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1.
Cells ; 10(12)2021 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-34944014

RESUMO

Information about mechanical strain in the extracellular space is conducted along collagen fibers connected with integrins and then transmitted within cells. An aim of the study is to verify the hypothesis that the stiffness of cardiac human fibroblast substrates exerts a regulatory effect on collagen metabolism via integrin α2ß1 and downstream signaling. The experiments were performed on human cardiac fibroblasts cultured on stiff or soft polyacrylamide gels. Extracellular and intracellular collagen content, metalloproteinase-1 (MMP-1), metalloproteinase-9 (MMP-9) and expression of the α1 chain of the procollagen type I gene (Col1A1) were elevated in cultures settled on soft substrate. The substrate stiffness did not modify tissue inhibitors of matrix metalloproteinase capacity (TIMPs 1-4). Integrin α2ß1 inhibition (TC-I 15) or α2 subunit silencing resulted in augmentation of collagen content within the culture. Expression of Col1A1 and Col3A1 genes was increased in TC-I 15-treated fibroblasts. Total and phosphorylated levels of both FAK and Src kinases were elevated in fibroblasts cultured on stiff substrate. Inhibition of FAK (FAK kinase inhibitor 14) or Src kinase (AZM 47527) increased collagen content within the culture. The substrate stiffness exerted a regulatory influence on collagen metabolism via integrin α2ß1 and its downstream signaling (FAK and Src kinases) in cardiac fibroblasts.


Assuntos
Colágeno/metabolismo , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrina alfa2beta1/metabolismo , Miocárdio/citologia , Quinases da Família src/metabolismo , Fenômenos Biomecânicos , Linhagem Celular , Humanos , Metaloproteinases da Matriz/metabolismo , Modelos Biológicos , Fosforilação , Subunidades Proteicas/metabolismo , Sistemas do Segundo Mensageiro , Especificidade por Substrato , Inibidores Teciduais de Metaloproteinases/metabolismo
2.
Front Immunol ; 12: 687044, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34630380

RESUMO

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.


Assuntos
Cofilina 1/metabolismo , AMP Cíclico/metabolismo , Macrófagos/microbiologia , Proteínas dos Microfilamentos/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium bovis/patogenicidade , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/patogenicidade , Fagossomos/microbiologia , Tuberculose/microbiologia , Animais , Linhagem Celular , Interações Hospedeiro-Patógeno , Concentração de Íons de Hidrogênio , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium bovis/imunologia , Mycobacterium smegmatis/imunologia , Mycobacterium tuberculosis/imunologia , Fagossomos/imunologia , Fagossomos/metabolismo , Sistemas do Segundo Mensageiro , Tuberculose/imunologia , Tuberculose/metabolismo
3.
Int J Mol Sci ; 22(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34638713

RESUMO

The NO-cGMP signal transduction pathway plays a crucial role in tone regulation in hepatic sinusoids and peripheral blood vessels. In a cirrhotic liver, the key enzymes endothelial NO synthase (eNOS), soluble guanylate cyclase (sGC), and phosphodiesterase-5 (PDE-5) are overexpressed, leading to decreased cyclic guanosine-monophosphate (cGMP). This results in constriction of hepatic sinusoids, contributing about 30% of portal pressure. In contrast, in peripheral arteries, dilation prevails with excess cGMP due to low PDE-5. Both effects eventually lead to circulatory dysfunction in progressed liver cirrhosis. The conventional view of portal hypertension (PH) pathophysiology has been described using the "NO-paradox", referring to reduced NO availability inside the liver and elevated NO production in the peripheral systemic circulation. However, recent data suggest that an altered availability of cGMP could better elucidate the contrasting findings of intrahepatic vasoconstriction and peripheral systemic vasodilation than mere focus on NO availability. Preclinical and clinical data have demonstrated that targeting the NO-cGMP pathway in liver cirrhosis using PDE-5 inhibitors or sGC stimulators/activators decreases intrahepatic resistance through dilation of sinusoids, lowering portal pressure, and increasing portal venous blood flow. These results suggest further clinical applications in liver cirrhosis. Targeting the NO-cGMP system plays a role in possible reversal of liver fibrosis or cirrhosis. PDE-5 inhibitors may have therapeutic potential for hepatic encephalopathy. Serum/plasma levels of cGMP can be used as a non-invasive marker of clinically significant portal hypertension. This manuscript reviews new data about the role of the NO-cGMP signal transduction system in pathophysiology of cirrhotic portal hypertension and provides perspective for further studies.


Assuntos
GMP Cíclico/metabolismo , Hipertensão Portal/metabolismo , Hipertensão Portal/terapia , Cirrose Hepática/metabolismo , Cirrose Hepática/terapia , Sistemas do Segundo Mensageiro , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Humanos , Hipertensão Portal/patologia , Cirrose Hepática/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo
4.
J Biol Chem ; 297(4): 101183, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34509475

RESUMO

Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2)-deficient mice develop spontaneous seizures in early life because of a marked reduction in M currents, which regulate neuronal membrane excitability. We have previously shown that hyper-SUMOylation of the Kv7.2 and Kv7.3 channels is critically involved in the regulation of the M currents conducted by these potassium voltage-gated channels. Here, we show that hyper-SUMOylation of the Kv7.2 and Kv7.3 proteins reduced binding to the lipid secondary messenger PIP2. CaM1 has been shown to be tethered to the Kv7 subunits via hydrophobic motifs in its C termini and implicated in the channel assembly. Mutation of the SUMOylation sites on Kv7.2 and Kv7.3 specifically resulted in decreased binding to CaM1 and enhanced CaM1-mediated assembly of Kv7.2 and Kv7.3, whereas hyper-SUMOylation of Kv7.2 and Kv7.3 inhibited channel assembly. SENP2-deficient mice exhibited increased acetylcholine levels in the brain and the heart tissue because of increases in the vagal tone induced by recurrent seizures. The SENP2-deficient mice develop seizures followed by a period of sinus pauses or atrioventricular conduction blocks. Chronic administration of the parasympathetic blocker atropine or unilateral vagotomy significantly prolonged the life of the SENP2-deficient mice. Furthermore, we showed that retigabine, an M-current opener, reduced the transcription of SUMO-activating enzyme SAE1 and inhibited SUMOylation of the Kv7.2 and Kv7.3 channels, and also prolonged the life of SENP2-deficient mice. Taken together, the previously demonstrated roles of PIP2, CaM1, and retigabine on the regulation of Kv7.2 and Kv7.3 channel function can be explained by their roles in regulating SUMOylation of this critical potassium channel.


Assuntos
Cisteína Endopeptidases/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Sistemas do Segundo Mensageiro , Sumoilação , Motivos de Aminoácidos , Animais , Encéfalo/metabolismo , Cisteína Endopeptidases/genética , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Camundongos , Camundongos Mutantes , Miocárdio/metabolismo , Convulsões/genética , Convulsões/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo
5.
J Biol Chem ; 297(3): 101118, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34437901

RESUMO

cAMP is the indispensable second messenger regulating cell metabolism and function in response to extracellular hormones and neurotransmitters. cAMP is produced via the activation of G protein-coupled receptors located at both the cell surface and inside the cell. Recently, Tsvetanova et al. explored cAMP generation in distinct locations and the impact on respective cell functions. Using a phospho-proteomic analysis, they provide insight into the unique role of localized cAMP production in cellular phospho-responses.


Assuntos
AMP Cíclico , Proteômica , Receptores Acoplados a Proteínas G , Sistemas do Segundo Mensageiro , Transdução de Sinais
6.
Cells ; 10(8)2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34440720

RESUMO

The vascular hypothesis used to explain the pathophysiology of Alzheimer's disease (AD) suggests that a dysfunction of the cerebral microvasculature could be the beginning of alterations that ultimately leads to neuronal damage, and an abnormal increase of the blood-brain barrier (BBB) permeability plays a prominent role in this process. It is generally accepted that, in physiological conditions, cyclic AMP (cAMP) plays a key role in maintaining BBB permeability by regulating the formation of tight junctions between endothelial cells of the brain microvasculature. It is also known that intracellular cAMP signaling is highly compartmentalized into small nanodomains and localized cAMP changes are sufficient at modifying the permeability of the endothelial barrier. This spatial and temporal distribution is maintained by the enzymes involved in cAMP synthesis and degradation, by the location of its effectors, and by the existence of anchor proteins, as well as by buffers or different cytoplasm viscosities and intracellular structures limiting its diffusion. This review compiles current knowledge on the influence of cAMP compartmentalization on the endothelial barrier and, more specifically, on the BBB, laying the foundation for a new therapeutic approach in the treatment of AD.


Assuntos
Doença de Alzheimer/metabolismo , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Humanos , Degeneração Neural , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Sistemas do Segundo Mensageiro
7.
Nature ; 597(7874): 109-113, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34261127

RESUMO

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'-5']pA[3'-5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells1-5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6-10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.


Assuntos
Drosophila melanogaster/metabolismo , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/metabolismo , RNA de Cadeia Dupla/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Sistemas do Segundo Mensageiro , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/imunologia , Drosophila melanogaster/virologia , Feminino , Humanos , Imunidade Inata , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Nucleotidiltransferases/química , Nucleotidiltransferases/imunologia , RNA de Cadeia Dupla/análise , RNA de Cadeia Dupla/imunologia , Receptores de Reconhecimento de Padrão/química , Receptores de Reconhecimento de Padrão/imunologia , Vírus/imunologia
8.
Biochem Biophys Res Commun ; 568: 110-115, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34214875

RESUMO

The phosphoinositides phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2] function as second messengers and have been implicated in cancerogenesis. The signalling events downstream of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are mediated through a complex network of phosphoinositide binding effector proteins and phosphatases. In this study, we compared the phosphoinositide effector proteins AKT1, TAPP1, TAPP2, VAV1 and P-REX1 and the phosphoinositide phosphatases PTEN, SHIP1 and INPP4B for their binding affinities to PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2 using Surface Plasmon Resonance. Our results demonstrate that all measured proteins except P-REX1 and VAV1 showed high affinity phosphoinositide binding with KD values in the nM to sub-nM range. Within the effector proteins, AKT1 showed the highest affinity for both PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Of the phosphoinositide phosphatases PTEN displayed the highest affinity towards PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The SHIP1 mutant E452K detected in carcinoma patients had a 100-fold increased affinity to PtdIns(3,4)P2 but not to PtdIns(3,4,5)P3 compared to SHIP1 WT. Distinct mutations in phosphoinositide binding proteins like the patient-derived SHIP1E452K mutant may be involved in the upregulation of PI(3,4)P2 -mediated signalling in tumor cells due to phosphoinositide trapping. Our results add further information to the complex hierarchy of phosphoinositide binding proteins helping to elucidate their functional role in cellular signal transduction.


Assuntos
PTEN Fosfo-Hidrolase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sistemas do Segundo Mensageiro , Humanos , Modelos Moleculares , Ligação Proteica , Transdução de Sinais
9.
PLoS One ; 16(6): e0253701, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34181669

RESUMO

Hyperinfection and disseminated infection by the parasitic nematode Strongyloides stercoralis can be induced by iatrogenic administration of steroids and immunosuppression and lead to an elevated risk of mortality. Responses of free-living stages of S. stercoralis to the therapeutic corticosteroid dexamethasone (DXM) were investigated using RNA-seq transcriptomes of DXM-treated female and male worms. A total of 17,950 genes representing the transcriptome of these free-living adult stages were obtained, among which 199 and 263 were differentially expressed between DXM-treated females and DXM-treated males, respectively, compared with controls. According to Gene Ontology analysis, differentially expressed genes from DXM-treated females participate in developmental process, multicellular organismal process, cell differentiation, carbohydrate metabolic process and embryonic morphogenesis. Others are involved in signaling and signal transduction, including cAMP, cGMP-dependent protein kinase pathway, endocrine system, and thyroid hormone pathway, as based on Kyoto Encyclopedia of Genes and Genomes analysis. The novel findings warrant deeper investigation of the influence of DXM on growth and other pathways in this neglected tropical disease pathogen, particularly in a setting of autoimmune and/or allergic disease, which may require the clinical use of steroid-like hormones during latent or covert strongyloidiasis.


Assuntos
Dexametasona/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Strongyloides stercoralis/metabolismo , Transcriptoma/efeitos dos fármacos , Animais , Feminino , Masculino
10.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946838

RESUMO

In eukaryotic cells, ultimate specificity in activation and action-for example, by means of second messengers-of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple-sometimes opposite-cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER-PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.


Assuntos
Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Sítios de Ligação , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Sistemas do Segundo Mensageiro/fisiologia , Análise Espaço-Temporal , Moléculas de Interação Estromal/metabolismo , Canais de Cátion TRPC/metabolismo
11.
J Biol Chem ; 296: 100771, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33989637

RESUMO

The KdpDE two-component system regulates potassium homeostasis and virulence in various bacterial species. The KdpD histidine kinases (HK) of this system contain a universal stress protein (USP) domain which binds to the second messenger cyclic-di-adenosine monophosphate (c-di-AMP) for regulating transcriptional output from this two-component system in Firmicutes such as Staphylococcus aureus. However, the structural basis of c-di-AMP specificity within the KdpD-USP domain is not well understood. Here, we resolved a 2.3 Å crystal structure of the S. aureus KdpD-USP domain (USPSa) complexed with c-di-AMP. Binding affinity analyses of USPSa mutants targeting the observed USPSa:c-di-AMP structural interface enabled the identification of the sequence residues that are required for c-di-AMP specificity. Based on the conservation of these residues in other Firmicutes, we identified the binding motif, (A/G/C)XSXSX2N(Y/F), which allowed us to predict c-di-AMP binding in other KdpD HKs. Furthermore, we found that the USPSa domain contains structural features distinct from the canonical standalone USPs that bind ATP as a preferred ligand. These features include inward-facing conformations of its ß1-α1 and ß4-α4 loops, a short α2 helix, the absence of a triphosphate-binding Walker A motif, and a unique dual phospho-ligand binding mode. It is therefore likely that USPSa-like domains in KdpD HKs represent a novel subfamily of the USPs.


Assuntos
Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Histidina Quinase/metabolismo , Proteínas Quinases/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Histidina Quinase/química , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas Quinases/química , Sistemas do Segundo Mensageiro , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química
12.
Nat Commun ; 12(1): 1986, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790266

RESUMO

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Algoritmos , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Cromatografia Líquida , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Espectrometria de Massas , Fósforo-Oxigênio Liases/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Temperatura
13.
J Bacteriol ; 203(13): e0004621, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33846117

RESUMO

Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while, its overexpression from an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible promoter showed a positive effect on both swimming and swarming motility and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes, as well as that of the biofilm-related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE c-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, and hence not many examples of these type of signaling proteins have been reported in the literature. Here, we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.


Assuntos
Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Diester Fosfórico Hidrolases/metabolismo , Vibrio parahaemolyticus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GMP Cíclico/química , GMP Cíclico/genética , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Sistemas do Segundo Mensageiro , Vibrio parahaemolyticus/genética
14.
Infect Immun ; 89(6)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33846120

RESUMO

Relapsing fever (RF), caused by spirochetes of the genus Borrelia, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. Cyclic di-AMP (c-di-AMP), synthesized by diadenylate cyclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium Borrelia turicatae, a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole-genome sequencing was performed on cdaA mutants, and single-nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium Borrelia burgdorferi Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia/fisiologia , Fósforo-Oxigênio Liases/metabolismo , Febre Recorrente/microbiologia , Animais , Proteínas de Bactérias/genética , Borrelia/patogenicidade , AMP Cíclico/metabolismo , Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno , Camundongos , Mutação , Fósforo-Oxigênio Liases/genética , Febre Recorrente/metabolismo , Sistemas do Segundo Mensageiro , Virulência/genética
15.
mBio ; 12(2)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33832972

RESUMO

The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic ß-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity.IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in ß-lactam resistance, however, is unclear despite being first identified a decade ago. Here, we demonstrate that the overaccumulation of potassium or free amino acids leads to cefuroxime sensitivity in Lactococcus lactis mutants partially defective in c-di-AMP synthesis. It was shown that c-di-AMP negatively affects the levels of the most abundant free amino acids (glutamate and aspartate) in L. lactis Regulation of these major free anions was found to occur via the glutamine transporter GlnPQ, whose activity increased in response to intracellular potassium levels, which are under c-di-AMP control. Evidence is also presented showing that they are major osmolytes that enhance osmoresistance and cell lysis. The regulatory reach of c-di-AMP can be extended to include the main free anions in bacteria.


Assuntos
Antibacterianos/farmacologia , Cefuroxima/farmacologia , AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Lactococcus lactis/metabolismo , Potássio/metabolismo , Sistemas do Segundo Mensageiro
16.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800808

RESUMO

Plants are subject to different types of stress, which consequently affect their growth and development. They have developed mechanisms for recognizing and processing an extracellular signal. Second messengers are transient molecules that modulate the physiological responses in plant cells under stress conditions. In this sense, it has been shown in various plant models that membrane lipids are substrates for the generation of second lipid messengers such as phosphoinositide, phosphatidic acid, sphingolipids, and lysophospholipids. In recent years, research on lipid second messengers has been moving toward using genetic and molecular approaches to reveal the molecular setting in which these molecules act in response to osmotic stress. In this sense, these studies have established that second messengers can transiently recruit target proteins to the membrane and, therefore, affect protein conformation, activity, and gene expression. This review summarizes recent advances in responses related to the link between lipid second messengers and osmotic stress in plant cells.


Assuntos
Lipídeos/fisiologia , Pressão Osmótica/fisiologia , Plantas/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Cálcio/metabolismo , Glicolipídeos/fisiologia , Modelos Biológicos , Fosfolipídeos/fisiologia , Proteínas de Plantas/metabolismo , Estresse Salino/fisiologia
17.
J Diabetes Res ; 2021: 5123241, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33708999

RESUMO

Insulin resistance is a state of impaired responsiveness to insulin action. This condition not only results in deficient glucose uptake but increases the risk for cardiovascular diseases (CVD), stroke, and obesity. The present work investigates the molecular mechanisms of high carbohydrate and fat diet in inducing prediabetic hyperinsulinemia and effect of exercise on InsR signaling events, muscular AChE, and lactate dehydrogenase activity. Adult male Wistar rats were divided into the control (C) diet group, high-carbohydrate diet (HCD) group, high-fat diet (HFD) group, and HCD and HFD groups with exercise (HCD Ex and HFD Ex, respectively). Acetyl choline esterase activity, lactate dehydrogenase activity, total lactate levels, IRS1 phosphorylations, and Glut4 expression patterns were studied in the muscle tissue among these groups. High carbohydrate and fat feeding led to hyperinsulinemic status with reduced acetylcholine esterase (AChE) activity and impaired phosphorylation of IRS1 along with increased lactate concentrations in the muscle. Exercise significantly upregulated phosphoinositide 3 kinase (PI3K) docking site phosphorylation and downregulated the negative IRS1 phosphorylations thereby increasing the glucose transporter (GLUT) expressions and reducing the lactate accumulation. Also, the levels of second messengers like IP3 and cAMP were increased with exercise. Increased second messenger levels induce calcium release thereby activating the downstream pathway promoting the translocation of GLUT4 to the plasma membrane. Our results showed that the metabolic and signaling pathway dysregulations seen during diet-induced hyperinsulinemia, a metabolic condition seen during the early stages in the development of prediabetes, were improved with vigorous physical exercise. Thus, exercise can be considered as an excellent management approach over drug therapy for diabetes and its complications.


Assuntos
Glicemia/metabolismo , Dieta Hiperlipídica , Carboidratos da Dieta , Terapia por Exercício , Hiperinsulinismo/terapia , Resistência à Insulina , Insulina/sangue , Contração Muscular , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Acetilcolinesterase/metabolismo , Animais , Modelos Animais de Doenças , Proteínas Ligadas por GPI/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Hiperinsulinismo/sangue , Hiperinsulinismo/fisiopatologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Ratos Wistar , Sistemas do Segundo Mensageiro
18.
FASEB J ; 35(4): e21475, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33772870

RESUMO

Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes are not entirely understood. Herein we present the report of a c-di-GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c-di-GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS-PdtaR (Rv1626) two-component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems' approach of transcriptomics, unbiased metabolomics, and lipid analyses.


Assuntos
Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Histidina Quinase/metabolismo , Bactérias , Proteínas de Bactérias/metabolismo , Simulação de Acoplamento Molecular/métodos , Mycobacterium/metabolismo , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia
19.
Sci Signal ; 14(675)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758061

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , Sinalização do Cálcio/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , NADP/análogos & derivados , SARS-CoV-2/fisiologia , Marcadores de Afinidade , Animais , Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Química Click/métodos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transcriptoma , Internalização do Vírus
20.
Blood ; 137(5): 678-689, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33538796

RESUMO

Thrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1-deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild-type (WT) but not TSP-1-/- platelets ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1-deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation, and arrest under flow by prostacyclin (PGI2). Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1-/- platelets. By contrast, incubation of TSP-1-/- platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1-/- platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1-/- mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This scenario suggests that the release of TSP-1 regulates hemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.


Assuntos
Plaquetas/fisiologia , AMP Cíclico/fisiologia , Transtornos Hemorrágicos/genética , Hemostasia/fisiologia , Trombospondina 1/fisiologia , Animais , Tempo de Sangramento , Plaquetas/efeitos dos fármacos , Antígenos CD36/deficiência , Antígenos CD36/fisiologia , Células Cultivadas , Cloretos/toxicidade , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Grânulos Citoplasmáticos/metabolismo , Epoprostenol/fisiologia , Compostos Férricos/toxicidade , Humanos , Iloprosta/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Transfusão de Plaquetas , Sistemas do Segundo Mensageiro/fisiologia , Trombose/induzido quimicamente , Trombose/prevenção & controle , Trombospondina 1/deficiência , Trombospondina 1/farmacologia
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