RESUMEN
Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the 'necrosome'), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.
Asunto(s)
Muerte Celular/genética , Complejos Multiproteicos/metabolismo , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Aminoácidos/metabolismo , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Ratones , Complejos Multiproteicos/genética , Necrosis/genética , Necrosis/metabolismo , Fosforilación , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
The death receptor ligands are involved in many physiological and pathological processes involving triggering of apoptosis, inflammation, proliferation, and activation. The expression of these molecules is reported to be tightly regulated at the transcriptional level. However, over the last few years, an increasing number of data demonstrated that the control of transcription is only one of the mechanisms that manage the expression of the death receptor ligands. Thus, this review is focused on posttranslational regulation of the three main members of this family, namely FasL, TNF-alpha, and TRAIL. We discuss here the importance of distribution, storage, and degranulation of these molecules, as well as their shedding by proteases on the control of death receptor ligands expression and activity.
Asunto(s)
Procesamiento Proteico-Postraduccional , Receptores de Muerte Celular/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Ligandos , Transporte de ProteínasRESUMEN
Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E(2) (PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE(2) in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.
Asunto(s)
Células Dendríticas/metabolismo , Dinoprostona/biosíntesis , Proteína Ligando Fas/genética , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Complejo CD3/metabolismo , Muerte Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoprotección/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.
Asunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/química , Metaloendopeptidasas/farmacología , Metaloproteasas/farmacología , Secuencia de Aminoácidos , Animales , Benchmarking , Factores de Coagulación Sanguínea , Células Cultivadas , Venenos de Crotálidos/farmacología , Células Endoteliales/efectos de los fármacos , Hemorragia/inducido químicamente , Humanos , Metaloendopeptidasas/química , Metaloproteasas/química , Ratones , Datos de Secuencia Molecular , Veneno de Bothrops JararacaAsunto(s)
Toxicología , Toxicología , Venenos , Intoxicación , Toxinas Biológicas , Toxicología , Bioquímica , Alergia e InmunologíaRESUMEN
Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40microg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-xL. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.