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1.
Nat Commun ; 10(1): 5766, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852900

RESUMO

Cognitive performance in people varies according to time-of-day, with memory retrieval declining in the late afternoon-early evening. However, functional roles of local brain circadian clocks in memory performance remains unclear. Here, we show that hippocampal clock controlled by the circadian-dependent transcription factor BMAL1 regulates time-of-day retrieval profile. Inducible transgenic dominant negative BMAL1 (dnBMAL1) expression in mouse forebrain or hippocampus disrupted retrieval of hippocampal memories at Zeitgeber Time 8-12, independently of retention delay, encoding time and Zeitgeber entrainment cue. This altered retrieval profile was associated with downregulation of hippocampal Dopamine-cAMP signaling in dnBMAL1 mice. These changes included decreases in Dopamine Receptors (D1-R and D5-R) and GluA1-S845 phosphorylation by PKA. Consistently, pharmacological activation of cAMP-signals or D1/5Rs rescued impaired retrieval in dnBMAL1 mice. Importantly, GluA1 S845A knock-in mice showed similar retrieval deficits with dnBMAL1 mice. Our findings suggest mechanisms underlying regulation of retrieval by hippocampal clock through D1/5R-cAMP-PKA-mediated GluA1 phosphorylation.


Assuntos
Relógios Circadianos/fisiologia , Hipocampo/metabolismo , Rememoração Mental/fisiologia , Receptores de AMPA/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Feminino , Técnicas de Introdução de Genes , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Transgênicos , Modelos Animais , Fosforilação/fisiologia
2.
Nat Commun ; 10(1): 4905, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659167

RESUMO

Therapeutic activation of mitochondrial function has been suggested as an effective strategy to combat aging. Hydralazine is an FDA-approved drug used in the treatment of hypertension, heart failure and cancer. Hydralazine has been recently shown to promote lifespan in C. elegans, rotifer and yeast through a mechanism which has remained elusive. Here we report cAMP-dependent protein kinase (PKA) as the direct target of hydralazine. Using in vitro and in vivo models, we demonstrate a mechanism in which binding and stabilization of a catalytic subunit of PKA by hydralazine lead to improved mitochondrial function and metabolic homeostasis via the SIRT1/SIRT5 axis, which underlies hydralazine's prolongevity and stress resistance benefits. Hydralazine also protects mitochondrial metabolism and function resulting in restoration of health and lifespan in C. elegans under high glucose and other stress conditions. Our data also provide new insights into the mechanism(s) that explain various other known beneficial effects of hydralazine.


Assuntos
Envelhecimento/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hidralazina/administração & dosagem , Sirtuína 1/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Feminino , Humanos , Longevidade/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Sirtuína 1/genética
3.
Nat Commun ; 10(1): 3984, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484930

RESUMO

Cyclic nucleotide-binding (CNB) domains allosterically regulate the activity of proteins with diverse functions, but the mechanisms that enable the cyclic nucleotide-binding signal to regulate distant domains are not well understood. Here we use optical tweezers and molecular dynamics to dissect changes in folding energy landscape associated with cAMP-binding signals transduced between the two CNB domains of protein kinase A (PKA). We find that the response of the energy landscape upon cAMP binding is domain specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, whereas the other triggers inter-domain interactions that promote the active conformation. Inter-domain interactions occur in a stepwise manner, beginning in intermediate-liganded states between apo and cAMP-bound domains. Moreover, we identify a cAMP-responsive switch, the N3A motif, whose conformation and stability depend on cAMP occupancy. This switch serves as a signaling hub, amplifying cAMP-binding signals during PKA activation.


Assuntos
Regulação Alostérica , Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transdução de Sinais , Algoritmos , Sítio Alostérico , Animais , Sítios de Ligação , Bovinos , AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática , Simulação de Dinâmica Molecular , Pinças Ópticas , Ligação Proteica
4.
Mol Med Rep ; 20(2): 1837-1845, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257533

RESUMO

Hypoxic preconditioning (HPC) is neuroprotective against ischaemic brain injury; however, the roles of potential anti­apoptotic signals in this process have not been assessed. To elucidate the molecular mechanisms involved in HPC­induced neuroprotection, the effects of HPC on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element­binding protein (CREB) signalling pathway and apoptosis in Sprague­Dawley pups (postnatal day 7) treated with propofol were investigated. Western blot and histological analyses demonstrated that HPC exerts multiple effects on the hippocampus, including the upregulation of cAMP and phosphorylation of CREB. These effects were partially blocked by intracerebroventricular injection of the protein kinase antagonist H89 (5 µmol/5 µl). Notably, the level of cleaved caspase­3 was significantly downregulated by treatment with the cAMP agonist Sp­cAMP (20 nmol/5 µl). The results indicate that propofol increased the level of cleaved caspase­3 and Bax by suppressing the activity of cAMP­dependent proteins and Bcl­2; thus, HPC prevents propofol from triggering apoptosis via the cAMP/PKA/CREB signalling pathway.


Assuntos
Lesões Encefálicas/terapia , Precondicionamento Isquêmico/métodos , Neurônios/metabolismo , Neuroproteção/genética , Animais , Animais Recém-Nascidos , Apoptose/genética , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Lesões Encefálicas/prevenção & controle , AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Neurônios/patologia , Fosforilação/genética , Propofol/toxicidade , Ratos , Transdução de Sinais/genética , Lobo Temporal/metabolismo , Lobo Temporal/patologia
5.
Nat Rev Cardiol ; 16(10): 612-622, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31186538

RESUMO

G protein-coupled receptors (GPCRs) are critical cellular sensors that mediate numerous physiological processes. In the heart, multiple GPCRs are expressed on various cell types, where they coordinate to regulate cardiac function by modulating critical processes such as contractility and blood flow. Under pathological settings, these receptors undergo aberrant changes in expression levels, localization and capacity to couple to downstream signalling pathways. Conventional therapies for heart failure work by targeting GPCRs, such as ß-adrenergic receptor and angiotensin II receptor antagonists. Although these treatments have improved patient survival, heart failure remains one of the leading causes of mortality worldwide. GPCR kinases (GRKs) are responsible for GPCR phosphorylation and, therefore, desensitization and downregulation of GPCRs. In this Review, we discuss the GPCR signalling pathways and the GRKs involved in the pathophysiology of heart disease. Given that increased expression and activity of GRK2 and GRK5 contribute to the loss of contractile reserve in the stressed and failing heart, inhibition of overactive GRKs has been proposed as a novel therapeutic approach to treat heart failure.


Assuntos
Quinases de Receptores Acoplados a Proteína G/antagonistas & inibidores , Quinases de Receptores Acoplados a Proteína G/metabolismo , Cardiopatias/tratamento farmacológico , Cardiopatias/fisiopatologia , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Catecolaminas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Humanos , Contração Muscular , Miócitos Cardíacos , Fragmentos de Peptídeos/genética , Receptores Adrenérgicos/metabolismo , Proteínas Recombinantes/genética , Transdução de Sinais/genética , beta-Arrestinas/metabolismo
6.
PLoS Genet ; 15(5): e1008176, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31150381

RESUMO

Gene expression changes in neural systems are essential for environment-induced behavioral plasticity in animals; however, neuronal signaling pathways mediating the effect of external stimuli on transcriptional changes are largely unknown. Recently, we have demonstrated that the neuropeptide F (NPF)/nitric oxide (NO) signaling pathway plays a regulatory role in phase-related locomotor plasticity in the migratory locust, Locusta migratoria. Here, we report that a conserved transcription factor, cAMP response element-binding protein B (CREB-B), is a key mediator involved in the signaling pathway from NPF2 to NOS in the migratory locust, triggering locomotor activity shift between solitarious and gregarious phases. We find that CREB-B directly activates brain NOS expression by interacting with NOS promoter region. The phosphorylation at serine 110 site of CREB-B dynamically changes in response to population density variation and is negatively controlled by NPF2. The involvement of CREB-B in NPF2-regulated locomotor plasticity is further validated by RNAi experiment and behavioral assay. Furthermore, we reveal that protein kinase A mediates the regulatory effects of NPF2 on CREB-B phosphorylation and NOS transcription. These findings highlight a precise signal cascade underlying environment-induced behavioral plasticity.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Locomoção/genética , Locusta migratoria/genética , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/genética , Gafanhotos/genética , Gafanhotos/metabolismo , Locusta migratoria/metabolismo , Plasticidade Neuronal/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico/fisiologia , Fosforilação , Regiões Promotoras Genéticas/genética , Interferência de RNA , Serina/metabolismo , Transdução de Sinais
7.
Mol Cell ; 75(1): 39-52.e4, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31078384

RESUMO

Ryanodine receptors (RyRs) are intracellular Ca2+ release channels controlling essential cellular functions. RyRs are targeted by cyclic AMP (cAMP)-dependent protein kinase A (PKA), a controversial regulation implicated in disorders ranging from heart failure to Alzheimer's. Using crystal structures, we show that the phosphorylation hotspot domain of RyR2 embraces the PKA catalytic subunit, with an extensive interface not seen in PKA complexes with peptides. We trapped an intermediary open-form PKA bound to the RyR2 domain and an ATP analog, showing that PKA can engage substrates in an open form. Phosphomimetics or prior phosphorylation at nearby sites in RyR2 either enhance or reduce the activity of PKA. Finally, we show that a phosphomimetic at S2813, a well-known target site for calmodulin-dependent kinase II, induces the formation of an alpha helix in the phosphorylation domain, resulting in increased interactions and PKA activity. This shows that the different phosphorylation sites in RyR2 are not independent.


Assuntos
Cálcio/química , Proteínas Quinases Dependentes de AMP Cíclico/química , AMP Cíclico/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Sítios de Ligação , Cálcio/metabolismo , Clonagem Molecular , Cristalografia por Raios X , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Camundongos , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Especificidade por Substrato , Termodinâmica
8.
PLoS One ; 14(4): e0214803, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973898

RESUMO

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, chronological aging, and stress responses in the fission yeast, Schizosaccharomyces pombe. We demonstrated here that Pka1 is responsible for normal growth in the presence of the microtubule-destabilization drug TBZ and proper chromosome segregation. The deletion of the pka1 gene resulted in the TBZ-sensitive phenotype and chromosome mis-segregation. We isolated the mal3 gene as a multi-copy suppressor of the TBZ-sensitive phenotype in the pka1Δ strains. Overexpression of the CH domain (1-143) or the high-affinity microtubule binding mutant (1-143 Q89R) of Mal3 rescued the TBZ-sensitive phenotype in the pka1Δ and mal3Δ strains, while the EB1 domain (135-308) and the mutants defective in microtubule binding (1-143 Q89E) failed to do so in the same strains. Chromosome mis-segregation caused by TBZ in the pka1Δ or mal3Δ strains was suppressed by the overexpression of the Mal3 CH domain (1-143), Mal3 CH domain with the coiled-coil domain (1-197), or full-length Mal3. Overexpression of EB1 orthologs from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, or Homo sapiens suppressed the TBZ-sensitive phenotype in the pka1Δ strains, indicating their conserved functions. These findings suggest that Pka1 and the microtubule binding of the Mal3 CH domain play a role in the maintenance of proper chromosome segregation.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Animais , Segregação de Cromossomos , Genes Fúngicos , Genes Supressores , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mutação , Fenótipo , Domínios Proteicos , Schizosaccharomyces/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/química , Moduladores de Tubulina/farmacologia
9.
Life Sci ; 225: 107-116, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928409

RESUMO

AIMS: To explore the effect and mechanism of 1, 25-(OH)2D3 on Schwann cell apoptosis induced by advanced glycation end products. MAIN METHODS: Schwann cells, isolated from rodent sciatic nerve were incubated with AGE-modified bovine serum albumin(AGE) to mimic diabetic conditions and 1,25-(OH)2D3 was used as protector. Cell apoptosis was detected by PI/Annexin-V staining, caspase 3 activity assay and western blotting for caspase 3 and PARP. The activation of protein kinase A (PKA) and nuclear factor kappa-B (NF-κB) was evaluated by western blot. Immunofluorescent staining was used for intercellular location of NF-κB. Cytokine secretion was evaluated by enzyme-linked immunosorbent assay. KEY FINDINGS: Schwann cell apoptosis accelerated after incubating with AGE. However, if combining 1,25-(OH)2D3 with AGE, apoptosis decreased significantly. 1,25-(OH)2D3 enhanced PKA activity, but inhibited AGE-induced nuclear translocation of NF-κB. Furthermore, PKA activator (8-bromoadenoside cyclic adenoside monophosphate, 8-Br-cAMP) or NF-κB inhibitor (caffeic acid phenethyl ester, CAPE) could reduce the apoptosis, decreased cleaved caspase 3 and cleaved PARP, suggesting the involvement of PKA and NF-κB pathways in the protection of 1,25-(OH)2D3 on Schwann cells. Moreover, 8-Br-cAMP and CAPE could inhibit AGE-induced secretion of interleukin(IL)-1ß, prostaglandin E2(PEG2) and cyclooxygenase 2(COX2). Interestingly, 8-Br-cAMP decreased phospho-NF-κB and inhibited nucleus translocation of NF-κB. It hinted at the regulation of PKA to NF-κB. Finally, a pre-treatment of H-89 (an inhibitor of PKA) could block the protection of 1,25-(OH)2D3 on cell apoptosis. In conclusion, 1,25-(OH)2D3 could protect Schwann cell against AGE-induced apoptosis through PKA/NF-κB pathway. SIGNIFICANCE: These findings provide experimental rationales for using vitamin D for diabetic neuropathy.


Assuntos
Apoptose/efeitos dos fármacos , Calcitriol/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , NF-kappa B/metabolismo , Substâncias Protetoras/farmacologia , Células de Schwann/efeitos dos fármacos , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/genética , Ratos , Ratos Wistar , Células de Schwann/metabolismo , Células de Schwann/patologia , Transdução de Sinais , Vitaminas/farmacologia
10.
Appl Microbiol Biotechnol ; 103(12): 4899-4915, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31030285

RESUMO

Coenzyme Q (CoQ) is an essential component of the electron transport system that produces ATP in nearly all living cells. CoQ10 is a popular commercial food supplement around the world, and demand for efficient production of this molecule has increased in recent years. In this study, we explored CoQ10 production in the fission yeast Schizosaccharomyces pombe. We found that CoQ10 level was higher in stationary phase than in log phase, and that it increased when the cells were grown in a low concentration of glucose, in maltose, or in glycerol/ethanol medium. Because glucose signaling is mediated by cAMP, we evaluated the involvement of this pathway in CoQ biosynthesis. Loss of Pka1, the catalytic subunit of cAMP-dependent protein kinase, increased production of CoQ10, whereas loss of the regulatory subunit Cgs1 decreased production. Manipulation of other components of the cAMP-signaling pathway affected CoQ10 production in a consistent manner. We also found that glycerol metabolism was controlled by the cAMP/PKA pathway. CoQ10 production by the S. pombe ∆pka1 reached 0.98 mg/g dry cell weight in medium containing a non-fermentable carbon source [2% glycerol (w/v) and 1% ethanol (w/v) supplemented with 0.5% casamino acids (w/v)], twofold higher than the production in wild-type cells under normal growth conditions. These findings demonstrate that carbon source, growth phase, and the cAMP-signaling pathway are important factors in CoQ10 production in S. pombe.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Fúngicas/genética , Glucose/química , Schizosaccharomyces/metabolismo , Ubiquinona/análogos & derivados , Domínio Catalítico , Meios de Cultura/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Maltose/química , Transdução de Sinais , Ubiquinona/biossíntese , Ubiquinona/metabolismo
11.
J Agric Food Chem ; 67(13): 3651-3661, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30875211

RESUMO

Forkhead/winged helix transcription factor O-class member 1 (FoxO1) is a key mediator of insulin and glucagon signaling in control of glucose homeostasis. Although epigallocatechin gallate (EGCG) has attracted interest owing to its potential to combat hyperglycemic diabetes, molecular mechanisms underlying its antihyperglycemic effect, in particular the effect on FoxO1, is poorly understand. This study aims to assess the impact of EGCG on the glucagon signaling pathway in regulating glucose metabolism. Primary hepatocytes from wild-type (WT), liver-specific FoxO1 knock out (FKO), and FoxO1-S273D knock-in (KI) mice were isolated, cultured, and treated with EGCG and/or glucagon. Our data showed the treatment of 10 µM EGCG for 6 h decreased hepatic glucose production by 20 and 23% in WT and FKO primary hepatocytes, respectively. EGCG repressed both gluconeogenesis and glycogenolysis in primary hepatocytes, coupled with activating AMPK. In addition, EGCG decreased mitochondrial oxygen consumption. We further investigated the effects of EGCG on glucagon-stimulated cAMP/PKA signaling pathway. EGCG reduced p-PKA-T197/T-PKA and p-CREB-S133/T-CREB levels by 39 and 20%, blocked p-FoxO1-S273, and suppressed nuclear FoxO1 translocation, suggesting that FoxO1 and CREB were possible downstream targets. A novel mechanism of EGCG in restraining hepatic glucose production (HGP) is through antagonizing glucagon signaling and suppressing FoxO1 via Ser273. EGCG may serve as a promising compound for regulating glucose homeostasis.


Assuntos
Catequina/análogos & derivados , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Forkhead Box O1/metabolismo , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Catequina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Regulação para Baixo/efeitos dos fármacos , Proteína Forkhead Box O1/genética , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos
12.
Proc Natl Acad Sci U S A ; 116(14): 7043-7052, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894498

RESUMO

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.


Assuntos
Citomegalovirus/fisiologia , Células Epiteliais/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Tropismo Viral/fisiologia , Células A549 , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Virais/genética
13.
PLoS Genet ; 15(3): e1008037, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30856163

RESUMO

Microbes can be metabolically engineered to produce biofuels and biochemicals, but rerouting metabolic flux toward products is a major hurdle without a systems-level understanding of how cellular flux is controlled. To understand flux rerouting, we investigated a panel of Saccharomyces cerevisiae strains with progressive improvements in anaerobic fermentation of xylose, a sugar abundant in sustainable plant biomass used for biofuel production. We combined comparative transcriptomics, proteomics, and phosphoproteomics with network analysis to understand the physiology of improved anaerobic xylose fermentation. Our results show that upstream regulatory changes produce a suite of physiological effects that collectively impact the phenotype. Evolved strains show an unusual co-activation of Protein Kinase A (PKA) and Snf1, thus combining responses seen during feast on glucose and famine on non-preferred sugars. Surprisingly, these regulatory changes were required to mount the hypoxic response when cells were grown on xylose, revealing a previously unknown connection between sugar source and anaerobic response. Network analysis identified several downstream transcription factors that play a significant, but on their own minor, role in anaerobic xylose fermentation, consistent with the combinatorial effects of small-impact changes. We also discovered that different routes of PKA activation produce distinct phenotypes: deletion of the RAS/PKA inhibitor IRA2 promotes xylose growth and metabolism, whereas deletion of PKA inhibitor BCY1 decouples growth from metabolism to enable robust fermentation without division. Comparing phosphoproteomic changes across ira2Δ and bcy1Δ strains implicated regulatory changes linked to xylose-dependent growth versus metabolism. Together, our results present a picture of the metabolic logic behind anaerobic xylose flux and suggest that widespread cellular remodeling, rather than individual metabolic changes, is an important goal for metabolic engineering.


Assuntos
Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Anaerobiose , Biocombustíveis , Biomassa , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Evolução Molecular Direcionada , Fermentação , Perfilação da Expressão Gênica , Genes Fúngicos , Glucose/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas , Modelos Biológicos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia de Sistemas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Food Chem Toxicol ; 126: 211-222, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30817943

RESUMO

Camellia euphlebia is a Chinese folk medicine, known for its multiple pharmacological properties. Our previous studies have demonstrated its antidepressant activity by several animal models of depression. The possible underlying mechanism was further explored by investigating the neuroprotective effect of Camellia euphlebia extract (CEE) on corticosterone-induced apoptosis in neuronally differentiated PC12 cells. The results of methyl-thiazolyl-tetrazolium assay, lactate dehydrogenase release assay, Hoechst 33342 staining, propidium iodide staining, AV-FITC/PI double staining and DNA fragmentation analysis consistently indicated that pretreatment of PC12 cells with CEE at 20-80 µg/mL significantly reversed 300 µmol/L corticosterone-induced apoptosis in a dose dependent manner. Furthermore, intracellular mitochondrial membrane potential, reactive oxygen species accumulation, calcium level, Bcl-2/Bax ratio, caspase activity were assessed, and the results indicated that CEE exhibited its anti-apoptotic effect through the regulation of mitochondrial apoptosis pathway. Additionally, CEE increased the cyclic adenosine monophosphate-dependent protein kinase (PKA) level, which phosphorylated cAMP response element binding protein (CREB), and finally elevated the mRNA expression of brain-derived neurotrophic factor (BDNF) gene. It is speculated that the antidepressant effect of CEE in vivo may be associated with the cytoprotection of neuron damaged by corticosterone, and the cellular mechanism involves the mitochondrial-mediated apoptosis and PKA-CREB-BDNF signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Camellia/química , Corticosterona/toxicidade , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
Nat Commun ; 10(1): 799, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778078

RESUMO

Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. For signaling enzymes, substrate recognition, binding, and product release are often rate-determining steps in which enthalpy-entropy compensation plays a crucial role. While the nature of enthalpic interactions can be inferred from structural data, the molecular origin and role of entropy in enzyme catalysis remains poorly understood. Using thermocalorimetry, NMR, and MD simulations, we studied the conformational landscape of the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous phosphoryl transferase involved in a myriad of cellular processes. Along the enzymatic cycle, the kinase exhibits positive and negative cooperativity for substrate and nucleotide binding and product release. We found that globally coordinated changes of conformational entropy activated by ligand binding, together with synchronous and asynchronous breathing motions of the enzyme, underlie allosteric cooperativity along the kinase's cycle.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Calorimetria/métodos , Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/genética , Entropia , Modelos Moleculares , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
16.
PLoS Genet ; 15(2): e1007976, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30742618

RESUMO

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence.


Assuntos
Candida albicans/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Prolina/metabolismo , Proteínas ras/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Animais , Candida albicans/genética , Candida albicans/patogenicidade , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Fúngicas/genética , Humanos , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Macrófagos/microbiologia , Camundongos , Mitocôndrias/metabolismo , Morfogênese , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Células RAW 264.7 , Transdução de Sinais , Virulência/fisiologia , Proteínas ras/genética
17.
Biochim Biophys Acta Mol Cell Res ; 1866(5): 828-838, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30769008

RESUMO

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.


Assuntos
Movimento Celular , Conexina 43/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Colforsina/farmacologia , Conexina 43/genética , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Células Endoteliais/citologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Células HeLa , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/genética
18.
J Cell Biol ; 218(3): 855-870, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30792230

RESUMO

Both menin and glucagon-like peptide 1 (GLP-1) pathways play central yet opposing role in regulating ß cell function, with menin suppressing, and GLP-1 promoting, ß cell function. However, little is known as to whether or how GLP-1 pathway represses menin function. Here, we show that GLP-1 signaling-activated protein kinase A (PKA) directly phosphorylates menin at the serine 487 residue, relieving menin-mediated suppression of insulin expression and cell proliferation. Mechanistically, Ser487-phosphorylated menin gains increased binding affinity to nuclear actin/myosin IIa proteins and gets sequestrated from the Ins1 promoter. This event leads to reduced binding of repressive epigenetic histone modifiers suppressor variegation 3-9 homologue protein 1 (SUV39H1) and histone deacetylases 1 (HDAC1) at the locus and subsequently increased Ins1 gene transcription. Ser487 phosphorylation of menin also increases expression of proproliferative cyclin D2 and ß cell proliferation. Our results have uncovered a previously unappreciated physiological link in which GLP-1 signaling suppresses menin function through phosphorylation-triggered and actin/myosin cytoskeletal protein-mediated derepression of gene transcription.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Transcrição Genética , Ativação Transcricional , Animais , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Células HEK293 , Humanos , Células Secretoras de Insulina/citologia , Metiltransferases , Camundongos , Ratos , Ratos Wistar , Proteínas Repressoras , Fatores de Transcrição/genética
19.
PLoS Biol ; 17(2): e3000154, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30794532

RESUMO

Cyclic nucleotide signalling is a major regulator of malaria parasite differentiation. Phosphodiesterase (PDE) enzymes are known to control cyclic GMP (cGMP) levels in the parasite, but the mechanisms by which cyclic AMP (cAMP) is regulated remain enigmatic. Here, we demonstrate that Plasmodium falciparum phosphodiesterase ß (PDEß) hydrolyses both cAMP and cGMP and is essential for blood stage viability. Conditional gene disruption causes a profound reduction in invasion of erythrocytes and rapid death of those merozoites that invade. We show that this dual phenotype results from elevated cAMP levels and hyperactivation of the cAMP-dependent protein kinase (PKA). Phosphoproteomic analysis of PDEß-null parasites reveals a >2-fold increase in phosphorylation at over 200 phosphosites, more than half of which conform to a PKA substrate consensus sequence. We conclude that PDEß plays a critical role in governing correct temporal activation of PKA required for erythrocyte invasion, whilst suppressing untimely PKA activation during early intra-erythrocytic development.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Transdução de Sinais/genética , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Eritrócitos/parasitologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hidrólise , Merozoítos/enzimologia , Merozoítos/genética , Merozoítos/crescimento & desenvolvimento , Fosfoproteínas/classificação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteoma/classificação , Proteoma/genética , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Esquizontes/enzimologia , Esquizontes/genética , Esquizontes/crescimento & desenvolvimento , Fatores de Tempo
20.
Curr Genet ; 65(4): 941-952, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30796472

RESUMO

Cellulase production in filamentous fungi is repressed by various carbon sources. In our preliminary survey in Aspergillus nidulans, degree of de-repression differed depending on carbon sources in a mutant of creA, encoding the transcriptional repressor for carbon catabolite repression (CCR). To further understand mechanisms of CCR of cellulase production, we compared the effects of creA deletion with deletion of protein kinase A (pkaA) and G (ganB) genes, which constitute a nutrient sensing and signaling pathway. In plate culture with carboxymethyl cellulose and D-glucose, deletion of pkaA and ganB, but not creA, led to significant de-repression of cellulase production. In submerged culture with cellobiose and D-glucose or 2-deoxyglucose, both creA or pkaA single deletion led to partial de-repression of cellulase genes with the highest level by their double deletion, while ganB deletion caused de-repression comparable to that of the creA/pkaA double deletion. With ball-milled cellulose and D-glucose, partial de-repression was detected by deletion of creA but not of pkaA or ganB. The creA/pkaA or creA/ganB double deletion led to earlier expression than the creA deletion. Furthermore, the effect of each deletion with D-xylose or L-arabinose as the repressing carbon source was significantly different from that with D-glucose, D-fructose, and D-mannose. Consequently, this study revealed that PkaA and GanB participate in CreA-independent CCR and that contribution of CreA, PkaA, and GanB in CCR differs depending on the inducers, repressing carbon sources, and culture conditions (plate or submerged). Further study of CreA-independent mechanisms is needed to fully understand CCR in filamentous fungi.


Assuntos
Celulase/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Fúngicas/genética , Proteínas Repressoras/genética , Aspergillus nidulans/genética , Carbono/metabolismo , Repressão Catabólica/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Proteínas Repressoras/metabolismo
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