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1.
Nutrients ; 14(22)2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36432463

RESUMO

Hypertension (HTN) is a major risk factor for cardiovascular disease (CVD) and cognitive decline. Elevations in blood pressure (BP) leading to HTN can be found in young adults with increased prevalence as people age. Oats are known to decrease CVD risk via an established effect of ß-glucan on the attenuation of blood cholesterol. Many past studies on CVD and oats have also reported a decrease in BP; however, a thorough assessment of oats and BP has not been conducted. Moreover, oats deliver several beneficial dietary components with putative beneficial effects on BP or endothelial function, such as ß-glucan, γ-amino butyric acid (GABA), and phytochemicals such as avenanthramides. We conducted a comprehensive search for systematic reviews, meta-analyses, and clinical intervention studies on oats and BP and identified 18 randomized controlled trials (RCTs) and three meta-analyses that supported the role of oats in decreasing BP. Emerging data also suggest oat consumption may reduce the use of anti-hypertensive medications. The majority of these studies utilized whole oats or oat bran, which include a vast array of oat bioactives. Therefore, we also extensively reviewed the literature on these bioactives and their putative effect on BP-relevant mechanisms. The data suggest several oat components, such as GABA, as well as the delivery of high-quality plant protein and fermentable prebiotic fiber, may contribute to the anti-HTN effect of oats. In particular, GABA is enhanced in oat sprouts, which suggests this food may be particularly beneficial for healthy BP management.


Assuntos
Doenças Cardiovasculares , Hipertensão , beta-Glucanas , Humanos , Avena , Pressão Sanguínea , Revisões Sistemáticas como Assunto , Grão Comestível , Fibras na Dieta/farmacologia , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Hipertensão/prevenção & controle , Hipertensão/tratamento farmacológico , beta-Glucanas/farmacologia , Doenças Cardiovasculares/tratamento farmacológico , Ácido gama-Aminobutírico
2.
J Nutr ; 151(9): 2655-2666, 2021 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-34236436

RESUMO

BACKGROUND: High-molecular-weight (MW) oat ß-glucan (OBG), consumed at 3-4 g/d, in solid foods reduces LDL cholesterol by a median of ∼6.5%. OBJECTIVES: We evaluated the effect of a beverage providing 3 g/d high-MW OBG on reduction of LDL cholesterol (primary endpoint) when compared with placebo. METHODS: We performed a parallel-design, randomized clinical trial at a contract research organization; participants, caregivers, and outcome assessors were blinded to treatment allocation. Participants with LDL cholesterol between 3.0 and 5.0 mmol/L, inclusive [n = 538 screened, n = 260 ineligible, n = 23 lost, n = 48 withdrawn (product safety); n = 207 randomly assigned, n = 7 dropped out, n = 9 withdrawn (protocol violation); n = 191 analyzed; n = 72 (37.7%) male, mean ± SD age: 43.3 ± 14.3 y, BMI: 29.7 ± 5.2 kg/m2], were randomly assigned to consume, 3 times daily for 4 wk, 1 g OBG (n = 104, n = 96 analyzed) or rice powder (Control, n = 103, n = 95 analyzed) mixed into 250 mL water. Treatment effects were assessed as change from baseline and differences analyzed using a 2-sided t test via ANOVA with baseline characteristics as covariates. RESULTS: After 4 wk, change from baseline least-squares-mean LDL cholesterol on OBG (-0.195 mmol/L) was less than on Control (0.012 mmol/L) by mean: 0.207 mmol/L (95% CI: 0.318, 0.096 mmol/L; P = 0.0003); the following secondary endpoints were also reduced as follows: total cholesterol (TC) (0.226 mmol/L; 95% CI: 0.361, 0.091 mmol/L; P = 0.001), TC:HDL cholesterol ratio (0.147; 95% CI: 0.284, 0.010; P = 0.036), non-HDL cholesterol (0.194 mmol/L; 95% CI: 0.314, 0.073 mmol/L; P = 0.002), and Framingham cardiovascular disease (CVD) risk (0.474; 95% CI: 0.900, 0.049, P = 0.029). Changes in HDL cholesterol, triglycerides, glucose, and insulin did not differ between treatment groups (P > 0.05). Lipid treatment effects were not significantly modified by age, sex, BMI, or hypertension treatment. There were no major adverse events, but both treatments transiently increased gastrointestinal symptoms. CONCLUSIONS: Consuming a beverage containing 1 g high-MW OBG 3 times daily for 4 wk significantly reduced LDL cholesterol by ∼6% and CVD risk by ∼8% in healthy adults with LDL cholesterol between 3 and 5 mmol/L.This trial was registered at clinicaltrials.gov as NCT03911427.


Assuntos
Doenças Cardiovasculares , Adulto , Bebidas , Doenças Cardiovasculares/prevenção & controle , Colesterol , HDL-Colesterol , LDL-Colesterol , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Triglicerídeos , beta-Glucanas
3.
Nutrients ; 13(4)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33804909

RESUMO

Optimal nutrition is the foundation for the development and maintenance of a healthy immune system. An optimal supply of nutrients is required for biosynthesis of immune factors and immune cell proliferation. Nutrient deficiency/inadequacy and hidden hunger, which manifests as depleted nutrients reserves, increase the risk of infectious diseases and aggravate disease severity. Therefore, an adequate and balanced diet containing an abundant diversity of foods, nutrients, and non-nutrient chemicals is paramount for an optimal immune defense against infectious diseases, including cold/flu and non-communicable diseases. Some nutrients and foods play a larger role than others in the support of the immune system. Oats are a nutritious whole grain and contain several immunomodulating nutrients. In this narrative review, we discuss the contribution of oat nutrients, including dietary fiber (ß-glucans), copper, iron, selenium, and zinc, polyphenolics (ferulic acid and avenanthramides), and proteins (glutamine) in optimizing the innate and adaptive immune system's response to infections directly by modulating the innate and adaptive immunity and indirectly by eliciting changes in the gut microbiota and related metabolites.


Assuntos
Avena/imunologia , Dieta/métodos , Imunidade/imunologia , Nutrientes/administração & dosagem , Nutrientes/imunologia , Fibras na Dieta/administração & dosagem , Humanos
4.
Oncotarget ; 8(47): 82352-82365, 2017 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-29137269

RESUMO

Functional significance of co-expressed erythropoietin (EPO) and its receptor (EPOR) in non-small cell lung cancer (NSCLC) had been under debate. In this study, co-overexpression of EPO/EPOR was confirmed to be positively associated with poor survival in NSCLC. The serum EPO in 14 of 35 enrolled NSCLC patients were found elevated significantly and decreased to normal level after tumor resection. With primary tumor cell culture and patient-derived tumor xenograft (PDX) mouse model, the EPO secretion from the tumors of these 14 patients was verified. Then, we proved the patient derived serum EPO was functionally active and had growth promotion effect in EPO/EPOR overexpressed but not in EPO/EPOR under-expressed NSCLC cells. We also illustrated EPO promoted NSCLC cell proliferation through an EPOR/Jak2/Stat5a/cyclinD1 pathway. In xenograft mouse model, we proved local application of EPO neutralizing antibody and short hairpin RNA (shRNA) against EPOR effectively inhibited the growth of EPO/EPOR overexpressed NSCLC cells and prolonged survivals of the mice. Finally, EPO/EPOR/Jak2/Stat5a/cyclinD1 signaling was found to be a mediator of hypoxia induced growth in EPO/EPOR overexpressed NSCLC. Our results illustrated a subgroup of NSCLC adapt to hypoxia through self-sustainable EPO/EPOR signaling and suggest local blockage of EPO/EPOR as potential therapeutic method in this distinct NSCLC population.

5.
Biochemistry ; 55(12): 1909-17, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26950759

RESUMO

The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183. ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligonucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Animais , Proteína Quinase 1 Ativada por Mitógeno/química , Mutação/fisiologia , Oligonucleotídeos/química , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Ratos
6.
ACS Chem Biol ; 11(4): 1128-36, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26828310

RESUMO

Novel strategies are needed to modulate ß-cell differentiation and function as potential ß-cell replacement or restorative therapies for diabetes. We previously demonstrated that small molecules based on the isoxazole scaffold drive neuroendocrine phenotypes. The nature of the effects of isoxazole compounds on ß-cells was incompletely defined. We find that isoxazole induces genes that support neuroendocrine and ß-cell phenotypes and suppresses genes important for proliferation. Isoxazole alters ß-cell metabolites and protects glucose-responsive signaling pathways under lipotoxic conditions. Finally, we show that isoxazole improves glycemia in a mouse model of ß-cell regeneration. Isoxazole is a prime candidate to alter cell fate in different contexts.


Assuntos
Proliferação de Células/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Isoxazóis/farmacologia , Humanos , Ilhotas Pancreáticas/citologia
7.
Biochem Biophys Res Commun ; 465(3): 414-420, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26260319

RESUMO

Calbindin-D28k, a key regulator of calcium homeostasis plays a cytoprotective role in various tissues. We used serum free (SFM) and charcoal stripped serum (csFBS) culture media as models of cellular stress to modulate calbindin D28k expression and identify regulatory cis-elements and trans-acting factors in kidney and beta cells. The murine calbindin-D28k promoter activity was significantly upregulated under SFM or csFBS condition. Promoter analysis revealed evolutionary conserved regulatory cis-elements and deletion of 23 nt from +117/+139 as critical for basal transcription. Bioinformatics analysis of the promoter revealed conserved NFAT and TFII regulators elements. Forced expression of NFAT stimulated promoter activity. Inhibition of NFAT transcriptional activity by FK506 attenuated calbindin-D28k expression. TFII-I was shown to be necessary for basal promoter activity and to act cooperatively with NFAT. Using chromatin immunoprecipitation (ChIP) assays, NFAT was shown to bind to both proximal and distal promoter regions. ChIP assays also revealed recruitment of TFII to the -36/+139 region. Knockdown of TFII-I decreased promoter activity. In summary, calbindin-D28k expression during serum deprivation is partly regulated by NFAT and TF-II. This regulation may be important in vivo during ischemia and growth factor withdrawal to regulate cellular function and maintenance.


Assuntos
Calbindina 1/genética , Fatores de Transcrição NFATC/genética , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição TFII/genética , Animais , Sequência de Bases , Sítios de Ligação , Meios de Cultura Livres de Soro , Cães , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica
8.
J Biol Chem ; 289(13): 9182-94, 2014 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-24554722

RESUMO

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)(+) ratio.


Assuntos
Complexos de ATP Sintetase/metabolismo , Cálcio/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , NADP/metabolismo , Oxirredução/efeitos dos fármacos , Ratos
9.
Proc Natl Acad Sci U S A ; 108(51): 20713-8, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22143803

RESUMO

New drugs for preserving and restoring pancreatic ß-cell function are critically needed for the worldwide epidemic of type 2 diabetes and the cure for type 1 diabetes. We previously identified a family of neurogenic 3,5-disubstituted isoxazoles (Isx) that increased expression of neurogenic differentiation 1 (NeuroD1, also known as BETA2); this transcription factor functions in neuronal and pancreatic ß-cell differentiation and is essential for insulin gene transcription. Here, we probed effects of Isx on human cadaveric islets and MIN6 pancreatic ß cells. Isx increased the expression and secretion of insulin in islets that made little insulin after prolonged ex vivo culture and increased expression of neurogenic differentiation 1 and other regulators of islet differentiation and insulin gene transcription. Within the first few hours of exposure, Isx caused biphasic activation of ERK1/2 and increased bulk histone acetylation. Although there was little effect on histone deacetylase activity, Isx increased histone acetyl transferase activity in nuclear extracts. Reconstitution assays indicated that Isx increased the activity of the histone acetyl transferase p300 through an ERK1/2-dependent mechanism. In summary, we have identified a small molecule with antidiabetic activity, providing a tool for exploring islet function and a possible lead for therapeutic intervention in diabetes.


Assuntos
Células Secretoras de Insulina/citologia , Insulina/metabolismo , Isoxazóis/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Células HEK293 , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo
10.
J Biol Chem ; 286(16): 13869-78, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21345792

RESUMO

Hypoxia-inducible factors (HIFs) are stress-responsive transcriptional regulators of cellular and physiological processes involved in oxygen metabolism. Although much is understood about the molecular machinery that confers HIF responsiveness to oxygen, far less is known about HIF isoform-specific mechanisms of regulation, despite the fact that HIF-1 and HIF-2 exhibit distinct biological roles. We recently determined that the stress-responsive genetic regulator sirtuin 1 (Sirt1) selectively augments HIF-2 signaling during hypoxia. However, the mechanism by which Sirt1 maintains activity during hypoxia is unknown. In this report, we demonstrate that Sirt1 gene expression increases in a HIF-dependent manner during hypoxia in Hep3B and in HT1080 cells. Impairment of HIF signaling affects Sirt1 deacetylase activity as decreased HIF-1 signaling results in the appearance of acetylated HIF-2α, which is detected without pharmacological inhibition of Sirt1. We also find that Sirt1 augments HIF-2 mediated, but not HIF-1 mediated, transcriptional activation of the isolated Sirt1 promoter. These data in summary reveal a bidirectional link of HIF and Sirt1 signaling during hypoxia.


Assuntos
Regulação da Expressão Gênica , Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Sirtuína 1/biossíntese , Acetilação , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Ativação Transcricional
11.
Cell ; 143(6): 867-9, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21145453

RESUMO

Understanding how signaling pathways are interconnected is vital for characterizing mechanisms of normal development and disease pathogenesis. In this issue, Van Wageningen et al. (2010) examine phosphorylation networks in Sacharromyces cerevisiae with genome-wide expression profiling to identify recurring themes in signaling redundancy.

12.
Cell ; 139(3): 462-3, 2009 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-19879834

RESUMO

Control of gene expression depends on a myriad of protein-DNA interactions, and the number of proteins involved just got larger. In this issue, Hu et al. (2009) identify hundreds of human proteins that bind to DNA, including many surprises such as the protein kinase ERK2 (MAPK1) that now appears to control gene expression directly.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica , Humanos
13.
Science ; 324(5932): 1289-93, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-19498162

RESUMO

To survive in hostile environments, organisms activate stress-responsive transcriptional regulators that coordinately increase production of protective factors. Hypoxia changes cellular metabolism and thus activates redox-sensitive as well as oxygen-dependent signal transducers. We demonstrate that Sirtuin 1 (Sirt1), a redox-sensing deacetylase, selectively stimulates activity of the transcription factor hypoxia-inducible factor 2 alpha (HIF-2alpha) during hypoxia. The effect of Sirt1 on HIF-2alpha required direct interaction of the proteins and intact deacetylase activity of Sirt1. Select lysine residues in HIF-2alpha that are acetylated during hypoxia confer repression of Sirt1 augmentation by small-molecule inhibitors. In cultured cells and mice, decreasing or increasing Sirt1 activity or levels affected expression of the HIF-2alpha target gene erythropoietin accordingly. Thus, Sirt1 promotes HIF-2 signaling during hypoxia and likely other environmental stresses.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Transdução de Sinais , Sirtuínas/metabolismo , Acetilação , Substituição de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Linhagem Celular Tumoral , Eritropoetina/genética , Regulação da Expressão Gênica , Humanos , Rim/metabolismo , Fígado/embriologia , Fígado/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredução , Sirtuína 1 , Sirtuínas/genética
14.
Invest Ophthalmol Vis Sci ; 49(6): 2714-20, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18281611

RESUMO

PURPOSE: To characterize the effect of HIF-2alpha haploinsufficiency on retinal neovascularization and angiogenic signaling in neonatal mice. METHODS: Retinal samples were obtained from HIF-2alpha-haploinsufficient (Epas1+/-) and wild-type (Epas1+/+) neonatal mice subjected to an oxygen-induced retinopathy (OIR) protocol. Histologic and molecular studies were performed immediately, 12 hours, or 5 days after initiation of the hypoxia phase of the OIR protocol. Molecular profiling was performed in mouse brain endothelial cells maintained in normoxia or hypoxia. Transfection studies assessed the response of isolated promoter regions from proangiogenic genes to HIF-1alpha or -2alpha overexpression. RESULTS: Epas1+/- mice exhibited no significant differences in retinal vasculature during normal development but had reduced retinal neovascularization in an OIR protocol. Multiple proangiogenic factors were induced during the hypoxia phase in Epas1+/+ OIR retinal samples, whereas Epas1+/- OIR retinal samples had absent or blunted induction of these same factors. Several, but not all, proangiogenic factors were induced in mouse brain endothelial cells after hypoxia. In transfection assays, most proangiogenic promoter regions were preferentially activated by HIF-2alpha relative to HIF-1alpha. CONCLUSIONS: HIF-2alpha deficiency results in reduced neovascularization and blunted inducibility of multiple proangiogenic factors in the retinas of mice with OIR. The authors propose that HIF-2alpha is a master regulator of proangiogenic factors in retinal vascular endothelial cells, the predominant cell type of the retina in which HIF-2alpha is expressed. Future studies will address whether the molecular and functional roles for HIF-2alpha identified from these studies can be generalized to other pathophysiological states involving neovascularization.


Assuntos
Proteínas Angiogênicas/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica/fisiologia , Hipóxia/metabolismo , Neovascularização Retiniana/prevenção & controle , Retinopatia da Prematuridade/prevenção & controle , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Códon sem Sentido , Feminino , Genes Reporter , Humanos , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Oxigênio/toxicidade , Plasmídeos , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/patologia , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
15.
J Biol Chem ; 282(16): 11750-6, 2007 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-17322295

RESUMO

Mice lacking Epas1, encoding the transcription factor Hypoxia-inducible Factor 2alpha (HIF-2alpha), exhibit an apparent mitochondrial disease state. Similarities between knock-outs of Epas1 and of Sod2, encoding the mitochondrial antioxidant enzyme manganese superoxide dismutase, led to the identification of Sod2 as a HIF-2alpha target gene. However, Sod2 levels in Epas1(-)(/)(-) liver are intermediate between that of Sod(+)(/)(-) and Sod2(-)(/)(-) mice, which have subtle or severe phenotypes, respectively. This suggests that additional HIF-2alpha target genes besides Sod2 contribute to the Epas1(-)(/)(-) mitochondrial disease state. To define the nature of the mitochondrial defect in Epas1(-)(/)(-) liver, we performed biophysical, biochemical, and molecular studies. In the setting of decreased Sod2 levels and increased oxidative stress, we found reduced respiration, sensitized mitochondrial permeability transition pore opening, intact electron transport chain activities, and impaired mitochondrial aconitase activity. Mitochondrial aconitase protein levels were preserved, whereas mRNA and protein levels for frataxin, the oxidative stress-regulated mitochondrial aconitase chaperone protein, were markedly reduced in Epas1(-)(/)(-) livers. The mouse Fxn promoter was preferentially activated by HIF-2alpha through a consensus HIF-responsive enhancer element. In summary, the studies reveal that Fxn, like Sod2, is a nuclear-encoded, mitochondrial-localized HIF-2alpha target gene required for optimal mitochondrial homeostasis. These findings expand upon the previously defined role of HIF-2alpha in the cellular response to oxidative stress and identify a novel link of HIF-2alpha with mitochondrial homeostasis.


Assuntos
Aconitato Hidratase/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Regulação da Expressão Gênica , Proteínas de Ligação ao Ferro/metabolismo , Animais , Antioxidantes/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosforilação , Frataxina
16.
Proc Natl Acad Sci U S A ; 101(17): 6675-80, 2004 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-15096613

RESUMO

Hemoglobins are ubiquitous in Eukarya and Bacteria but, until now, have not been found in Archaea. A phylogenetic analysis of the recently revealed microbial family of globin-coupled heme-based sensors suggests that these sensors descended from an ancient globin-only progenitor, or a protoglobin (Pgb). Here, we report the discovery and characterization of two Pgbs from the Archaea: ApPgb from the obligately aerobic hyperthermophile Aeropyrum pernix, and MaPgb from the strictly anaerobic methanogen Methanosarcina acetivorans. Both ApPgb and MaPgb bind molecular oxygen, nitric oxide, and carbon monoxide by means of a heme moiety that is coordinated to the protein through the F8 histidine (histidine 120). We postulate that these archaeal globins are the ancestors of contemporary hemoglobins.


Assuntos
Archaea/química , Hemoglobinas/química , Sequência de Aminoácidos , Sequência de Bases , Monóxido de Carbono/metabolismo , Primers do DNA , Hemoglobinas/genética , Hemoglobinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta
17.
Biochemistry ; 42(25): 7701-8, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12820879

RESUMO

To evaluate the contributions of the G(beta)-2 arginine to signal transduction in oxygen-sensing heme-PAS domains, we replaced this residue with alanine in Bradyrhizobium japonicum FixL and examined the results on heme-domain structure, ligand binding, and kinase regulation. In the isolated R220A BjFixL heme-PAS domain, the iron-histidine bond was increased in length by 0.31 A, the heme flattened even without a ligand, and the interaction of a presumed regulatory loop (the FG loop) with the helix of heme attachment was weakened. Binding of carbon monoxide was similar for ferrous BjFixL and R220A BjFixL. In contrast, the level of binding of oxygen was dramatically lower (K(d) approximately 1.5 mM) for R220A BjFixL, and this was manifested as 60- and 3-fold lower on- and off-rate constants, respectively. Binding of cyanide followed the same pattern as binding of oxygen. The catalytic activity was 3-4-fold higher in the "on-state" unliganded forms of R220A BjFixL than in the corresponding BjFixL species. Cyanide regulation of this activity was strongly impaired, but some inhibition was nevertheless preserved. Carbon monoxide and nitric oxide regulation, although weak in BjFixL, were abolished from R220A BjFixL. We conclude that the G(beta)-2 arginine assists in the binding of oxygen to BjFixL but does not accomplish this by stabilizing the oxy form. This arginine is not absolutely required for regulation, although it is important for shifting a pre-existing kinase equilibrium toward the inactive state on binding of regulatory ligands. These findings support a regulatory model in which the heme-PAS domain operates as an ensemble that couples to the kinase rather than a mechanism driven by a single central switch.


Assuntos
Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Hemeproteínas/metabolismo , Bradyrhizobium/metabolismo , Cristalização , Histidina Quinase , Ligantes , Fosforilação , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/fisiologia
18.
Science ; 298(5602): 2385-7, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12446832

RESUMO

Neuronal PAS domain protein 2 (NPAS2) is a mammalian transcription factor that binds DNA as an obligate dimeric partner of BMAL1 and is implicated in the regulation of circadian rhythm. Here we show that both PAS domains of NPAS2 bind heme as a prosthetic group and that the heme status controls DNA binding in vitro. NPAS2-BMAL1 heterodimers, existing in either the apo (heme-free) or holo (heme-loaded) state, bound DNA avidly under favorably reducing ratios of the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate. Low micromolar concentrations of carbon monoxide inhibited the DNA binding activity of holo-NPAS2 but not that of apo-NPAS2. Upon exposure to carbon monoxide, inactive BMAL1 homodimers were formed at the expense of NPAS2-BMAL1 heterodimers. These results indicate that the heterodimerization of NPAS2, and presumably the expression of its target genes, are regulated by a gas through the heme-based sensor described here.


Assuntos
Monóxido de Carbono/metabolismo , DNA/metabolismo , Heme/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Monóxido de Carbono/farmacologia , Ritmo Circadiano , Dimerização , Sequências Hélice-Alça-Hélice , Heme/química , Ligantes , Mioglobina/metabolismo , NADP/metabolismo , Oxirredução , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta
19.
Biochemistry ; 41(26): 8414-21, 2002 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-12081490

RESUMO

The EcDos protein belongs to a group of heme-based sensors that detect their ligands with a heme-binding PAS domain. Among these various heme-PAS proteins, EcDos is unique in having its heme iron coordinated at both axial positions to residues of the protein. To achieve its high affinities for ligands, one of the axial heme-iron residues in EcDos must be readily displaceable. Here we present evidence from mutagenesis, ligand-binding measurements, and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopies about the nature of the displaceable residue in the heme-PAS domain of EcDos, i.e., EcDosH. The magnetic circular dichroism spectra in the near-infrared region establish histidine-methionine coordination in met-EcDos. To determine whether in deoxy-EcDos coordination of the sixth axial position is also to methionine, methionine 95 was substituted with isoleucine. This substitution caused the ferrous heme iron to change from an exclusively hexacoordinate low-spin form (EcDosH) to an exclusively pentacoordinate high-spin form (M95I EcDosH). This was accompanied by a modest acceleration of the dissociation rates of ligands but a dramatic increase (60-1300-fold) in the association rate constants for binding of O(2), CO, and NO. As a result, the affinity for O(2) was enhanced 10-fold in M95I EcDosH, but the partition constant M = [K(d)(O(2))/K(d)(CO)] between CO and O(2) was raised to about 30 from the extraordinarily low EcDosH value of 1. Thus a major consequence of the increased O(2) affinity of this sensor was the loss of its unusually strong ligand discrimination.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Heme/metabolismo , Hemeproteínas/química , Hemeproteínas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Diester Fosfórico Hidrolases , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Análise Espectral Raman
20.
Biochemistry ; 41(19): 6170-7, 2002 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-11994013

RESUMO

Phosphorylation of the transcription factor RmFixJ is the key step in the hypoxic induction of Sinorhizobium meliloti nitrogen fixation genes. Oxygen regulates this process by binding reversibly to RmFixL, a heme protein kinase whose deoxy form catalyzes the phosphoryl transfer from ATP to RmFixJ. Here we present the first quantitative measure of the extent by which various heme ligands inhibit the turnover of RmFixJ to phospho-RmFixJ. We also quantitate the inhibition by ligands of the reaction of RmFixL with ATP, in the absence of RmFixJ, to form phospho-RmFixL, i.e., the "autophosphorylation". Phospho-RmFixL formed from autophosphorylation will transfer its phosphoryl group to RmFixJ in an oxygen-independent "phosphotransfer." Here we show that the mode of substrate presentation, i.e., simultaneous versus sequential, influences the regulation of phosphoryl transfer by heme status. Inhibition factors for O(2), CO, NO, CN(-), and imidazole in the presence of RmFixJ are drastically different from the inhibition of autophosphorylation by the same ligands. Oxidation of the heme iron in unliganded RmFixL is known to have no effect on either of the sequential reactions; yet oxidation causes a 100-fold decrease in RmFixJ turnover when ATP and RmFixJ are presented simultaneously. The profound difference between the regulation of isolated RmFixL versus the complex of RmFixL with RmFixJ shows that interaction of a response regulator with its histidine-kinase partner need not be limited to the enzymatic regions of the histidine kinase, but can extend also to its sensory domain.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hemeproteínas/química , Hemeproteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Dimerização , Heme/química , Histidina Quinase , Cinética , Ligantes , Modelos Biológicos , Oxirredução , Oxigênio/metabolismo , Fosforilação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Sinorhizobium meliloti/metabolismo
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