RESUMO
Colorectal cancer (CRC) is one of the common malignancies with a global trend of increasing incidence and mortality. There is an urgent need to identify new predictive markers and therapeutic targets for the treatment of CRC. Protease-activated receptors (PARs) are a class of G-protein-coupled receptors, with currently identified subtypes including PAR1, PAR2, PAR3 and PAR4. Increasingly, studies suggest that PARs play an important role in the growth and metastasis of CRC. By targeting multiple signaling pathways may contribute to the pathogenesis of CRC. In this review, we first describe recent studies on the role of PARs in CRC inflammation-cancer transformation, focusing on the important role of PARs in signaling pathways associated with inflammation-cancer transformation, and summarize the progress of research on PARs-targeted drugs.
Assuntos
Neoplasias , Receptores Ativados por Proteinase , Humanos , Receptores Ativados por Proteinase/metabolismo , Receptores de Trombina/metabolismo , Transdução de Sinais , InflamaçãoRESUMO
Protease activated receptors (PARs) are among the first receptors shown to transactivate other receptors: noticeably, these interactions are not limited to members of the same family, but involve receptors as diverse as receptor kinases, prostanoid receptors, purinergic receptors and ionic channels among others. In this review, we will focus on the evidence for PAR interactions with members of their own family, as well as with other types of receptors. We will discuss recent evidence as well as what we consider as emerging areas to explore; from the signalling pathways triggered, to the physiological and pathological relevance of these interactions, since this additional level of molecular cross-talk between receptors and signaling pathways is only beginning to be explored and represents a novel mechanism providing diversity to receptor function and play important roles in physiology and disease.
Assuntos
Receptores Ativados por Proteinase , Transdução de Sinais , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.
Assuntos
Venenos de Crotalídeos/efeitos adversos , Edema/induzido quimicamente , Proteínas de Répteis/efeitos adversos , Serina Proteases/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Crotalus/metabolismo , Edema/metabolismo , Edema/patologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Camundongos , Modelos Moleculares , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores Ativados por Proteinase/metabolismo , Proteínas de Répteis/química , Proteínas de Répteis/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , Venenos de Serpentes , Fosfolipases Tipo C/metabolismoRESUMO
Proteinase-activated receptors 1 (PAR1) and 2 (PAR2) are the most highly expressed members of the PAR family in the periodontium. These receptors regulate periodontal inflammatory and repair processes through their activation by endogenous and bacterial enzymes. PAR1 is expressed by the periodontal cells such as human gingival fibroblasts, gingival epithelial cells, periodontal ligament cells, osteoblasts, and monocytic cells and can be activated by thrombin, matrix metalloproteinase 1 (MMP-1), MMP-13, fibrin, and gingipains from Porphyromonas gingivalis. PAR2 is expressed by neutrophils, osteoblasts, oral epithelial cells, and human gingival fibroblasts, and its possible activators in the periodontium are gingipains, neutrophil proteinase 3, and mast cell tryptase. The mechanisms through which PARs can respond to periodontal enzymes and result in appropriate immune responses have until recently been poorly understood. This review discusses recent findings that are beginning to identify a cardinal role for PAR1 and PAR2 on periodontal tissue metabolism.
Assuntos
Periodontite/metabolismo , Periodontite/fisiopatologia , Periodonto/metabolismo , Receptor PAR-1/metabolismo , Receptores Ativados por Proteinase/metabolismo , Adesinas Bacterianas/metabolismo , Animais , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Células Epiteliais , Fibroblastos , Regulação da Expressão Gênica , Cisteína Endopeptidases Gingipaínas , Gengiva/citologia , Gengiva/metabolismo , Humanos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Periodontite/genética , Porphyromonas gingivalis , Receptor PAR-1/agonistas , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/genética , Receptores Ativados por Proteinase/agonistas , Receptores Ativados por Proteinase/antagonistas & inibidores , Receptores Ativados por Proteinase/genéticaRESUMO
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.
Assuntos
Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Paracoccidioides , Receptores Ativados por Proteinase/metabolismo , Células A549 , Linhagem Celular , Sobrevivência Celular/imunologia , Endopeptidases/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Paracoccidioides/enzimologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacosRESUMO
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 kcat/KM 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 kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM 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-1, -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.
Assuntos
Venenos de Crotalídeos/química , Crotalus , Receptores Ativados por Proteinase/metabolismo , Serina Proteases/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Coagulantes/química , Citosol/metabolismo , Hidrólise , Masculino , Camundongos , Receptores Ativados por Proteinase/antagonistas & inibidores , Transdução de Sinais , América do Sul , Trombina/química , Tripsina/metabolismoRESUMO
Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.
Assuntos
Retinopatia Diabética/tratamento farmacológico , Inibidores de Serina Proteinase/uso terapêutico , alfa 1-Antitripsina/uso terapêutico , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Hipóxia Celular , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Retinopatia Diabética/fisiopatologia , Radicais Livres , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , NF-kappa B/metabolismo , Neutrófilos/fisiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Substâncias Protetoras/metabolismo , Receptores Ativados por Proteinase/metabolismo , Inibidores de Serina Proteinase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , alfa 1-Antitripsina/metabolismoRESUMO
Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.
Assuntos
Humanos , Animais , Inibidores de Serina Proteinase/uso terapêutico , alfa 1-Antitripsina/uso terapêutico , Retinopatia Diabética/tratamento farmacológico , Hipóxia Celular , Inibidores de Serina Proteinase/metabolismo , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , alfa 1-Antitripsina/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Óxido Nítrico Sintase/antagonistas & inibidores , Substâncias Protetoras/metabolismo , Receptores Ativados por Proteinase/metabolismo , Retinopatia Diabética/fisiopatologia , Radicais Livres , Inflamação/metabolismo , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Neutrófilos/fisiologiaRESUMO
Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.
Assuntos
Cisteína Proteases/química , Cisteína Proteases/metabolismo , Fibrinolíticos/química , Folhas de Planta/enzimologia , Agregação Plaquetária/efeitos dos fármacos , Contagem de Células , Cisteína Proteases/farmacologia , Dipeptídeos/química , Fibrinolíticos/metabolismo , Fibrinolíticos/farmacologia , Corantes Fluorescentes , Humanos , Cinética , Receptores Ativados por Proteinase/química , Receptores Ativados por Proteinase/metabolismo , Trombina/metabolismoRESUMO
OBJECTIVE AND DESIGN: Although proteinase-activated receptor (PAR)-4 has been implicated in inflammation, its role in regulating eosinophil recruitment in response to chemoattractants has not yet been demonstrated. To investigate the contribution of proteinases and PAR-4 activation to eosinophil migration in response to eotaxin-1 or leukotriene B(4) (LTB(4)), the effects of aprotinin or PAR-4 antagonist trans-cinnamoyl-YPGKF-NH(2) (tcY-NH(2)) on eosinophil migration induced by these chemoattractants were investigated. METHODS: BALB/c mice were pretreated with aprotinin or tcY-NH(2) (30 µg/mouse) prior to intrapleural injection of LTB(4) or eotaxin-1 and the number of infiltrating eosinophils was determined 48 h later. RESULTS: Aprotinin (1 mg/kg) inhibited eosinophil recruitment induced by eotaxin-1 (p < 0.01), but not that induced by LTB(4). Moreover, tcY-NH(2) treatment inhibited eosinophil recruitment in response to eotaxin-1 (p < 0.01 by ANOVA/Tukey post-test). CONCLUSION: These data suggest that aprotinin-inhibited proteinases participate in eosinophil migration induced by eotaxin-1 and that PAR-4 activation plays an important role in regulating this migration.