RESUMO
Niemann-Pick disease, type C1 (NPC1) is an inborn error of metabolism that results in endolysosomal accumulation of unesterified cholesterol. Clinically, NPC1 manifests as cholestatic liver disease in the newborn or as a progressive neurogenerative condition characterized by cerebellar ataxia and cognitive decline. Currently there are no FDA approved therapies for NPC1. Thus, understanding the pathological processes that contribute to neurodegeneration will be important in both developing and testing potential therapeutic interventions. Neuroinflammation and necroptosis contribute to the NPC1 pathological cascade. Receptor Interacting Protein Kinase 1 and 3 (RIPK1 and RIPK3), are protein kinases that play a central role in mediating neuronal necroptosis. Our prior work suggested that pharmacological inhibition of RIPK1 had a significant but modest beneficial effect; however, the inhibitors used in that study had suboptimal pharmacokinetic properties. In this work we evaluated both pharmacological and genetic inhibition of RIPK1 kinase activity. Lifespan in both Npc1-/- mice treated with GSK'547, a RIPK1 inhibitor with better pharmacokinetic properties, and Npc1-/-:Ripk1kd/kd double mutant mice was significantly increased. In both cases the increase in lifespan was modest, suggesting that the therapeutic potential of RIPK1 inhibition, as a monotherapy, is limited. We thus investigated the potential of combining RIPK1 inhibition with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) therapy HPßCD has been shown to slow neurological disease progression in NPC1 mice, cats and patients. HPßCD appeared to have an additive positive effect on the pathology and survival of Npc1-/-:Ripk1kd/kd mice. RIPK1 and RIPK3 are both critical components of the necrosome, thus we were surprised to observe no increase survival in Npc1-/-;Ripk3-/- mice compared to Npc1-/- mice. These data suggest that although necroptosis is occurring in NPC1, the observed effects of RIPK1 inhibition may be related to its RIPK3-independent role in neuroinflammation and cytokine production.
Assuntos
2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Apoptose , Necrose , Doença de Niemann-Pick Tipo C/terapia , Proteínas/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Terapia Combinada , Modelos Animais de Doenças , Excipientes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidoresRESUMO
Receptor interacting protein kinase 1 (RIPK1) participates in several cell signaling complexes that promote cell activation and cell death. Stimulation of RIPK1 in the absence of caspase signaling induces regulated necrosis (necroptosis), which promotes an inflammatory response. Understanding of the mechanisms through which RIPK1 promotes inflammation has been unclear. Herein we have evaluated the impact of a K45A mutation of RIPK1 on necroptosis of macrophages and the activation of inflammatory response. We show that K45A mutation of RIPK1 results in attenuated necroptosis of macrophages in response to stimulation with LPS, TNFα and IFNß in the absence of caspase signaling. Impairment in necroptosis correlated with poor phosphorylation of RIPK1, RIPK3 and reduced trimerization of MLKL. Furthermore, K45A mutation of RIPK1 resulted in poor STAT1 phosphorylation (at S727) and expression of RANTES and MIP-1α following TNF-R engagement in the absence of caspase activation. Our results further indicate that in the absence of stimulation by pathogen-associated molecular patterns (PAMPs), cellular inhibitors of apoptotic proteins (cIAPs) prevent the K45-dependent auto-phosphorylation of RIPK1, leading to resistance against necroptosis. Finally, RIPK1(K45A) mice displayed attenuated inflammatory response in vivo as they were significantly resistant against endotoxin shock, but highly susceptible against a challenge with Salmonella typhimurium. This correlated with reduced expression of IL-1ß and ROS, and poor processing of caspase 8 by RIPK1(K45A) macrophages. Overall, these results indicate that K45 mediated kinase activity of RIPK1 is not only important for necroptosis but it also has a key role in promoting cytokine signaling and host response to inflammatory stimuli.
Assuntos
Apoptose/genética , Citocinas/metabolismo , Inflamação/patologia , Lisina/genética , Macrófagos/enzimologia , Mutação/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Endotoxinas , Inflamação/enzimologia , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/patologia , Camundongos , Necrose , Fosforilação , Proteínas Quinases/metabolismo , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Transcrição STAT1/metabolismo , Salmonella typhimurium/fisiologia , Choque Séptico/patologia , Transdução de SinaisRESUMO
Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD(+)) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD(+) promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD(+)-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD(+) does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.
Assuntos
NAD/metabolismo , Necrose/genética , Sirtuína 2/genética , Sirtuínas/genética , Apoptose/genética , Linhagem Celular , Citoplasma/metabolismo , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Humanos , Ligantes , NAD/genética , Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Sirtuína 2/metabolismo , Sirtuínas/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms. In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma. Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors. Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3. Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion. Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling. Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present. Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis. Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.
Assuntos
Melanoma/genética , Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Apoptose , Humanos , Necrose/enzimologiaRESUMO
Apoptosis is a key mechanism for metazoans to eliminate unwanted cells. Resistance to apoptosis is a hallmark of many cancer cells and a major roadblock to traditional chemotherapy. Recent evidence indicates that inhibition of caspase-dependent apoptosis sensitizes many cancer cells to a form of non-apoptotic cell death termed necroptosis. This has led to widespread interest in exploring necroptosis as an alternative strategy for anti-cancer therapy. Here we show that in human colon cancer tissues, the expression of the essential necroptosis adaptors receptor interacting protein kinase (RIPK)1 and RIPK3 is significantly decreased compared with adjacent normal colon tissues. The expression of RIPK1 and RIPK3 was suppressed by hypoxia, but not by epigenetic DNA modification. To explore the role of necroptosis in chemotherapy-induced cell death, we used inhibitors of RIPK1 or RIPK3 kinase activity, and modulated their expression in colon cancer cell lines using short hairpin RNAs. We found that RIPK1 and RIPK3 were largely dispensable for classical chemotherapy-induced cell death. Caspase inhibitor and/or second mitochondria-derived activator of caspase mimetic, which sensitize cells to RIPK1- and RIPK3-dependent necroptosis downstream of tumor necrosis factor receptor-like death receptors, also did not alter the response of cancer cells to chemotherapeutic agents. In contrast to the RIPKs, we found that cathepsins are partially responsible for doxorubicin or etoposide-induced cell death. Taken together, these results indicate that traditional chemotherapeutic agents are not efficient inducers of necroptosis and that more potent pathway-specific drugs are required to fully harness the power of necroptosis in anti-cancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Necrose/induzido quimicamente , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Catepsinas/metabolismo , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Células HCT116 , Células HT29 , Humanos , Técnicas In Vitro , Células MCF-7 , Fenilalanina/análogos & derivados , Piperazinas , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Compostos de Tosil , Compostos de Vinila/farmacologiaRESUMO
Necroptosis and signaling regulated by RIP1 kinase activity is emerging as a key driver of inflammation in a variety of disease settings. A significant amount has been learned about how RIP1 regulates necrotic cell death through the use of the RIP1 kinase inhibitor Necrostatin-1 (Nec-1). Nec-1 has been a transformational tool for exploring the function of RIP1 kinase activity; however, its utility is somewhat limited by moderate potency, off-target activity against indoleamine-2,3-dioxygenase (IDO), and poor pharmacokinetic properties. These limitations of Nec-1 have driven an effort to identify next-generation tools to study RIP1 function, and have led to the identification of 7-Cl-O-Nec-1 (Nec-1s), which has improved pharmacokinetic properties and lacks IDO inhibitory activity. Here we describe the characterization of GSK'963, a chiral small-molecule inhibitor of RIP1 kinase that is chemically distinct from both Nec-1 and Nec-1s. GSK'963 is significantly more potent than Nec-1 in both biochemical and cellular assays, inhibiting RIP1-dependent cell death with an IC50 of between 1 and 4 nM in human and murine cells. GSK'963 is >10 000-fold selective for RIP1 over 339 other kinases, lacks measurable activity against IDO and has an inactive enantiomer, GSK'962, which can be used to confirm on-target effects. The increased in vitro potency of GSK'963 also translates in vivo, where GSK'963 provides much greater protection from hypothermia at matched doses to Nec-1, in a model of TNF-induced sterile shock. Together, we believe GSK'963 represents a next-generation tool for examining the function of RIP1 in vitro and in vivo, and should help to clarify our current understanding of the role of RIP1 in contributing to disease pathogenesis.
RESUMO
An increasing incidence of enteric disorders clinically suggestive of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time reverse transcriptase-polymerase chain reaction assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37% of the intestinal samples collected from diseased turkey flocks. The full-length spike (S) gene of these viruses was amplified, cloned and sequenced from three samples. The French S sequences shared 98% identity at both the nucleotide and amino acid levels, whereas they were at most 65% and 60% identical with North American (NA) TCoV and at most 50% and 37% identical with IBV at the nucleotide and amino acid levels, respectively. Higher divergence with NA TCoV was observed in the S1-encoding domain. Phylogenetic analysis based on the S gene revealed that the newly detected viruses form a sublineage genetically related with, but significantly different from, NA TCoV. Additionally, the RNA-dependent RNA polymerase gene and the N gene, located on the 5' and 3' sides of the S gene in the coronavirus genome, were partially sequenced. Phylogenetic analysis revealed that both the NA TCoV and French TCoV (Fr TCoV) lineages included some IBV relatives, which were however different in the two lineages. This suggested that different recombination events could have played a role in the evolution of the NA and Fr TCoV. The present results provide the first S sequence for a European TCoV. They reveal extensive genetic variation in TCoV and suggest different evolutionary pathways in North America and Europe.
Assuntos
Coronavirus do Peru/genética , Enterite Transmissível dos Perus/virologia , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Coronavirus do Peru/isolamento & purificação , Coronavirus do Peru/patogenicidade , Enterite Transmissível dos Perus/epidemiologia , França/epidemiologia , Variação Genética , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Filogenia , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Sensibilidade e Especificidade , Alinhamento de Sequência , Análise de Sequência de DNA , Glicoproteína da Espícula de Coronavírus , Perus , Proteínas do Envelope Viral/química , Proteínas Virais/genéticaRESUMO
Light availability and infestation by the green spruce aphid (Elatobium abietinum) are key factors affecting the growth of Sitka spruce (Picea sitchensis) seedlings under a mature tree canopy, but their combined effect on seedling growth has not previously been quantified. A controlled outdoor experiment in which light levels (high light (HL): 100%, intermediate light (IL): 24%) and aphid infestation (absence/presence) were manipulated was conducted over 2 years to look at the effects on seedling growth and biomass distribution patterns. Aphid population assessments showed a significantly increased population density under IL, with three to four times higher cumulative aphid densities than that under HL. Defoliation rates of infested seedlings were directly related to aphid density. Total seedling biomass was strongly reduced in IL, and aphid infestation caused additional reductions in the biomass of particular components of the seedlings. Dry weight (DW) of older (≥1-year-old) needles in infested trees was significantly decreased in both years. Total root DW at the end of the second year was significantly affected by aphid infestation, and the reduction (14-18%) was similar in IL and HL treatments despite large differences in aphid density. Biomass distribution patterns in infested trees were similar to that of uninfested trees within each light treatment, indicating that the relative decreases in root biomass were accompanied by similar reductions in distribution to the above-ground parts of the seedlings. Leader extension growth of infested seedlings was reduced by 15-17% compared with uninfested seedlings under IL, whereas only a 2-3% reduction in leader extension of infested seedlings under HL was observed. The results showed that the response of seedlings to E. abietinum were primarily dependent on the light environment. The significant reduction caused by aphids on the total DW of older needles and roots, and on leader extension growth, does suggest the potential for effects to accumulate over time.
Assuntos
Afídeos/crescimento & desenvolvimento , Ecossistema , Picea/parasitologia , Plântula/parasitologia , Animais , Afídeos/efeitos da radiação , Biomassa , Interações Hospedeiro-Parasita , Luz , Picea/crescimento & desenvolvimento , Picea/efeitos da radiação , Folhas de Planta/parasitologia , Raízes de Plantas/parasitologia , Densidade Demográfica , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiação , Fatores de TempoRESUMO
Mass spectral fingerprints of detergents in Austrian, Hungarian, Uruguayan, and Chilean gasolines have been obtained using electrospray ionization mass spectrometry. Polymers or copolymers were observed based on ether motifs (inter-peak spacings of 44, 58 and 72 u) in all samples. Austrian gasoline was found to also contain polymers based on isobutene-amine.
RESUMO
In dairy goats and ewes the use of equine Chorionic Gonadotropin (eCG) as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination (AI). Treatment for induction and synchronization of ovulation consists of a progestagen delivered by vaginal sponge, followed by an eCG injection. In some females, the first injection of eCG induces a humoral response with high concentrations of anti-eCG antibodies in contrast to other females displaying a very low concentration of anti-eCG antibodies. Females eliciting a low response were also poor responders after the following treatments. Conversely, high responders at the first treatment systematically yielded high immune responses upon the following treatment. By a molecular genetic approach using microsatellites we showed that the anti-eCG immune response phenotypes were associated with MHC class II polymorphism. Females with high residual antibody concentrations at the time of eCG injection exhibited a much lower kidding rate than other females did. Lower fertility of these females, inseminated at a fixed time after eCG treatment (43H for goats and 55H for ewes), might be due to the delay in estrus occurrence and the pre ovulatory LH surge. Consequently, under field conditions old females selected for AI are only those with low residual anti-eCG antibody concentrations and old females with high residual antibody concentration are culled from AI breeding because of their low fertility during the previous year. So we have undertaken comparative studies to establish if the anti-eCG immune response is correlated with the global immunity in animals.
Assuntos
Gonadotropina Coriônica/imunologia , Cabras/imunologia , Indução da Ovulação/veterinária , Ovinos/imunologia , Animais , Anticorpos/sangue , Formação de Anticorpos , Cruzamento , Feminino , Cabras/genética , Antígenos de Histocompatibilidade Classe II/genética , Cavalos , Imunidade/genética , Inseminação Artificial/veterinária , Indução da Ovulação/métodos , Polimorfismo Genético , Ovinos/genéticaRESUMO
In addition to promoting cell survival, neurotrophins also can elicit apoptosis in restricted cell types. Recent results indicate that nerve growth factor (NGF) can induce Schwann cell death via engagement of the p75 neurotrophin receptor. Here we describe a novel interaction between the p75 receptor and receptor-interacting protein 2, RIP2 (RICK/CARDIAK), that accounts for the ability of neurotrophins to choose between a survival-versus-death pathway. RIP2, an adaptor protein with a serine threonine kinase and a caspase recruitment domain (CARD), is highly expressed in dissociated Schwann cells and displays an endogenous association with p75. RIP2 binds to the death domain of p75 via its CARD domain in an NGF-dependent manner. The introduction of RIP2 into Schwann cells deficient in RIP2 conferred NGF-dependent nuclear transcription factor-kappaB (NF-kappaB) activity and decreased the cell death induced by NGF. Conversely, the expression of a dominant-negative version of RIP2 protein resulted in a loss of NGF-induced NF-kappaB induction and increased NGF-mediated cell death. These results indicate that adaptor proteins like RIP2 can provide a bifunctional switch for cell survival or cell death decisions mediated by the p75 neurotrophin receptor.
Assuntos
Caspases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Fator 2 Ativador da Transcrição , Animais , Apoptose/fisiologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Genes Dominantes , Glutationa Transferase/genética , Proteínas de Fluorescência Verde , Humanos , Rim/citologia , Rim/metabolismo , Ligantes , Proteínas Luminescentes/genética , NF-kappa B/metabolismo , Fator de Crescimento Neural/farmacologia , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas/genética , Proteínas/metabolismo , Ratos , Receptor de Fator de Crescimento Neural , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores , Receptores de Fator de Crescimento Neural/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Fator 6 Associado a Receptor de TNF , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-kappaB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-kappaB induction. Dominant-negative versions of CARD4 block activation of NF-kappaB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Regulação da Expressão Gênica/fisiologia , Lipopolissacarídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Shigella flexneri/fisiologia , Transdução de Sinais/fisiologia , Proteínas de Transporte/genética , Linhagem Celular , Genes Reporter , Células HeLa , Humanos , Quinase I-kappa B , Interleucina-1/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Lipopolissacarídeos/administração & dosagem , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microinjeções , Proteína Adaptadora de Sinalização NOD1 , Testes de Precipitina , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Shigella flexneri/patogenicidade , Fator 2 Associado a Receptor de TNF , Receptores Toll-Like , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The CED4/Apaf-1 family of proteins functions as critical regulators of apoptosis and NF-kappaB signaling pathways. A novel human member of this family, called CARD12, was identified that induces apoptosis when expressed in cells. CARD12 is most similar in structure to the CED4/Apaf-1 family member CARD4, and is comprised of an N-terminal caspase recruitment domain (CARD), a central nucleotide-binding site (NBS), and a C-terminal domain of leucine-rich repeats (LRR). The CARD domain of CARD12 interacts selectively with the CARD domain of ASC, a recently identified proapoptotic protein. In addition, CARD12 coprecipitates caspase-1, a caspase that participates in both apoptotic signaling and cytokine processing. CARD12 may assemble with proapoptotic CARD proteins to coordinate the activation of downstream apoptotic and inflammatory signaling pathways.
Assuntos
Apoptose , Proteínas de Caenorhabditis elegans , Proteínas de Ligação ao Cálcio/genética , Proteínas de Helminto/genética , Proteínas/genética , Animais , Especificidade de Anticorpos , Fator Apoptótico 1 Ativador de Proteases , Caspase 1/metabolismo , Linhagem Celular , Chlorocebus aethiops , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Bases de Dados Factuais , Expressão Gênica , Genes Reporter , Humanos , Immunoblotting , Rim/citologia , Rim/metabolismo , Dados de Sequência Molecular , Família Multigênica , Especificidade de Órgãos , Estrutura Terciária de Proteína/fisiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Células VeroRESUMO
The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-kappaB signaling complexes. We report here that CARD protein 11 (CARD11) and CARD protein 14 (CARD14) are novel CARD-containing proteins that belong to the membrane-associated guanylate kinase (MAGUK) family, a class of proteins that functions as molecular scaffolds for the assembly of multiprotein complexes at specialized regions of the plasma membrane. CARD11 and CARD14 have homologous structures consisting of an N-terminal CARD domain, a central coiled-coil domain, and a C-terminal tripartite domain comprised of a PDZ domain, an Src homology 3 domain, and a GUK domain with homology to guanylate kinase. The CARD domains of both CARD11 and CARD14 associate specifically with the CARD domain of BCL10, a signaling protein that activates NF-kappaB through the IkappaB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-kappaB and induce the phosphorylation of BCL10. These findings suggest that CARD11 and CARD14 are novel MAGUK family members that function as upstream activators of BCL10 and NF-kappaB signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Guanilato Ciclase/metabolismo , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD , Guanilato Ciclase/química , Guanilato Ciclase/genética , Guanilato Quinases , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , Núcleosídeo-Fosfato Quinase/genética , Fosforilação , Testes de Precipitina , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
BCL10 belongs to the caspase recruitment domain (CARD) family of proteins that regulate apoptosis and NF-kappaB signaling pathways. Analysis of BCL10-deficient mice has revealed that BCL10 mediates NF-kappaB activation by antigen receptors in B and T cells. We recently identified a subclass of CARD proteins (CARD9, CARD11, and CARD14) that may function to connect BCL10 to multiple upstream signaling pathways. We report here that CARD10 is a novel BCL10 interactor that belongs to the membrane-associated guanylate kinase family, a class of proteins that function to organize signaling complexes at plasma membranes. When expressed in cells, CARD10 binds to BCL10 and signals the activation of NF-kappaB through its N-terminal effector CARD domain. We propose that CARD10 functions as a molecular scaffold for the assembly of a BCL10 signaling complex that activates NF-kappaB.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Proteína 10 de Linfoma CCL de Células B , Sítios de Ligação , Proteínas Adaptadoras de Sinalização CARD , Linhagem Celular , Membrana Celular/metabolismo , Genes Reporter , Guanilato Quinases , Humanos , Mamíferos , Dados de Sequência Molecular , Núcleosídeo-Fosfato Quinase/química , Especificidade de Órgãos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transcrição Gênica , Transfecção , Domínios de Homologia de srcRESUMO
BCL10/CLAP is an activator of apoptosis and NF-kappaB signaling pathways and has been implicated in B cell lymphomas of mucosa-associated lymphoid tissue. Although its role in apoptosis remains to be determined, BCL10 likely activates NF-kappaB through the IKK complex in response to upstream stimuli. The N-terminal caspase recruitment domain (CARD) of BCL10 has been proposed to function as an activation domain that mediates homophilic interactions with an upstream CARD-containing NF-kappaB activator. To identify upstream signaling partners of BCL10, we performed a mammalian two-hybrid analysis and identified CARD9 as a novel CARD-containing protein that interacts selectively with the CARD activation domain of BCL10. When expressed in cells, CARD9 binds to BCL10 and activates NF-kappaB. Furthermore, endogenous CARD9 is found associated with BCL10 suggesting that both proteins form a pre-existing signaling complex within cells. CARD9 also self-associates and contains extensive coiled-coil motifs that may function as oligomerization domains. We propose here that CARD9 is an upstream activator of BCL10 and NF-kappaB signaling.