicações , Síndrome de Cockayne/induzido quimicamente , Síndrome de Cockayne/complicações , Síndrome de Cockayne/genética , Síndrome de Cockayne/patologia , Enzimas Reparadoras do DNA/deficiência , Modelos Animais de Doenças , Feminino , Formaldeído/metabolismo , Fator 15 de Diferenciação de Crescimento/antagonistas & inibidores , Fator 15 de Diferenciação de Crescimento/biossíntese , Fator 15 de Diferenciação de Crescimento/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , Insuficiência Renal/complicações , Transcrição Gênica/genética
3.
Cell Rep ; 34(13): 108922, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33789111

RESUMO

Hematopoietic stem cells (HSCs) regenerate blood cells upon hematopoietic injuries. During homeostasis, HSCs are maintained in a low reactive oxygen species (ROS) state to prevent exhaustion. However, the role of nitric oxide (NO) in controlling HSC regeneration is still unclear. Here, we find increased NO during HSC regeneration with an accumulation of protein aggregation. S-nitrosoglutathione reductase (GSNOR)-deleted HSCs exhibit a reduced reconstitution capacity and loss of self-renewal after chemotherapeutic injury, which is resolved by inhibition of NO synthesis. Deletion of GSNOR enhances protein S-nitrosylation, resulting in an accumulation of protein aggregation and activation of unfolded protein response (UPR). Treatment of taurocholic acid (TCA), a chemical chaperone, rescues the regeneration defect of Gsnor-/- HSCs after 5-fluorouracil (5-FU) treatment. Deletion of C/EBP homologous protein (Chop) restores the reconstitution capacity of Gsnor-/- HSCs. These findings establish a link between S-nitrosylation and protein aggregation in HSC in the context of blood regeneration.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas/metabolismo , Proteostase , Regeneração , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/metabolismo , Sobrevivência Celular , Fluoruracila/farmacologia , Deleção de Genes , Óxido Nítrico/metabolismo , Nitrosação , Fenótipo , Agregados Proteicos , Fator de Transcrição CHOP/metabolismo
4.
J Clin Invest ; 131(4)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33586683

RESUMO

The relationship between adiposity and metabolic health is well established. However, very little is known about the fat depot, known as paracardial fat (pCF), located superior to and surrounding the heart. Here, we show that pCF remodels with aging and a high-fat diet and that the size and function of this depot are controlled by alcohol dehydrogenase 1 (ADH1), an enzyme that oxidizes retinol into retinaldehyde. Elderly individuals and individuals with obesity have low ADH1 expression in pCF, and in mice, genetic ablation of Adh1 is sufficient to drive pCF accumulation, dysfunction, and global impairments in metabolic flexibility. Metabolomics analysis revealed that pCF controlled the levels of circulating metabolites affecting fatty acid biosynthesis. Also, surgical removal of the pCF depot was sufficient to rescue the impairments in cardiometabolic flexibility and fitness observed in Adh1-deficient mice. Furthermore, treatment with retinaldehyde prevented pCF remodeling in these animals. Mechanistically, we found that the ADH1/retinaldehyde pathway works by driving PGC-1α nuclear translocation and promoting mitochondrial fusion and biogenesis in the pCF depot. Together, these data demonstrate that pCF is a critical regulator of cardiometabolic fitness and that retinaldehyde and its generating enzyme ADH1 act as critical regulators of adipocyte remodeling in the pCF depot.


Assuntos
Tecido Adiposo/enzimologia , Álcool Desidrogenase/metabolismo , Mitocôndrias Cardíacas/metabolismo , Obesidade/enzimologia , Pericárdio/enzimologia , Tecido Adiposo/patologia , Álcool Desidrogenase/deficiência , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Metabolômica , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Obesidade/genética , Obesidade/patologia , Pericárdio/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Retinaldeído/metabolismo , Transdução de Sinais/genética
5.
Sci Rep ; 10(1): 9206, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32514067

RESUMO

Candida albicans is a commensal yeast able to cause life threatening invasive infections particularly in immunocompromised patients. Despite the availability of antifungal treatments, mortality rates are still unacceptably high and drug resistance is increasing. We, therefore, generated the Ca37 monoclonal antibody against the C. albicans alcohol dehydrogenase (Adh) 1. Our data showed that Ca37 was able to detect C. albicans cells, and it bound to Adh1 in yeast and Adh2 in hyphae among the cell wall-associated proteins. Moreover, Ca37 was able to inhibit candidal growth following 18 h incubation time and reduced the minimal inhibitory concentration of amphotericin B or fluconazole when used in combination with those antifungals. In addition, the antibody prolonged the survival of C. albicans infected-Galleria mellonella larvae, when C. albicans was exposed to antibody prior to inoculating G. mellonella or by direct application as a therapeutic agent on infected larvae. In conclusion, the Ca37 monoclonal antibody proved to be effective against C. albicans, both in vitro and in vivo, and to act together with antifungal drugs, suggesting Adh proteins could be interesting therapeutic targets against this pathogen.


Assuntos
Álcool Desidrogenase/imunologia , Anticorpos Monoclonais/farmacologia , Candida albicans/enzimologia , Proteínas Fúngicas/imunologia , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Anfotericina B/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Candidíase/veterinária , Fluconazol/farmacologia , Hifas/enzimologia , Larva/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Virulência
6.
Cell Death Dis ; 10(5): 354, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043586

RESUMO

Neuronal nitric oxide synthase (nNOS) plays a crucial role in the maintenance of correct skeletal muscle function due, at least in part, to S-nitrosylation of specific protein targets. Similarly, we recently provided evidence for a muscular phenotype in mice lacking the denitrosylase S-nitrosoglutathione reductase (GSNOR). Here, we demonstrate that nNOS and GSNOR are concomitantly expressed during differentiation of C2C12. They colocalizes at the sarcolemma and co-immunoprecipitate in cells and in myofibers. We also provide evidence that GSNOR expression decreases in mouse models of muscular dystrophies and of muscle atrophy and wasting, i.e., aging and amyotrophic lateral sclerosis, suggesting a more general regulatory role of GSNOR in skeletal muscle homeostasis.


Assuntos
Envelhecimento/genética , Álcool Desidrogenase/genética , Homeostase/genética , Desenvolvimento Muscular/genética , Distrofias Musculares/genética , Óxido Nítrico Sintase Tipo I/genética , Envelhecimento/metabolismo , Álcool Desidrogenase/antagonistas & inibidores , Álcool Desidrogenase/deficiência , Animais , Diferenciação Celular , Linhagem Celular Transformada , Modelos Animais de Doenças , Proteínas Associadas à Distrofina/genética , Proteínas Associadas à Distrofina/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Mioblastos/citologia , Mioblastos/enzimologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sarcolema/enzimologia , Transdução de Sinais , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
7.
Cell Death Differ ; 25(11): 1967-1979, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29511342

RESUMO

Metabolically reactive formaldehyde is a genotoxin and a carcinogen. Mice lacking the main formaldehyde-detoxifying gene Adh5 combined with the loss of the Fanconi anemia (FA) DNA repair pathway rapidly succumbed to bone marrow failure (BMF) primarily due to the extensive ablation of the hematopoietic stem cell (HSC) pool. However, the mechanism by which formaldehyde mediates these toxic effects is still unknown. We uncover a detrimental role of tetrahydrofolic acid (THF) in cells lacking Adh5 or the FA repair pathway. We show that Adh5- or FA-deficient cells are hypersensitive to formaldehyde and to THF, presenting DNA damage and genome instability. THF cytotoxicity involved imbalance of the nucleotide pool by deregulation of the thymidylate synthase (TYMS) enzyme, which stalled replication forks. In mice, THF exposure had widespread effects on hematopoiesis, affecting the frequency and the viability of myeloid- and lymphoid-committed precursor cells. Moreover, the hematopoietic stem and progenitor cells (HSPC) showed genomic instability, reduced colony-forming capacity and increased frequency of cycling and apoptotic HSCs upon THF exposure. Overall, our data reveal that the physiological pool of THF and formaldehyde challenge the stability of the genome of HSPCs that might lead to blood disorders.


Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Tetra-Hidrofolatos/toxicidade , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Galinhas , Proteínas de Grupos de Complementação da Anemia de Fanconi/deficiência , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Instabilidade Genômica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Timidilato Sintase/metabolismo
8.
Nature ; 553(7687): 171-177, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29323295

RESUMO

Haematopoietic stem cells renew blood. Accumulation of DNA damage in these cells promotes their decline, while misrepair of this damage initiates malignancies. Here we describe the features and mutational landscape of DNA damage caused by acetaldehyde, an endogenous and alcohol-derived metabolite. This damage results in DNA double-stranded breaks that, despite stimulating recombination repair, also cause chromosome rearrangements. We combined transplantation of single haematopoietic stem cells with whole-genome sequencing to show that this damage occurs in stem cells, leading to deletions and rearrangements that are indicative of microhomology-mediated end-joining repair. Moreover, deletion of p53 completely rescues the survival of aldehyde-stressed and mutated haematopoietic stem cells, but does not change the pattern or the intensity of genome instability within individual stem cells. These findings characterize the mutation of the stem-cell genome by an alcohol-derived and endogenous source of DNA damage. Furthermore, we identify how the choice of DNA-repair pathway and a stringent p53 response limit the transmission of aldehyde-induced mutations in stem cells.


Assuntos
Acetaldeído/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Etanol/metabolismo , Etanol/farmacologia , Instabilidade Genômica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Mutação , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades , Etanol/administração & dosagem , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Feminino , Deleção de Genes , Genes p53/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Autoantígeno Ku/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sequenciamento Completo do Genoma
9.
Alcohol Clin Exp Res ; 41(10): 1675-1685, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28792616

RESUMO

BACKGROUND: Chronic alcohol abuse, a major risk factor for such diseases as hepatitis and cirrhosis, impairs hepatic alcohol dehydrogenase (ADH; key ethanol [EtOH]-metabolizing enzyme). Therefore, differentially altered hepatic and plasma proteomes were identified in chronic EtOH feeding model of hepatic ADH-deficient (ADH- ) deer mice to understand the metabolic basis of alcoholic liver disease (ALD). METHODS: ADH- deer mice were fed 3.5 g% EtOH via Lieber-DeCarli liquid diet daily for 3 months and histology of the liver assessed. Liver and plasma proteins were separated by 2-dimensional gel electrophoresis. The proteins differentially expressed were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS: Histology of the liver showed panlobular steatosis and infiltration of T lymphocytes. Using the criteria of ≥1.5 for fold change (p-value ≤0.05) with expectation value (E ≤10-3 ) and protein score (≥64), 18 proteins in the livers and 5 in the plasma of EtOH-fed mice were differentially expressed and identified. Prolyl 4-hydroxylase, cytochrome b-5, endo A cytokeratin, ATP synthase, heat-shock 70 kD proteins, enoyl CoA hydratase, stress-70 protein, peroxiredoxin 1, and ornithine carbamoyl transferase were up-regulated in the livers. However, carbonic anhydrase 3, mitochondrial ATP synthase, aldolase 2, actin γ, laminin receptor, and carbamoyl phosphate synthase were down-regulated. Contrary to the increased expression of creatine kinase M-type, a decreased expression of serine protease inhibitor A3A precursor, sulfated glycoprotein-2 (clusterin), and apolipoprotein E isoforms were found in the plasma of EtOH group. CONCLUSIONS: Chronic EtOH feeding in ADH- deer mice causes steatosis and infiltration of T lymphocytes in the livers along with increased expression of proteins involved in endoplasmic reticulum (ER) stress, fibrosis, fatty acid ß oxidation and biogenesis, and decreased expression of proteins involved in ATP synthesis, carbohydrate metabolism, in cell regulation and architecture. Reduced expression of various carrier proteins as found in the plasma of EtOH group has a biomarker potential.


Assuntos
Álcool Desidrogenase/deficiência , Etanol/toxicidade , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Proteômica/métodos , Álcool Desidrogenase/genética , Animais , Etanol/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/genética , Masculino , Camundongos , Peromyscus
10.
Infect Immun ; 85(9)2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28674030

RESUMO

Artesunate remains the mainstay of treatment for cerebral malaria, but it is less effective in later stages of disease when the host inflammatory response and blood-brain barrier integrity dictate clinical outcomes. Nitric oxide (NO) is an important regulator of inflammation and microvascular integrity, and impaired NO bioactivity is associated with fatal outcomes in malaria. Endogenous NO bioactivity in mammals is largely mediated by S-nitrosothiols (SNOs). Based on these observations, we hypothesized that animals deficient in the SNO-metabolizing enzyme, S-nitrosoglutathione reductase (GSNOR), which exhibit enhanced S-nitrosylation, would have improved outcomes in a preclinical model of cerebral malaria. GSNOR knockout (KO) mice infected with Plasmodium berghei ANKA had significantly delayed mortality compared to WT animals (P < 0.0001), despite higher parasite burdens (P < 0.01), and displayed markedly enhanced survival versus the wild type (WT) when treated with the antimalarial drug artesunate (77% versus 38%; P < 0.001). Improved survival was associated with higher levels of protein-bound NO, decreased levels of CD4+ and CD8+ T cells in the brain, improved blood-brain barrier integrity, and improved coma scores, as well as higher levels of gamma interferon. GSNOR KO animals receiving WT bone marrow had significantly reduced survival following P. berghei ANKA infection compared to those receiving KO bone barrow (P < 0.001). Reciprocal transplants established that survival benefits of GSNOR deletion were attributable primarily to the T cell compartment. These data indicate a role for GSNOR in the host response to malaria infection and suggest that strategies to disrupt its activity will improve clinical outcomes by enhancing microvascular integrity and modulating T cell tissue tropism.


Assuntos
Álcool Desidrogenase/deficiência , Malária Cerebral/patologia , Plasmodium berghei/patogenicidade , Animais , Antimaláricos/administração & dosagem , Artemisininas/administração & dosagem , Artesunato , Modelos Animais de Doenças , Feminino , Malária Cerebral/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Análise de Sobrevida , Linfócitos T/imunologia , Resultado do Tratamento
11.
Pancreas ; 46(6): 806-812, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28609370

RESUMO

OBJECTIVES: The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. METHODS: Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. RESULTS: Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit ß and the proteins involved in cell motility, structure, and conformation. CONCLUSIONS: Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.


Assuntos
Álcool Desidrogenase/deficiência , Consumo de Bebidas Alcoólicas , Etanol , Fígado/enzimologia , Pâncreas/metabolismo , Pancreatite Alcoólica/metabolismo , Proteínas/metabolismo , Álcool Desidrogenase/genética , Animais , Modelos Animais de Doenças , Genótipo , Masculino , Camundongos Knockout , Pancreatite Alcoólica/genética , Peromyscus , Fenótipo , Proteômica/métodos , Fatores de Tempo
12.
ACS Synth Biol ; 6(4): 659-666, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28080037

RESUMO

The use of renewable feedstocks for producing biofuels and biobased chemicals by engineering metabolic pathways of yeast Saccharomyces cerevisiae has recently become an attractive option. Many researchers attempted to increase glucose consumption rate by overexpressing some glycolytic enzymes because most target biobased chemicals are derived through glycolysis. However, these attempts have met with little success. In this study, to create a S. cerevisiae strain with high glucose consumption rate, we used multicopy integration to develop a global metabolic engineering strategy. Among approximately 350 metabolically engineered strains, YPH499/dPdA3-34 exhibited the highest glucose consumption rate. This strain showed 1.3-fold higher cell growth rate and glucose consumption rate than the control strain. Real-time PCR analysis revealed that transcription levels of glycolysis-related genes such as HXK2, PFK1, PFK2, PYK2, PGI1, and PGK1 in YPH499/dPdA3-34 were increased. Our strategy is thus a promising approach to optimize global metabolic pathways in S. cerevisiae.


Assuntos
Engenharia Metabólica , Saccharomyces cerevisiae/metabolismo , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Dosagem de Genes , Glucose/metabolismo , Glicólise/genética , Hexoquinase/genética , Hexoquinase/metabolismo , Redes e Vias Metabólicas , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Piruvato Descarboxilase/deficiência , Piruvato Descarboxilase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Antioxid Redox Signal ; 26(4): 165-181, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27412893

RESUMO

AIM: Nitric oxide (NO) plays important, but incompletely defined roles in skeletal muscle. NO exerts its regulatory effects partly though S-nitrosylation, which is balanced by denitrosylation by enzymes such as S-nitrosoglutathione reductase (GSNOR), whose functions in skeletal muscle remain to be fully deciphered. RESULTS: GSNOR null (GSNOR-/-) tibialis anterior (TA) muscles showed normal growth and were stronger and more fatigue resistant than controls in situ. However, GSNOR-/- lumbrical muscles showed normal contractility and Ca2+ handling in vitro, suggesting important differences in GSNOR function between muscles or between in vitro and in situ environments. GSNOR-/- TA muscles exhibited normal mitochondrial content, and capillary densities, but reduced type IIA fiber content. GSNOR inhibition did not impact mitochondrial respiratory complex I, III, or IV activities. These findings argue that enhanced GSNOR-/- TA contractility is not driven by changes in mitochondrial content or activity, fiber type, or blood vessel density. However, loss of GSNOR led to RyR1 hypernitrosylation, which is believed to increase muscle force output under physiological conditions. cGMP synthesis by soluble guanylate cyclase (sGC) was decreased in resting GSNOR-/- muscle and was more responsive to agonist (DETANO, BAY 41, and BAY 58) stimulation, suggesting that GSNOR modulates cGMP production in skeletal muscle. INNOVATION: GSNOR may act as a "brake" on skeletal muscle contractile performance under physiological conditions by modulating nitrosylation/denitrosylation balance. CONCLUSIONS: GSNOR may play important roles in skeletal muscle contractility, RyR1 S-nitrosylation, fiber type specification, and sGC activity. Antioxid. Redox Signal. 26, 165-181.


Assuntos
Álcool Desidrogenase/deficiência , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , Fadiga Muscular/genética , Força Muscular/genética , Músculo Esquelético/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cálcio/metabolismo , GMP Cíclico/biossíntese , Genótipo , Hipertrofia , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/patologia , Neovascularização Fisiológica
14.
Circ Res ; 117(10): 846-57, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26338901

RESUMO

RATIONALE: S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. OBJECTIVE: To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. METHODS AND RESULTS: We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. CONCLUSIONS: Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations.


Assuntos
Biotina/metabolismo , Cisteína/metabolismo , Sondas Moleculares , Miocárdio/metabolismo , Compostos Nitrosos/metabolismo , Proteômica/métodos , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrosação , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
16.
J Am Heart Assoc ; 4(7)2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26178404

RESUMO

BACKGROUND: Mammalian heart regenerative activity is lost before adulthood but increases after cardiac injury. Cardiac repair mechanisms, which involve both endogenous cardiac stem cells (CSCs) and cardiomyocyte cell-cycle reentry, are inadequate to achieve full recovery after myocardial infarction (MI). Mice deficient in S-nitrosoglutathione reductase (GSNOR(-/-)), an enzyme regulating S-nitrosothiol turnover, have preserved cardiac function after MI. Here, we tested the hypothesis that GSNOR activity modulates cardiac cell proliferation in the post-MI adult heart. METHODS AND RESULTS: GSNOR(-/-) and C57Bl6/J (wild-type [WT]) mice were subjected to sham operation (n=3 GSNOR(-/-); n=3 WT) or MI (n=41 GSNOR(-/-); n=65 WT). Compared with WT, GSNOR(-/-) mice exhibited improved survival, cardiac performance, and architecture after MI, as demonstrated by higher ejection fraction (P<0.05), lower endocardial volumes (P<0.001), and diminished scar size (P<0.05). In addition, cardiomyocytes from post-MI GSNOR(-/-) hearts exhibited faster calcium decay and sarcomeric relaxation times (P<0.001). Immunophenotypic analysis illustrated that post-MI GSNOR(-/-) hearts demonstrated enhanced neovascularization (P<0.001), c-kit(+) CSC abundance (P=0.013), and a ≈3-fold increase in proliferation of adult cardiomyocytes and c-kit(+)/CD45(-) CSCs (P<0.0001 and P=0.023, respectively) as measured by using 5-bromodeoxyuridine. CONCLUSIONS: Loss of GSNOR confers enhanced post-MI cardiac regenerative activity, characterized by enhanced turnover of cardiomyocytes and CSCs. Endogenous denitrosylases exert an inhibitory effect over cardiac repair mechanisms and therefore represents a potential novel therapeutic target.


Assuntos
Células-Tronco Adultas/enzimologia , Álcool Desidrogenase/deficiência , Proliferação de Células , Infarto do Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Regeneração , Células-Tronco Adultas/patologia , Álcool Desidrogenase/genética , Animais , Biomarcadores/metabolismo , Sinalização do Cálcio , Células Cultivadas , Modelos Animais de Doenças , Homozigoto , Antígenos Comuns de Leucócito/deficiência , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/patologia , Neovascularização Fisiológica , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Volume Sistólico , Fatores de Tempo
17.
Appl Environ Microbiol ; 81(1): 453-60, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25362059

RESUMO

4-Hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3(2H)-furanone (HEMF) is an important flavor compound that contributes to the sensory properties of many natural products, particularly soy sauce and soybean paste. The compound exhibits a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. HEMF is produced by yeast species in soy sauce manufacturing; however, the enzymes involved in HEMF production remain unknown, hindering efforts to breed yeasts with high-level HEMF production. In this study, we identified high-level HEMF-producing mutants among a Saccharomyces cerevisiae gene deletion mutant collection. Fourteen deletion mutants were screened as high-level HEMF-producing mutants, and the ADH1 gene deletion mutant (adh1Δ) exhibited the maximum HEMF production capacity. Further investigations of the adh1Δ mutant implied that acetaldehyde accumulation contributes to HEMF production, agreeing with previous findings. Therefore, acetaldehyde might be a precursor for HEMF. The ADH1 gene deletion mutant of Zygosaccharomyces rouxii, which is the dominant strain of yeast found during soy sauce fermentation, also produces HEMF effectively, suggesting that acetaldehyde accumulation might be a benchmark for breeding industrial yeasts with excellent HEMF production abilities.


Assuntos
Furanos/metabolismo , Deleção de Genes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetaldeído/metabolismo , Álcool Desidrogenase/deficiência , Antioxidantes/metabolismo , Aromatizantes/metabolismo , Programas de Rastreamento , Proteínas de Saccharomyces cerevisiae
18.
Toxicol Appl Pharmacol ; 277(2): 109-17, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24625836

RESUMO

Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to <1.0% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 µg/g in the lungs of ethanol-fed mice as compared to 1.5 µg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 was observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease.


Assuntos
Álcool Desidrogenase/deficiência , Consumo de Bebidas Alcoólicas/efeitos adversos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Etanol/toxicidade , Fígado/enzimologia , Pulmão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Acetaldeído/sangue , Álcool Desidrogenase/genética , Consumo de Bebidas Alcoólicas/sangue , Consumo de Bebidas Alcoólicas/patologia , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Retículo Endoplasmático/metabolismo , Esterificação , Ésteres/metabolismo , Etanol/sangue , Ácidos Graxos/metabolismo , Genótipo , Pulmão/metabolismo , Pulmão/patologia , Masculino , Peromyscus , Fenótipo , Fatores de Tempo , Resposta a Proteínas não Dobradas/efeitos dos fármacos
19.
Nat Commun ; 4: 2632, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24153335

RESUMO

The growing availability of 'omics' data and high-quality in silico genome-scale metabolic models (GSMMs) provide a golden opportunity for the systematic identification of new metabolic drug targets. Extant GSMM-based methods aim at identifying drug targets that would kill the target cell, focusing on antibiotics or cancer treatments. However, normal human metabolism is altered in many diseases and the therapeutic goal is fundamentally different--to retrieve the healthy state. Here we present a generic metabolic transformation algorithm (MTA) addressing this issue. First, the prediction accuracy of MTA is comprehensively validated using data sets of known perturbations. Second, two predicted yeast lifespan-extending genes, GRE3 and ADH2, are experimentally validated, together with their associated hormetic effect. Third, we show that MTA predicts new drug targets for human ageing that are enriched with orthologs of known lifespan-extending genes and with genes downregulated following caloric restriction mimetic treatments. MTA offers a promising new approach for the identification of drug targets in metabolically related disorders.


Assuntos
Envelhecimento/metabolismo , Algoritmos , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Terapia de Alvo Molecular , Saccharomyces cerevisiae/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Oxirredutases do Álcool/deficiência , Oxirredutases do Álcool/genética , Restrição Calórica , Simulação por Computador , Eicosanoides/metabolismo , Genoma Fúngico , Genoma Humano , Hormese , Humanos , Longevidade/efeitos dos fármacos , Longevidade/genética , Redes e Vias Metabólicas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Estilbenos/farmacologia
20.
Microb Cell Fact ; 12: 57, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23758664

RESUMO

BACKGROUND: Polylactic acid (PLA), a biodegradable polymer, has the potential to replace (at least partially) traditional petroleum-based plastics, minimizing "white pollution". However, cost-effective production of optically pure L-lactic acid is needed to achieve the full potential of PLA. Currently, starch-based glucose is used for L-lactic acid fermentation by lactic acid bacteria. Due to its competition with food resources, an alternative non-food substrate such as cellulosic biomass is needed for L-lactic acid fermentation. Nevertheless, the substrate (sugar stream) derived from cellulosic biomass contains significant amounts of xylose, which is unfermentable by most lactic acid bacteria. However, the microorganisms that do ferment xylose usually carry out heterolactic acid fermentation. As a result, an alternative strain should be developed for homofermentative production of optically pure L-lactic acid using cellulosic biomass. RESULTS: In this study, an ethanologenic Escherichia coli strain, SZ470 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA), was reengineered for homofermentative production of L-lactic acid from xylose (1.2 mole xylose = > 2 mole L-lactic acid), by deleting the alcohol dehydrogenase gene (adhE) and integrating the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici. The resulting strain, WL203, was metabolically evolved further through serial transfers in screw-cap tubes containing xylose, resulting in the strain WL204 with improved anaerobic cell growth. When tested in 70 g L-1 xylose fermentation (complex medium), WL204 produced 62 g L-1 L-lactic acid, with a maximum production rate of 1.631 g L-1 h-1 and a yield of 97% based on xylose metabolized. HPLC analysis using a chiral column showed that an L-lactic acid optical purity of 99.5% was achieved by WL204. CONCLUSIONS: These results demonstrated that WL204 has the potential for homofermentative production of L-lactic acid using cellulosic biomass derived substrates, which contain a significant amount of xylose.


Assuntos
Escherichia coli/metabolismo , Ácido Láctico/biossíntese , Xilose/metabolismo , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Proteínas de Bactérias/genética , Biomassa , Fermentação , Engenharia Genética , L-Lactato Desidrogenase/genética , Ácido Láctico/síntese química , Pediococcus/enzimologia , Pediococcus/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Poliésteres , Polímeros/síntese química , Estereoisomerismo
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