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1.
BMC Bioinformatics ; 21(Suppl 14): 408, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32998723

RESUMO

BACKGROUND: Second messengers, c-di-GMP and (p)ppGpp, are vital regulatory molecules in bacteria, influencing cellular processes such as biofilm formation, transcription, virulence, quorum sensing, and proliferation. While c-di-GMP and (p)ppGpp are both synthesized from GTP molecules, they play antagonistic roles in regulating the cell cycle. In C. crescentus, c-di-GMP works as a major regulator of pole morphogenesis and cell development. It inhibits cell motility and promotes S-phase entry by inhibiting the activity of the master regulator, CtrA. Intracellular (p)ppGpp accumulates under starvation, which helps bacteria to survive under stressful conditions through regulating nucleotide levels and halting proliferation. (p)ppGpp responds to nitrogen levels through RelA-SpoT homolog enzymes, detecting glutamine concentration using a nitrogen phosphotransferase system (PTS Ntr). This work relates the guanine nucleotide-based second messenger regulatory network with the bacterial PTS Ntr system and investigates how bacteria respond to nutrient availability. RESULTS: We propose a mathematical model for the dynamics of c-di-GMP and (p)ppGpp in C. crescentus and analyze how the guanine nucleotide-based second messenger system responds to certain environmental changes communicated through the PTS Ntr system. Our mathematical model consists of seven ODEs describing the dynamics of nucleotides and PTS Ntr enzymes. Our simulations are consistent with experimental observations and suggest, among other predictions, that SpoT can effectively decrease c-di-GMP levels in response to nitrogen starvation just as well as it increases (p)ppGpp levels. Thus, the activity of SpoT (or its homologues in other bacterial species) can likely influence the cell cycle by influencing both c-di-GMP and (p)ppGpp. CONCLUSIONS: In this work, we integrate current knowledge and experimental observations from the literature to formulate a novel mathematical model. We analyze the model and demonstrate how the PTS Ntr system influences (p)ppGpp, c-di-GMP, GMP and GTP concentrations. While this model does not consider all aspects of PTS Ntr signaling, such as cross-talk with the carbon PTS system, here we present our first effort to develop a model of nutrient signaling in C. crescentus.


Assuntos
Caulobacter crescentus/fisiologia , Modelos Teóricos , Sistemas do Segundo Mensageiro , Pontos de Checagem do Ciclo Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Nitrogênio/metabolismo , Fosfotransferases/metabolismo , Sistemas do Segundo Mensageiro/fisiologia
2.
Nature ; 586(7829): 429-433, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32877915

RESUMO

Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP-AMP signalling during viral infection and anti-tumour immunity1-5. STING shares no structural homology with other known signalling proteins6-9, which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD+ cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIR-STING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGAS-STING pathway in prokaryotic defence against bacteriophages.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Evolução Molecular , Proteínas de Membrana , Sistemas do Segundo Mensageiro , Animais , Bactérias/química , Bactérias/virologia , Proteínas de Bactérias/química , Bacteriófagos , Cristalografia por Raios X , GMP Cíclico/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , NAD/metabolismo , Nucleotidiltransferases/metabolismo
3.
PLoS Genet ; 16(8): e1008505, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776934

RESUMO

Dynamic gene expression in neurons shapes fundamental processes in the nervous systems of animals. However, how neuronal activation by different stimuli can lead to distinct transcriptional responses is not well understood. We have been studying how microbial metabolites modulate gene expression in chemosensory neurons of Caenorhabditis elegans. Considering the diverse environmental stimuli that can activate chemosensory neurons of C. elegans, we sought to understand how specific transcriptional responses can be generated in these neurons in response to distinct cues. We have focused on the mechanism of rapid (<6 min) and selective transcriptional induction of daf-7, a gene encoding a TGF-ß ligand, in the ASJ chemosensory neurons in response to the pathogenic bacterium Pseudomonas aeruginosa. DAF-7 is required for the protective behavioral avoidance of P. aeruginosa by C. elegans. Here, we define the involvement of two distinct cyclic GMP (cGMP)-dependent pathways that are required for daf-7 expression in the ASJ neuron pair in response to P. aeruginosa. We show that a calcium-independent pathway dependent on the cGMP-dependent protein kinase G (PKG) EGL-4, and a canonical calcium-dependent signaling pathway dependent on the activity of a cyclic nucleotide-gated channel subunit CNG-2, function in parallel to activate rapid, selective transcription of daf-7 in response to P. aeruginosa metabolites. Our data suggest that fast, selective early transcription of neuronal genes require PKG in shaping responses to distinct microbial stimuli in a pair of C. elegans chemosensory neurons.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Células Quimiorreceptoras/metabolismo , GMP Cíclico/metabolismo , Pseudomonas aeruginosa/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(35): 21568-21575, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817552

RESUMO

The DNA sensor cGMP-AMP synthase (cGAS) senses cytosolic microbial or self DNA to initiate a MITA/STING-dependent innate immune response. cGAS is regulated by various posttranslational modifications at its C-terminal catalytic domain. Whether and how its N-terminal unstructured domain is regulated by posttranslational modifications remain unknown. We identified the acetyltransferase KAT5 as a positive regulator of cGAS-mediated innate immune signaling. Overexpression of KAT5 potentiated viral-DNA-triggered transcription of downstream antiviral genes, whereas a KAT5 deficiency had the opposite effects. Mice with inactivated Kat5 exhibited lower levels of serum cytokines in response to DNA virus infection, higher viral titers in the brains, and more susceptibility to DNA-virus-induced death. Mechanistically, KAT5 catalyzed acetylation of cGAS at multiple lysine residues in its N-terminal domain, which promoted its DNA-binding ability. Our findings suggest that KAT5-mediated cGAS acetylation at its N terminus is important for efficient innate immune response to DNA virus.


Assuntos
Infecções por Vírus de DNA/imunologia , Vírus de DNA/imunologia , Lisina Acetiltransferase 5/imunologia , Nucleotidiltransferases/imunologia , Acetilação , Animais , GMP Cíclico/metabolismo , Infecções por Vírus de DNA/genética , Infecções por Vírus de DNA/metabolismo , Vírus de DNA/genética , Feminino , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Interferon beta/imunologia , Lisina Acetiltransferase 5/genética , Lisina Acetiltransferase 5/metabolismo , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Proteínas Virais/metabolismo
5.
Nat Commun ; 11(1): 3532, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669539

RESUMO

Asexual proliferation of the Plasmodium parasites that cause malaria follows a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCα, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCα and find that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/enzimologia , Proteína Fosfatase 1/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Proliferação de Células , GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Fosfatidilcolinas/química , Domínios Proteicos , Proteoma , Ubiquitina-Proteína Ligases/metabolismo
6.
Nat Commun ; 11(1): 3533, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669552

RESUMO

Cyclic dinucleotides (CDNs) are second messengers conserved across all three domains of life. Within eukaryotes they mediate protective roles in innate immunity against malignant, viral, and bacterial disease, and exert pathological effects in autoimmune disorders. Despite their ubiquitous role in diverse biological contexts, CDN detection methods are limited. Here, using structure guided design of the murine STING CDN binding domain, we engineer a Förster resonance energy transfer (FRET) based biosensor deemed BioSTING. Recombinant BioSTING affords real-time detection of CDN synthase activity and inhibition. Expression of BioSTING in live human cells allows quantification of localized bacterial and eukaryotic CDN levels in single cells with low nanomolar sensitivity. These findings establish BioSTING as a powerful kinetic in vitro platform amenable to high throughput screens and as a broadly applicable cellular tool to interrogate the temporal and spatial dynamics of CDN signaling in a variety of infectious, malignant, and autoimmune contexts.


Assuntos
Técnicas Biossensoriais , Proteínas de Membrana/química , Nucleotídeos Cíclicos/análise , Transdução de Sinais , Animais , Bacillus subtilis/química , GMP Cíclico/metabolismo , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Imunidade Inata , Ligantes , Camundongos , Mutagênese , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes
7.
Proc Natl Acad Sci U S A ; 117(29): 17211-17220, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611811

RESUMO

The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger. For this, we developed a c-di-GMP-sequestering peptide (CSP) that was derived from a CheY-like c-di-GMP effector protein. CSP binds c-di-GMP with submicromolar affinity. The elucidation of the CSP⋅c-di-GMP complex structure by NMR identified a linear c-di-GMP-binding motif, in which a self-intercalated c-di-GMP dimer is tightly bound by a network of H bonds and π-stacking interactions involving arginine and aromatic residues. Structure-based mutagenesis yielded a variant with considerably higher, low-nanomolar affinity, which subsequently was shortened to 19 residues with almost uncompromised affinity. We demonstrate that endogenously expressed CSP intercepts c-di-GMP signaling and effectively inhibits biofilm formation in Pseudomonas aeruginosa, the most widely used model for serious biofilm-associated medical implications.


Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Peptídeos/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais , Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli , Modelos Moleculares , Mutagênese , Peptídeos/química , Peptídeos/genética , Mutação Puntual , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(30): 17984-17991, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661164

RESUMO

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium Caulobacter crescentus employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell-cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer-membrane pilus secretin CpaC stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell-cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell-cycle regulator cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and sensing by alterations in pilus activity stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle.


Assuntos
Fenômenos Fisiológicos Bacterianos , Caulobacter crescentus/fisiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Ciclo Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Replicação do DNA , Fímbrias Bacterianas/fisiologia , Modelos Biológicos , Mutação
9.
Proc Natl Acad Sci U S A ; 117(32): 19599-19603, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719136

RESUMO

We have used recent measurements of mammalian cone light responses and voltage-gated currents to calculate cone ATP utilization and compare it to that of rods. The largest expenditure of ATP results from ion transport, particularly from removal of Na+ entering outer segment light-dependent channels and inner segment hyperpolarization-activated cyclic nucleotide-gated channels, and from ATP-dependent pumping of Ca2+ entering voltage-gated channels at the synaptic terminal. Single cones expend nearly twice as much energy as single rods in darkness, largely because they make more synapses with second-order retinal cells and thus must extrude more Ca2+ In daylight, cone ATP utilization per cell remains high because cones never remain saturated and must continue to export Na+ and synaptic Ca2+ even in bright illumination. In mouse and human retina, rods greatly outnumber cones and consume more energy overall even in background light. In primates, however, the high density of cones in the fovea produces a pronounced peak of ATP utilization, which becomes particularly prominent in daylight and may make this part of the retina especially sensitive to changes in energy availability.


Assuntos
Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Fóvea Central/citologia , Fóvea Central/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico , Luz , Camundongos , Terminações Pré-Sinápticas/metabolismo , Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sódio/metabolismo
10.
PLoS Genet ; 16(6): e1008897, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32589664

RESUMO

The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Cólera/microbiologia , GMP Cíclico/análogos & derivados , Protease La/antagonistas & inibidores , Vibrio cholerae/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Protease La/genética , Protease La/isolamento & purificação , Protease La/metabolismo , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteólise , Proteômica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Virulência/genética
11.
Biochim Biophys Acta Rev Cancer ; 1874(1): 188383, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32535158

RESUMO

Androgen deprivation therapy (ADT) is the primary systemic therapy for treating locally advanced or metastatic prostate cancer (PCa). Despite its positive effect on PCa patient survival, ADT causes various adverse effects, including increased cardiovascular risk factors and cardiotoxicity. Lifespans extension, early use of ADT, and second-line treatment with next-generation androgen receptor pathway inhibitors would further extend the duration of ADT and possibly increase the risk of ADT-induced cardiotoxicity. Meanwhile, information on the molecular mechanisms underlying ADT-induced cardiotoxicity and measures to prevent it is limited, mainly due to the lack of specifically designed preclinical studies and clinical trials. This review article compiles up-to-date evidence obtained from observational studies and clinical trials, in order to gain new insights for deciphering the association between ADT use and cardiotoxicity. In addition, potential cardioprotective strategies involving GnRH receptors and second messenger cGMP are discussed.


Assuntos
Antagonistas de Androgênios/efeitos adversos , Antineoplásicos Hormonais/efeitos adversos , Doenças Cardiovasculares/epidemiologia , Neoplasias da Próstata/tratamento farmacológico , Antagonistas de Androgênios/administração & dosagem , Androgênios/metabolismo , Antineoplásicos Hormonais/administração & dosagem , Cardiotoxicidade/epidemiologia , Cardiotoxicidade/etiologia , Cardiotoxicidade/fisiopatologia , Cardiotoxicidade/prevenção & controle , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/fisiopatologia , Doenças Cardiovasculares/prevenção & controle , Ensaios Clínicos como Assunto , GMP Cíclico/metabolismo , Hormônio Liberador de Gonadotropina/agonistas , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Longevidade/fisiologia , Masculino , Estudos Observacionais como Assunto , Inibidores da Fosfodiesterase 5/farmacologia , Inibidores da Fosfodiesterase 5/uso terapêutico , Receptores LHRH/agonistas , Receptores LHRH/antagonistas & inibidores , Receptores LHRH/metabolismo , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Resultado do Tratamento
12.
Proc Natl Acad Sci U S A ; 117(25): 14220-14230, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513741

RESUMO

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Animais , Animais Geneticamente Modificados , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Humanos , Fosforilação , Tauopatias , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Peixe-Zebra , Proteínas tau/metabolismo
13.
Life Sci ; 256: 117960, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32534033

RESUMO

BACKGROUND: Intestinal GC-C/cGMP pathway may be involved in visceral hypersensitivity and fluid secretion in irritable bowel syndrome (IBS). The guanylcyclase C agonist linaclotide, approved for IBS- constipation, is contraindicated in children as it may cause severe diarrhea. In contrast, drugs increasing cGMP by inhibiting phosphodiesterase 5 (PDE-5) are well tolerated in children with pulmonary hypertension. Accordingly, we investigated whether beneficial effects of linaclotide in IBS might be shared by PDE-5inhibitor tadalafil without the severe diarrhea reported for linaclotide. Since depression is commonly comorbid with IBS and is implicated in its pathophysiology; and since tadalafil is absorbed systemically and crosses blood brain barrier, whereas linaclotide does not, impact of both drugs on behavioral changes in IBS was also investigated. METHODS: 72 rats were divided into 6groups (control naive, control tadalafil, control linaclotide, untreated IBS, IBS tadalafil, and IBS linaclotide-treated). IBS was induced by 0 to 4 °C intragastric saline for 14 days. RESULTS: Both drugs reduced visceral hypersensitivity and colonic C fos. Tadalafil, and to a greater extent, linaclotide increased colonic cGMP, fecal pellets (8.66 ± 4.6 (IBS),versus14.8 ± 3.3(tadalafil), 20 ± 1.2(linaclotide), fecal water content (29.8 ± 5.5 (IBS), versus 47.83 ± 12.6 (tadalafil), 63.58 ± 11.6 (linaclotide) and reduced intestinal transit time (% distance travelled: 29 ± 6.1(IBS), versus 40.58 + 7.5(tadalafil), 51.83 ± 8.3(linaclotide). Tadalafil, but not linaclotide, increased hippocampal cGMP, and improved behavioral tests scores compared to linaclotide (immobility time: 97.3 ± 12.5 s (IBS) versus 68 ± 12.8(tadalafil), 80 ± 17.06 (linaclotide). CONCLUSION: Systemic PDE-5 inhibitors might be alternatives to locally acting guanyl cyclase agonists in IBS, inducing less severe diarrhea and more beneficial effects on the associated behavioral changes.


Assuntos
Constipação Intestinal/complicações , Constipação Intestinal/fisiopatologia , Síndrome do Intestino Irritável/tratamento farmacológico , Síndrome do Intestino Irritável/fisiopatologia , Peptídeos/uso terapêutico , Tadalafila/uso terapêutico , Animais , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Colo/fisiopatologia , Constipação Intestinal/tratamento farmacológico , GMP Cíclico/metabolismo , Fezes/química , Trânsito Gastrointestinal/efeitos dos fármacos , Hipocampo/metabolismo , Intestino Grosso/metabolismo , Masculino , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos Wistar , Reflexo/efeitos dos fármacos , Natação , Tadalafila/farmacologia , Água
14.
Nat Struct Mol Biol ; 27(7): 625-634, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483338

RESUMO

Cyclic nucleotide-gated (CNG) channels convert cyclic nucleotide (CN) binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length Caenorhabditis elegans cyclic GMP-activated channel TAX-4, reconstituted in lipid nanodiscs. These structures, together with computational and functional analyses and a mutant channel structure, reveal a double-barrier hydrophobic gate formed by two S6 amino acids in the central cavity. cGMP binding produces global conformational changes that open the cavity gate located ~52 Å away but do not alter the structure of the selectivity filter-the commonly presumed activation gate. Our work provides mechanistic insights into the allosteric gating and regulation of CN-gated and nucleotide-modulated channels and CNG channel-related channelopathies.


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Proteínas de Caenorhabditis elegans/genética , Microscopia Crioeletrônica , GMP Cíclico/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/genética , Ligantes , Lipídeos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Conformação Proteica
15.
Proc Natl Acad Sci U S A ; 117(20): 10839-10847, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358188

RESUMO

Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from Spirochaeta thermophila Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B'-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B'-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.


Assuntos
Sítio Alostérico/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Spirochaeta/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Escherichia coli/metabolismo , Ativação do Canal Iônico/fisiologia , Modelos Moleculares , Nucleotídeos Cíclicos , Conformação Proteica
16.
Nat Rev Mol Cell Biol ; 21(9): 501-521, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32424334

RESUMO

The cGAS-STING signalling axis, comprising the synthase for the second messenger cyclic GMP-AMP (cGAS) and the cyclic GMP-AMP receptor stimulator of interferon genes (STING), detects pathogenic DNA to trigger an innate immune reaction involving a strong type I interferon response against microbial infections. Notably however, besides sensing microbial DNA, the DNA sensor cGAS can also be activated by endogenous DNA, including extranuclear chromatin resulting from genotoxic stress and DNA released from mitochondria, placing cGAS-STING as an important axis in autoimmunity, sterile inflammatory responses and cellular senescence. Initial models assumed that co-localization of cGAS and DNA in the cytosol defines the specificity of the pathway for non-self, but recent work revealed that cGAS is also present in the nucleus and at the plasma membrane, and such subcellular compartmentalization was linked to signalling specificity of cGAS. Further confounding the simple view of cGAS-STING signalling as a response mechanism to infectious agents, both cGAS and STING were shown to have additional functions, independent of interferon response. These involve non-catalytic roles of cGAS in regulating DNA repair and signalling via STING to NF-κB and MAPK as well as STING-mediated induction of autophagy and lysosome-dependent cell death. We have also learnt that cGAS dimers can multimerize and undergo liquid-liquid phase separation to form biomolecular condensates that could importantly regulate cGAS activation. Here, we review the molecular mechanisms and cellular functions underlying cGAS-STING activation and signalling, particularly highlighting the newly emerging diversity of this signalling pathway and discussing how the specificity towards normal, damage-induced and infection-associated DNA could be achieved.


Assuntos
Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Autofagia , AMP Cíclico/metabolismo , AMP Cíclico/fisiologia , GMP Cíclico/metabolismo , GMP Cíclico/fisiologia , Citosol/metabolismo , DNA/metabolismo , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas de Membrana/fisiologia , Nucleotídeos Cíclicos , Nucleotidiltransferases/genética , Transdução de Sinais
17.
Nat Rev Cardiol ; 17(11): 698-717, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32444692

RESUMO

Investigations into the mixed muscle-secretory phenotype of cardiomyocytes from the atrial appendages of the heart led to the discovery that these cells produce, in a regulated manner, two polypeptide hormones - the natriuretic peptides - referred to as atrial natriuretic factor or atrial natriuretic peptide (ANP) and brain or B-type natriuretic peptide (BNP), thereby demonstrating an endocrine function for the heart. Studies on the gene encoding ANP (NPPA) initiated the field of modern research into gene regulation in the cardiovascular system. Additionally, ANP and BNP were found to be the natural ligands for cell membrane-bound guanylyl cyclase receptors that mediate the effects of natriuretic peptides through the generation of intracellular cGMP, which interacts with specific enzymes and ion channels. Natriuretic peptides have many physiological actions and participate in numerous pathophysiological processes. Important clinical entities associated with natriuretic peptide research include heart failure, obesity and systemic hypertension. Plasma levels of natriuretic peptides have proven to be powerful diagnostic and prognostic biomarkers of heart disease. Development of pharmacological agents that are based on natriuretic peptides is an area of active research, with vast potential benefits for the treatment of cardiovascular disease.


Assuntos
Fator Natriurético Atrial/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Receptores Acoplados a Guanilato Ciclase/metabolismo , Animais , Apêndice Atrial/citologia , Fibrilação Atrial/metabolismo , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/fisiologia , Remodelamento Atrial , Biomarcadores/metabolismo , GMP Cíclico/metabolismo , Diabetes Mellitus/metabolismo , Fibrose , Regulação da Expressão Gênica no Desenvolvimento , Átrios do Coração/citologia , Humanos , Hipertensão/metabolismo , Metabolismo dos Lipídeos/fisiologia , Síndrome Metabólica/metabolismo , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Peptídeo Natriurético Encefálico/genética , Peptídeo Natriurético Encefálico/fisiologia , Obesidade/metabolismo , Fragmentos de Peptídeos/metabolismo , Prognóstico , Processamento de Proteína Pós-Traducional , Hipertensão Arterial Pulmonar/metabolismo , Vesículas Secretórias/metabolismo , Remodelação Ventricular , Equilíbrio Hidroeletrolítico/fisiologia
18.
Nat Commun ; 11(1): 1791, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286293

RESUMO

Cyclic di-GMP (c-di-GMP) is a second messenger that modulates multiple responses to environmental and cellular signals in bacteria. Here we identify CdbA, a DNA-binding protein of the ribbon-helix-helix family that binds c-di-GMP in Myxococcus xanthus. CdbA is essential for viability, and its depletion causes defects in chromosome organization and segregation leading to a block in cell division. The protein binds to the M. xanthus genome at multiple sites, with moderate sequence specificity; however, its depletion causes only modest changes in transcription. The interactions of CdbA with c-di-GMP and DNA appear to be mutually exclusive and residue substitutions in CdbA regions important for c-di-GMP binding abolish binding to both c-di-GMP and DNA, rendering these protein variants non-functional in vivo. We propose that CdbA acts as a nucleoid-associated protein that contributes to chromosome organization and is modulated by c-di-GMP, thus revealing a link between c-di-GMP signaling and chromosome biology.


Assuntos
Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , Segregação de Cromossomos , GMP Cíclico/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Myxococcus xanthus/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Cromossomos Bacterianos/metabolismo , GMP Cíclico/metabolismo , DNA Bacteriano/metabolismo , Loci Gênicos , Modelos Moleculares , Multimerização Proteica , Estrutura Secundária de Proteína , Transcrição Genética
19.
Life Sci ; 253: 117683, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32315727

RESUMO

OBJECTIVE: To explore the potential mechanism of KMUP-1 in the vascular calcification of chronic renal failure (CRF) through mediating NO/cGMP/PKG pathway, and provide novel insights into the CRF treatment. METHODS: CRF rats were treated by KMUP-1 with/without L-NNA (a NOS inhibitor) and then performed by ELISA, alizarin red staining, Von Kossa staining, Masson's trichrome, Sirius red staining and CD3 immunohistochemical staining. Simultaneously, vascular smooth muscle cells (VSMCs) were collected from rats to confirm the effect of KMUP-1 on vascular calcification in vitro via NO/cGMP/PKG pathway. Besides, protein and mRNA expressions were determined via Western blotting and qRT-PCR, respectively. RESULTS: CRF rats were elevated in 24-h urine protein, blood urea nitrogen (BUN), serum creatinine, Cys-C levels and inflammatory cytokines. Besides, CRF rats also showed increased calcium content and ALP level with up-regulated mRNA of osteogenic differentiation-related markers. Furthermore, the up-regulated expressions of eNOS and PKG, as well as down-regulated levels of NOx and cGMP were also found in CRF rats. However, renal failure and vascular calcification of CRF were improved significantly by KMUP-1 treatment via activation of NO/cGMP/PKG pathway. Moreover, KMUP-1 treatment attenuated calcified VSMCs, accompanied by the decreases in the calcified nodules, level of calcium and activity of ALP. In addition, either L-NNA treatment for CRF rats or the calcified VSMCs could antagonize the improving effect of KMUP-1. CONCLUSION: KMUP-1 can improve the renal function and vascular calcification in CRF rats at least in part by activating NO/cGMP/PKG pathway.


Assuntos
Falência Renal Crônica/tratamento farmacológico , Miócitos de Músculo Liso/efeitos dos fármacos , Piperidinas/farmacologia , Calcificação Vascular/tratamento farmacológico , Xantinas/farmacologia , Animais , Cálcio/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Modelos Animais de Doenças , Falência Renal Crônica/fisiopatologia , Masculino , Miócitos de Músculo Liso/patologia , Óxido Nítrico/metabolismo , Osteogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Calcificação Vascular/patologia
20.
PLoS Pathog ; 16(4): e1008281, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32236137

RESUMO

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.


Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Óperon , Polissacarídeos/metabolismo , Bacillus cereus/genética , Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Filogenia , Conformação Proteica
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