Navegando por Palavras-chave "Protease-activated receptors"

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    Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors
    (Elsevier B.V., 2014-03-01) Yonamine, Camila M. [UNIFESP]; Kondo, Marcia Y. [UNIFESP]; Nering, Marcela B. [UNIFESP]; Gouvea, Iuri E. [UNIFESP]; Okamoto, Debora [UNIFESP]; Andrade, Douglas [UNIFESP]; Silva, Jose Alberto A. da; Prieto da Silva, Alvaro R. B.; Yamane, Tetsuo; Juliano, Maria A. [UNIFESP]; Juliano, Luiz [UNIFESP]; Lapa, Antonio J. [UNIFESP]; Hayashi, Mirian A. F. [UNIFESP]; Lima-Landman, Maria Teresa R. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); IPEN; Inst Butantan; Univ Estado Amazonas
    Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. the serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. in the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. the k(cat)/K-M values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. for the sh and lg PAR-2 mimetic peptides the k(cat)/K-M values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. the k(cat)/K-M values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-I, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. the determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects. (C) 2013 Elsevier B.V. All rights reserved.
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    Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner
    (Springer, 2017) de Oliveira, Priscila [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Carmona, Adriana Karaoglanovic [UNIFESP]; Batista dos Santos, Saara Maria [UNIFESP]; Silva Campitelli de Barros, Bianca Carla [UNIFESP]; Maza, Paloma Korehisa [UNIFESP]; Puccia, Rosana [UNIFESP]; Suzuki, Erika [UNIFESP]
    Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.