RESUMEN
Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.
Asunto(s)
Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Artritis Reumatoide/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Anoctaminas/metabolismo , Artritis Reumatoide/patología , Membrana Celular/metabolismo , Células HEK293 , Células HT29 , Humanos , Neoplasias/patología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Human papillomaviruses (HPV) are causative agents of various tumours such as cervical cancer. HPV binding to the cell surface of keratinocytes leads to virus endocytosis at tetraspanin enriched microdomains. Complex interactions of the capsid proteins with host proteins as well as ADAM17-dependent ERK1/2 signal transduction enable the entry platform assembly of the oncogenic HPV type 16. Here, we studied the importance of tetraspanin CD9, also known as TSPAN29, in HPV16 infection of different epithelial cells. We found that both overexpression and loss of the tetraspanin decreased infection rates in cells with low endogenous CD9 levels, while reduction of CD9 expression in keratinocytes that exhibit high-CD9 protein amounts, led to an increase of infection. Therefore, we concluded that low-CD9 supports infection. Moreover, we found that changes in CD9 amounts affect the shedding of the ADAM17 substrate transforming growth factor alpha (TGFα) and the downstream phosphorylation of ERK. These effects correlate with those on infection rates suggesting that a specific CD9 optimum promotes ADAM17 activity, ERK signalling and virus infection. Together, our findings implicate that CD9 regulates HPV16 infection through the modulation of ADAM17 sheddase activity.
Asunto(s)
Proteína ADAM17/metabolismo , Sistema de Señalización de MAP Quinasas , Infecciones por Papillomavirus/metabolismo , Tetraspanina 29/metabolismo , Proteína ADAM17/genética , Endocitosis , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HaCaT , Células HeLa , Papillomavirus Humano 16 , Humanos , Queratinocitos/virología , Infecciones por Papillomavirus/virología , Tetraspanina 29/genética , Factor de Crecimiento Transformador alfa/metabolismo , Internalización del VirusRESUMEN
BACKGROUND: ADAM10 and ADAM17 are the best characterized members of the ADAM (A Disintegrin and Metalloproteinase) - family of transmembrane proteases. Both are involved diverse physiological and pathophysiological processes. ADAMs are known to be regulated by posttranslational mechanisms. However, emerging evidence indicates that the plasma membrane with its unique dynamic properties may additionally play an important role in controlling sheddase function. SCOPE OF REVIEW: Membrane events that could contribute to regulation of ADAM-function are summarized. MAJOR CONCLUSIONS: Surface expression of peptidolytic activity should be differentiated from ADAM-sheddase function since the latter additionally requires that the protease finds its substrate in the lipid bilayer. We propose that this is achieved through horizontal and vertical reorganization of membrane nanoarchitecture coordinately occurring at the sites of sheddase activation. Reshuffling of nanodomains thereby guides traffic of enzyme and substrate to each other. For ADAM17 phosphatidylserine exposure is required to then induce its shedding function. GENERAL SIGNIFICANCE: The novel concept that physicochemical properties of the lipid bilayer govern the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.
Asunto(s)
Proteína ADAM10/genética , Proteína ADAM17/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Proteínas de la Membrana/genética , Proteolisis , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismoRESUMEN
KEY POINTS: TMEM16 proteins can operate as Ca2+ -activated Cl- channels or scramble membrane phospholipids, which are both highly relevant mechanisms during disease. Overexpression of TMEM16A and TMEM16F were found to be partially active at 37°C and at resting intracellular Ca2+ concentrations. We show that TMEM16 Cl- currents and phospholipid scrambling can be activated by modification of plasma membrane phospholipids, through reactive oxygen species and phospholipase A2. While phospholipids and Cl- ions are likely to use the same pore within TMEM16F, TMEM16A only conducts Cl- ions. Lipid regulation of TMEM16 proteins is highly relevant during inflammation and regulated cell death such as apoptosis and ferroptosis. ABSTRACT: TMEM16/anoctamin (ANO) proteins form Ca2+ -activated ion channels or phospholipid scramblases. We found that both TMEM16A/ANO1 and TMEM16F/ANO6 produced Cl- currents when activated by intracellular Ca2+ , but only TMEM16F was able to expose phosphatidylserine to the outer leaflet of the plasma membrane. Mutations within TMEM16F or TMEM16A/F chimeras similarly changed Cl- currents and phospholipid scrambling, suggesting the same intramolecular pathway for Cl- and phospholipids. When overexpressed, TMEM16A and TMEM16F produced spontaneous Cl- currents at 37°C even at resting intracellular Ca2+ levels, which was abolished by inhibition of phospholipase A2 (PLA2 ). Connversely, activation of PLA2 or application of active PLA2 , as well as lipid peroxidation induced by reactive oxygen species (ROS) using staurosporine or tert-butyl hydroperoxide, enhanced ion currents by TMEM16A/F and in addition activated phospholipid scrambling by TMEM16F. Thus, TMEM16 proteins are activated by an increase in intracellular Ca2+ , or independent of intracellular Ca2+ , by modifications occurring in plasma and intracellular membrane phospholipids. These results may help to explain why regions distant to the TMEM16 pore and the Ca2+ binding sites control Cl- currents and phospholipid scrambling. Regulation of TMEM16 proteins through modification of membrane phospholipids occurs during regulated cell death such as apoptosis and ferroptosis. It contributes to inflammatory and nerve injury-induced hypersensitivity and generation of pain and therefore provides a regulatory mechanism that is particularly relevant during disease.
Asunto(s)
Anoctamina-1/metabolismo , Anoctaminas/metabolismo , Calcio/farmacología , Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Anoctamina-1/genética , Anoctaminas/genética , Apoptosis , Hormonas y Agentes Reguladores de Calcio/farmacología , Células HEK293 , Humanos , Transporte Iónico , Proteínas de Neoplasias/genética , Fosfolipasas A2/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Transducción de SeñalRESUMEN
BACKGROUND: ADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors. METHODS: We investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry. RESULTS: In CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation. CONCLUSIONS: We identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.
Asunto(s)
Proteínas ADAMTS/genética , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Metilación de ADN , Neoplasias Pulmonares/genética , Neoplasias de la Boca/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Proteínas ADAMTS/metabolismo , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Islas de CpG , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Células HT29 , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Neoplasias de la Boca/enzimología , Neoplasias de la Boca/patología , Regiones Promotoras Genéticas , Carcinoma de Células Escamosas de Cabeza y Cuello/enzimología , Carcinoma de Células Escamosas de Cabeza y Cuello/patologíaRESUMEN
ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10â nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20â nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Membrana Celular/enzimología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Animales , Células COS , Catálisis , Membrana Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , RatonesRESUMEN
The membrane-anchored metalloproteinase a disintegrin and metalloprotease 10 (ADAM10) is required for shedding of membrane proteins such as EGF, betacellulin, the amyloid precursor protein, and CD23 from cells. ADAM10 is constitutively active and can be rapidly and post-translationally enhanced by several stimuli, yet little is known about the underlying mechanism. Here, we use ADAM10-deficient cells transfected with wild type or mutant ADAM10 to address the role of its cytoplasmic and transmembrane domain in regulating ADAM10-dependent protein ectodomain shedding. We report that the cytoplasmic domain of ADAM10 negatively regulates its constitutive activity through an ER retention motif but is dispensable for its stimulated activity. However, chimeras with the extracellular domain of ADAM10 and the transmembrane domain of ADAM17 with or without the cytoplasmic domain of ADAM17 show reduced stimulated shedding of the ADAM10 substrate betacellulin, whereas the ionomycin-stimulated shedding of the ADAM17 substrates CD62-L and TGFα is not affected. Moreover, we show that influx of extracellular calcium activates ADAM10 but is not essential for its activation by APMA and BzATP. Finally, the rapid stimulation of ADAM10 is not significantly affected by incubation with proprotein convertase inhibitors for up to 8 h, arguing against a major role of increased prodomain removal in the rapid stimulation of ADAM10. Thus, the cytoplasmic domain of ADAM10 negatively influences constitutive shedding through an ER retention motif, whereas the cytoplasmic domain and prodomain processing are not required for the rapid activation of ADAM10-dependent shedding events.
Asunto(s)
Proteínas ADAM/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Citoplasma/enzimología , Proteínas de la Membrana/fisiología , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM10 , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteolisis , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
A wide variety of biological processes including differentiation, regeneration, and cancer progression are regulated by shedding of membrane-anchored proteins. One of the major sheddases is A Disintegrin And Metalloprotease-17 (ADAM17) whose extracellular region consists of a pro-, a catalytic, a disintegrin-, and a membrane-proximal domain (MPD) as well as a short juxtamembrane segment of 17 amino acid residues that has been named "Conserved ADAM-seventeeN Dynamic Interaction Sequence" (CANDIS). This segment is involved in substrate recognition. Key mediators of inflammation including interleukin-6 receptor (IL-6R) and tumor necrosis factor (TNF-α) are substrates of ADAM17. The shedding activity of ADAM17 is regulated by the conformation of the membrane-proximal domain preceding the CANDIS segment. Here, we show that CANDIS, besides being involved in substrate recognition, is able to interact with lipid bilayers in vitro and that this property could be involved in regulating ADAM17 shedding activity.
Asunto(s)
Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas ADAM/análisis , Proteínas ADAM/genética , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Línea Celular , Células Hep G2 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Datos de Secuencia Molecular , Mutación , Dominios y Motivos de Interacción de Proteínas , Especificidad por SustratoRESUMEN
BACKGROUND: Growing evidence suggests that disintegrin and metalloprotease (ADAM) 17 (ADAM17) and ADAM10 contribute to the pathogenesis of vascular diseases. ADAM17 promotes inflammatory processes by liberating tumor necrosis factor α, interleukin 6 receptor (IL-6R), and tumor necrosis factor receptor 1 (TNFR1). ADAM17 and ADAM10 modulate vascular permeability by cleaving endothelial adhesion molecules such as junctional adhesion molecule A (JAM-A) and vascular endothelial cadherin (VE-cadherin), respectively. OBJECTIVE: This study was designed to investigate whether a link might exist between the protective effects of fish oil (FO) supplementation against atherosclerosis and ADAM function. METHODS: Male LDL receptor knockout (LDLR(-/-)) mice and male wild-type (WT) mice were fed a Western diet (200 g/kg fat, 1.5 g/kg cholesterol) containing either 20% lard (LDLR(-/-)-lard and WT-lard groups) or 10% lard combined with 10% FO (LDLR(-/-)-FO and WT-FO groups) for 12 wk. Atherosclerotic lesion development and fatty acid composition of liver microsomes were evaluated. ADAM10 and ADAM17 expression was determined by quantitative real-time polymerase chain reaction and immunoblot analyses. Concentrations of soluble ADAM substrates in plasma and liver extracts were measured by ELISA. RESULTS: Diets supplemented with FO markedly reduced development of early atherosclerotic lesions in LDLR(-/-) mice (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 29.6 ± 6.1% vs. 22.5 ± 4.2%, P < 0.05). This was not accompanied by changes in expression of ADAM17 or ADAM10 in the aorta or liver. No dietary effects on circulating TNFR1 (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 1.22 ± 0.23 vs. 1.39 ± 0.28, P > 0.2) or IL-6R (1.06 ± 0.12 vs. 0.98 ± 0.09 fold of WT-lard group, P > 0.1), classical substrates of ADAM17 on macrophages, and neutrophil granulocytes were observed. However, a reduction in atherosclerotic lesions in the LDLR(-/-)-FO group was accompanied by a significant reduction in the circulating endothelial cell adhesion molecules JAM-A (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 1.42 ± 0.20 vs. 0.95 ± 0.56 fold of WT-lard group, P < 0.05), intercellular adhesion molecule 1 (1.15 ± 0.14 vs. 0.88 ± 0.17 fold of WT-lard group, P < 0.05), and VE-cadherin (0.88 ± 0.12 vs. 0.72 ± 0.15 fold of WT-lard group, P < 0.05), reflecting reduced ADAM activity in endothelial cells. CONCLUSION: FO exerted an antiatherogenic effect on male LDLR(-/-) mice that was accompanied by a reduced release of ADAM17 and ADAM10 substrates from endothelial cells. It is suggested that FO-decreased ADAM activity contributes to improved endothelial barrier function and thus counteracts intimal lipoprotein insudation and macrophage accumulation.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/prevención & control , Suplementos Dietéticos , Aceites de Pescado/farmacología , Proteínas de la Membrana/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Colesterol en la Dieta/administración & dosificación , Colesterol en la Dieta/efectos adversos , Dieta Occidental/efectos adversos , Grasas de la Dieta/administración & dosificación , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
Severe Plasmodium falciparum malaria evolves through the interplay among capillary sequestration of parasitized erythrocytes, deregulated inflammatory responses, and hemostasis dysfunction. After rupture, each parasitized erythrocyte releases not only infective merozoites, but also the digestive vacuole (DV), a membrane-bounded organelle containing the malaria pigment hemozoin. In the present study, we report that the intact organelle, but not isolated hemozoin, dually activates the alternative complement and the intrinsic clotting pathway. Procoagulant activity is destroyed by phospholipase C treatment, indicating a critical role of phospholipid head groups exposed at the DV surface. Intravenous injection of DVs caused alternative pathway complement consumption and provoked apathy and reduced nociceptive responses in rats. Ultrasonication destroyed complement-activating and procoagulant properties in vitro and rendered the DVs biologically inactive in vivo. Low-molecular-weight dextran sulfate blocked activation of both complement and coagulation and protected animals from the harmful effects of DV infusion. We surmise that in chronic malaria, complement activation by and opsonization of the DV may serve a useful function in directing hemozoin to phagocytic cells for safe disposal. However, when the waste disposal system of the host is overburdened, DVs may transform into a trigger of pathology and therefore represent a potential therapeutic target in severe malaria.
Asunto(s)
Coagulación Sanguínea/fisiología , Vía Alternativa del Complemento/fisiología , Eritrocitos/parasitología , Plasmodium falciparum/fisiología , Vacuolas/fisiología , Animales , Coagulación Sanguínea/efectos de los fármacos , Vía Alternativa del Complemento/efectos de los fármacos , Sulfato de Dextran/farmacología , Hemoproteínas/fisiología , Hemólisis , Humanos , Hipoestesia/etiología , Membranas Intracelulares/fisiología , Pulmón/parasitología , Malaria Falciparum/sangre , Malaria Falciparum/complicaciones , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Masculino , Monocitos/parasitología , Umbral del Dolor , Fagocitosis , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/ultraestructura , Ratas , Ratas Sprague-Dawley , Bazo/parasitologíaRESUMEN
The digestive vacuole (DV) of Plasmodium falciparum, which is released into the bloodstream upon rupture of each parasitized red blood cell (RBC), was recently discovered to activate the alternative complement pathway. In the present work, we show that C3- and C5-convertases assembling on the parasitic organelle are able to provoke deposition of activated C3 and C5b-9 on non-infected bystander erythrocytes. Direct contact of DVs with cells is mandatory for the effect, and bystander complement deposition occurs focally, possibly at the sites of contact. Complement opsonization promotes protracted erythrophagocytosis by human macrophages, an effect that is magnified when ring-stage infected RBCs with reduced CD55 and CD59, or paroxysmal nocturnal hemoglobinuria (PNH)-RBCs lacking these complement inhibitors are employed as targets. Bystander attack can also directly induce lysis of PNH-RBCs. Direct evidence for complement activation and bystander attack mediated by DVs was obtained through immunohistochemical analyses of brain paraffin sections from autopsies of patients who had died of cerebral malaria. C3d and the assembled C5b-9 complex could be detected in all sections, colocalizing with and often extending locally beyond massive accumulations of DVs that were identified under polarized light. This is the first demonstration that a complement-activating particle can mediate opsonization of bystander cells to promote their antibody-independent phagocytosis. The phenomenon may act in concert with other pathomechanisms to promote the development of anemia in patients with severe malaria.
Asunto(s)
Efecto Espectador , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Eritrocitos/inmunología , Fagocitosis , Plasmodium falciparum/inmunología , Vacuolas/inmunología , Encéfalo/patología , Eritrocitos/patología , Humanos , InmunohistoquímicaRESUMEN
Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent.
Asunto(s)
Proteínas ADAM/metabolismo , Queratinocitos/efectos de los fármacos , Meliteno/farmacología , Receptores Purinérgicos P2X7/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Adenosina Trifosfato/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Western Blotting , Cadherinas/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/citología , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Fosforilación/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Sequestration of parasitized erythrocytes and dysregulation of the coagulation and complement system are hallmarks of severe Plasmodium falciparum malaria. A link between these events emerged through the discovery that the parasite digestive vacuole (DV), which is released together with infective merozoites into the bloodstream, dually activates the intrinsic clotting and alternative complement pathway. Complement attack occurs exclusively on the membrane of the DVs, and the question followed whether DVs might be marked for uptake by polymorphonuclear granulocytes (PMNs). We report that DVs are indeed rapidly phagocytosed by PMNs after schizont rupture in active human serum. Uptake of malaria pigment requires an intact DV membrane and does not occur when the pigment is extracted from the organelle. Merozoites are not opsonized and escape phagocytosis in nonimmune serum. Antimalarial Abs mediate some uptake of the parasites, but to an extent that is not sufficient to markedly reduce reinvasion rates. Phagocytosis of DVs induces a vigorous respiratory burst that drives the cells into a state of functional exhaustion, blunting the production of reactive oxygen species (ROS) and microbicidal activity upon challenge with bacterial pathogens. Systemic overloading of PMNs with DVs may contribute to the enhanced susceptibility of patients with severe malaria toward invasive bacterial infections.
Asunto(s)
Neutrófilos/parasitología , Fagocitosis/fisiología , Plasmodium falciparum/patogenicidad , Vacuolas/fisiología , Animales , Recuento de Células Sanguíneas , Muerte Celular/inmunología , Eritrocitos/parasitología , Eritrocitos/patología , Humanos , Malaria Falciparum/sangre , Malaria Falciparum/inmunología , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Merozoítos/inmunología , Merozoítos/metabolismo , Merozoítos/patología , Merozoítos/fisiología , Modelos Biológicos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/fisiología , Fagocitosis/inmunología , Plasmodium falciparum/inmunología , Plasmodium falciparum/metabolismo , Plasmodium falciparum/ultraestructura , Especies Reactivas de Oxígeno/metabolismo , Especificidad por Sustrato , Factores de Tiempo , Vacuolas/metabolismo , Vacuolas/parasitologíaRESUMEN
ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through the cleavage of transmembrane substrates, including epidermal growth factor receptor (EGFR) ligands such as transforming growth factor (TGF)-alpha and Epiregulin (EREG). Several ADAM17 substrates are relevant to oncogenesis and tumor growth. We have presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. The scramblase Xkr8 is instrumental for calcium-independent exposure of PS in apoptotic cells. Xkr8 can be dually activated by caspase-3 and by kinases. In this investigation, we examined whether Xkr8 would modulate ADAM17 activity under apoptotic and non-apoptotic conditions. Overexpression of Xkr8 in HEK293T cells led to significantly increased caspase-dependent as well as PMA-induced release of EREG and TGF-alpha. Conversely, siRNA-mediated downregulation of Xkr8 in colorectal Caco-2 cancer cells led to decreased PS externalization upon induction of apoptosis, which was accompanied by reduced shedding of endogenously expressed EREG and reduced cell survival. We conclude that Xkr8 shares with conventional scramblases the propensity to upmodulate the ADAM-sheddase function. Liberation of growth factors could serve a rescue function in cells on the pathway to apoptotic death.
RESUMEN
The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine changes in enzyme activity, but correlate with changes in membrane fluidity as revealed by measurement of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and fluorescence recovery after photobleaching analyses. ELISA and immunoblot experiments conducted with granulocytes, endothelial cells, and keratinocytes revealed rapid increase of ectodomain shedding of ADAM10 and ADAM17 substrates upon membrane fluidization. Large amounts of unsaturated FFA may be liberated from cholesteryl esters in LDL that is entrapped in atherosclerotic lesions. Incubation of cells with thus modified LDL resulted in rapid cleavage of ADAM substrates with corresponding functional consequences on cell proliferation, cell migration, and endothelial permeability, events of high significance in atherogenesis. We propose that FFA represent critical regulators of ADAM function that may assume relevance in many biological settings through their influence on mobility of enzyme and substrate in lipid bilayers.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Movimiento Celular , Proliferación Celular , Ácidos Grasos Insaturados/metabolismo , Fluidez de la Membrana , Proteínas de la Membrana/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Aterosclerosis/genética , Aterosclerosis/mortalidad , Permeabilidad Capilar/genética , Adhesión Celular , Ésteres del Colesterol/genética , Ésteres del Colesterol/metabolismo , Células Endoteliales/metabolismo , Granulocitos/metabolismo , Células HEK293 , Humanos , Queratinocitos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Proteínas de la Membrana/genética , ConejosRESUMEN
Protein ectodomain shedding is crucial for cell-cell interactions because it controls the bioavailability of soluble tumor necrosis factor-α (TNFα) and ligands of the epidermal growth factor (EGF) receptor, and the release of many other membrane proteins. Various stimuli can rapidly trigger ectodomain shedding, yet much remains to be learned about the identity of the enzymes that respond to these stimuli and the mechanisms underlying their activation. Here, we demonstrate that the membrane-anchored metalloproteinase ADAM17, but not ADAM10, is the sheddase that rapidly responds to the physiological signaling pathways stimulated by thrombin, EGF, lysophosphatidic acid and TNFα. Stimulation of ADAM17 is swift and quickly reversible, and does not depend on removal of its inhibitory pro-domain by pro-protein convertases, or on dissociation of an endogenous inhibitor, TIMP3. Moreover, activation of ADAM17 by physiological stimuli requires its transmembrane domain, but not its cytoplasmic domain, arguing against inside-out signaling via cytoplasmic phosphorylation as the underlying mechanism. Finally, experiments with the tight binding hydroxamate inhibitor DPC333, used here to probe the accessibility of the active site of ADAM17, demonstrate that this inhibitor can quickly bind to ADAM17 in stimulated, but not quiescent cells. These findings support the concept that activation of ADAM17 involves a rapid and reversible exposure of its catalytic site.
Asunto(s)
Proteínas ADAM/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Proteína ADAM17 , Animales , Células COS , Dominio Catalítico , Células Cultivadas , Chlorocebus aethiops , Regulación hacia Abajo , Humanos , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Ratones , Fosforilación , Transducción de Señal , TransfecciónRESUMEN
Membrane-perturbating proteins and peptides are widespread agents in biology. Pore-forming bacterial toxins represent major virulence factors of pathogenic microorganisms. Membrane-damaging peptides constitute important antimicrobial effectors of innate immunity. Membrane perturbation can incur multiple responses in mammalian cells. The present discussion will focus on the interplay between membrane-damaging agents and the function of cell-bound metalloproteinases of the ADAM family. These transmembrane enzymes have emerged as the major proteinase family that mediate the proteolytic release of membrane-associated proteins, a process designated as "shedding". They liberate a large spectrum of functionally active molecules including inflammatory cytokines, growth factor receptors and cell adhesion molecules, thereby regulating such vital cellular functions as cell-cell adhesion, cell proliferation and cell migration. ADAM activation may constitute part of the cellular recovery machinery on the one hand, but likely also promotes inflammatory processes on the other. The mechanisms underlying ADAM activation and the functional consequences thereof are currently the subject of intensive research. Attention here is drawn to the possible involvement of purinergic receptors and ceramide generation in the context of ADAM activation following membrane perturbation by membrane-active agents.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Toxinas Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Adhesión Celular , Movimiento Celular , Proliferación CelularRESUMEN
Proteolytic ectodomain release is a key mechanism for regulating the function of many cell surface proteins. The sheddases ADAM10 and ADAM17 are the best-characterized members of the family of transmembrane disintegrin-like metalloproteinase. Constitutive proteolytic activities are low but can be abruptly upregulated via inside-out signaling triggered by diverse activating events. Emerging evidence indicates that the plasma membrane itself must be assigned a dominant role in upregulation of sheddase function. Data are discussed that tentatively identify phospholipid scramblases as central players during these events. We propose that scramblase-dependent externalization of the negatively charged phospholipid phosphatidylserine (PS) plays an important role in the final activation step of ADAM10 and ADAM17. In this manuscript, we summarize the current knowledge on the interplay of cell membrane changes, PS exposure, and proteolytic activity of transmembrane proteases as well as the potential consequences in the context of immune response, infection, and cancer. The novel concept that scramblases regulate the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface.
RESUMEN
Ca2+-activated Cl- channels (TMEM16, also known as anoctamins) perform important functions in cell physiology, including modulation of cell proliferation and cancer growth. Many members, including TMEM16F/ANO6, additionally act as Ca2+-activated phospholipid scramblases. We recently presented evidence that ANO6-dependent surface exposure of phosphatidylserine (PS) is pivotal for the disintegrin-like metalloproteases ADAM10 and ADAM17 to exert their sheddase function. Here, we compared the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, and 10) on ADAM sheddase activity. Similar to ANO6, overexpression of ANO4 and ANO9 led to increased release of ADAM10 and ADAM17 substrates, such as betacellulin, TGFα, and amphiregulin (AREG), upon ionophore stimulation in HEK cells. Inhibitor experiments indicated that ANO4/ANO9-mediated enhancement of TGFα-cleavage broadened the spectrum of participating metalloproteinases. Annexin V-staining demonstrated increased externalisation of PS in ANO4/ANO9-overexpressing cells. Competition experiments with the soluble PS-headgroup phosphorylserine indicated that the ANO4/ANO9 effects were due to increased PS exposure. Overexpression of ANO4 or ANO9 in human cervical cancer cells (HeLa), enhanced constitutive shedding of the growth factor AREG and increased cell proliferation. We conclude that ANO4 and ANO9, by virtue of their scramblase activity, may play a role as important regulators of ADAM-dependent cellular functions.
RESUMEN
Despite partial sequence identity and structural similarity, human ß-defensin 3 (HBD3) kills Staphylococcus aureus with a 4- to 8-fold higher efficiency than human ß-defensin 2 (HBD2), whereas the activities against Escherichia coli are identical. The design and characterization of HBD2/HBD3 chimeric peptides revealed that distinct molecular regions are responsible for their divergent killing properties. Two of the chimeras killed both E. coli and S. aureus with an even higher efficacy than the wild-type molecules. Moreover, one of these two chimeras maintained its high killing activities in the presence of physiologic salt concentrations. Due to the broad spectrum of their antimicrobial activities against many human multidrug-resistant pathogens, these two designer peptides of human origin represent promising templates for a new class of antibiotics.