RESUMO
Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Venenos de Crotalídeos/enzimologia , Crotalinae , Metaloproteases/farmacologia , Glicoproteínas da Membrana de Plaquetas/biossíntese , Sequência de Aminoácidos , Animais , Crotalinae/metabolismo , Matriz Extracelular , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/isolamento & purificação , Modelos Moleculares , Filogenia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/química , Conformação Proteica , Proteólise , Relação Estrutura-AtividadeRESUMO
ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU-driven NF-κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF-κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR-dependent NF-κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR-/- or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU-induced NF-κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF-κB inhibitor Bay 11-7082, or transfection with IκBα negative-dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11-7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF-κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.
Assuntos
Proteínas de Bactérias/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Pseudomonas aeruginosa/patogenicidade , Receptores Acoplados a Proteínas G/genética , Fator de Transcrição RelA/metabolismo , Animais , Azepinas/farmacologia , Toxinas Bacterianas/metabolismo , Linhagem Celular , Ativação Enzimática , Feminino , Regulação da Expressão Gênica , Humanos , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Fator de Ativação de Plaquetas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/biossíntese , Regiões Promotoras Genéticas , Ligação Proteica , Infecções por Pseudomonas/patologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/biossíntese , Transdução de Sinais/genética , Triazóis/farmacologiaRESUMO
Melanoma cells express the platelet-activating factor receptor (PAFR) and, thus, respond to PAF, a bioactive lipid produced by both tumour cells and those in the tumour microenvironment such as macrophages. Here, we show that treatment of a human melanoma SKmel37 cell line with cisplatin led to increased expression of PAFR and its accumulation. In the presence of exogenous PAF, melanoma cells were significantly more resistant to cisplatin-induced cell death. Inhibition of PAFR-dependent signalling pathways by a PAFR antagonist (WEB2086) showed chemosensitisation of melanoma cells in vitro. Nude mice were inoculated with SKmel37 cells and treated with cisplatin and WEB2086. Animals treated with both agents showed significantly decreased tumour growth compared to the control group and groups treated with only one agent. PAFR accumulation and signalling are part of a prosurvival program of melanoma cells, therefore constituting a promising target for combination therapy for melanomas.