RESUMEN

As a component of the apoptosome, a caspase-activating complex, Apaf-1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. We report here the identification of a novel Apaf-1 interacting protein, hepatocellular carcinoma antigen 66 (HCA66) that is able to modulate selectively Apaf-1-dependent apoptosis through its direct association with the CED4 domain of Apaf-1. Expression of HCA66 was able to potentiate Apaf-1, but not receptor-mediated apoptosis, by increasing downstream caspase activity following cytochrome c release from the mitochondria. Conversely, cells depleted of HCA66 were severely impaired for apoptosome-dependent apoptosis. Interestingly, expression of the Apaf-1-interacting domain of HCA66 had the opposite effect of the full-length protein, interfering with the Apaf-1 apoptotic pathway. Using a cell-free system, we showed that reduction of HCA66 expression was associated with a diminished amount of caspase-9 in the apoptosome, resulting in a lower ability of the apoptosome to activate caspase-3. HCA66 maps to chromosome 17q11.2 and is among the genes heterozygously deleted in neurofibromatosis type 1 (NF1) microdeletion syndrome patients. These patients often have a distinct phenotype compared to other NF1 patients, including a more severe tumour burden. Our results suggest that reduced expression of HCA66, owing to haploinsufficiency of HCA66 gene, could render NF1 microdeleted patients-derived cells less susceptible to apoptosis.

Asunto(s)

Antígenos de Neoplasias/fisiología , Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Neurofibromatosis 1/fisiopatología , Neurofibromina 1/genética , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Factor Apoptótico 1 Activador de Proteasas/genética , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Proteínas Portadoras/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Línea Celular , Células Cultivadas , Cromatografía en Gel , Eliminación de Gen , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Síndrome , Transfección

RESUMEN

Caspase-14 is a recent addition to the caspase family of aspartate proteases involved in apoptotic processes. Human caspase-14 appears to be only weakly processed during apoptosis, and it does not cleave classical caspase substrates. Post partum, caspase-14 is prominently expressed by human keratinocytes and reportedly participates in terminal differentiation of complex epithelia. Here we provide evidence challenging the view that caspase-14 expression or processing is linked exclusively to terminal keratinocyte differentiation. We demonstrate that caspase-14 expression extended to multiple cell lines derived from simple epithelia of the breast, prostate, and stomach. In keratinocytes and breast epithelial cells, caspase-14 expression was upregulated in high-density cultures and during forced suspension culture. These effects were primarily due to transcriptional activation as indicated by reporter gene assays using a 2 kb caspase-14 promoter fragment. Importantly, caspase-14 was not cleaved during forced suspension culture of either cell type although this treatment induced caspase-dependent apoptosis (anoikis). Forced expression of caspase-14 in immortalized human keratinocytes had no effect on cell death in forced suspension nor was the transfected caspase-14 processed in this setting. In contrast to postconfluent and forced suspension culture, terminal differentiation of keratinocytes induced in vitro by Ca2+ treatment was not associated with increased caspase-14 expression or promoter activity. Our results indicate that (1) caspase-14 is expressed not only in complex but also simple epithelia; (2) cells derived from complex and simple epithelia upregulate caspase-14 expression in conditions of high cell density or lack of matrix interaction and; (3) in both cell types this phenomenon is due to transcriptional regulation.

Asunto(s)

Caspasas/genética , Diferenciación Celular/genética , Células Epiteliales/enzimología , Epitelio/enzimología , Regulación Enzimológica de la Expresión Génica/genética , Genes Reguladores/genética , Especificidad de Anticuerpos/inmunología , Mama/citología , Mama/enzimología , Mama/crecimiento & desarrollo , Caspasa 14 , Adhesión Celular/fisiología , Compartimento Celular/fisiología , Ciclo Celular/fisiología , Células Cultivadas , Células Epidérmicas , Epidermis/enzimología , Epidermis/crecimiento & desarrollo , Células Epiteliales/citología , Epitelio/crecimiento & desarrollo , Matriz Extracelular/enzimología , Humanos , Recién Nacido , Queratinocitos/citología , Queratinocitos/enzimología , Masculino , Regiones Promotoras Genéticas/genética , Próstata/citología , Próstata/enzimología , Próstata/crecimiento & desarrollo

RESUMEN

Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. Here we describe the identification of a human Apaf-1-related protein, named Ipaf that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. Our results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli.

Asunto(s)

Caspasa 1/metabolismo , Proteínas/química , Proteínas/fisiología , Secuencia de Aminoácidos , Apoptosis , Factor Apoptótico 1 Activador de Proteasas , Caspasa 9 , Caspasas/metabolismo , Línea Celular , Células Cultivadas , ADN Complementario/metabolismo , Activación Enzimática , Precursores Enzimáticos/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Microscopía Confocal , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal , Distribución Tisular , Transfección , Células Tumorales Cultivadas

RESUMEN

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.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Proteína 10 de la LLC-Linfoma de Células B , Sitios de Unión , Proteínas Adaptadoras de Señalización CARD , Línea Celular , Membrana Celular/metabolismo , Genes Reporteros , Guanilato-Quinasas , Humanos , Mamíferos , Datos de Secuencia Molecular , Nucleósido-Fosfato Quinasa/química , Especificidad de Órganos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Transcripción Genética , Transfección , Dominios Homologos src

RESUMEN

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.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Guanilato Ciclasa/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Proteína 10 de la LLC-Linfoma de Células B , Proteínas Adaptadoras de Señalización CARD , Guanilato Ciclasa/química , Guanilato Ciclasa/genética , Guanilato-Quinasas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Familia de Multigenes , Nucleósido-Fosfato Quinasa/genética , Fosforilación , Pruebas de Precipitina , Unión Proteica , Homología de Secuencia de Aminoácido

RESUMEN

vCLAP, the E10 gene product of equine herpesvirus-2, is a caspase-recruitment domain (CARD)-containing protein that has been shown to induce both apoptosis and NF-kappaB activation in mammalian cells. vCLAP has a cellular counterpart, Bcl10/cCLAP, which is also an activator of apoptosis and NF-kappaB. Recent studies demonstrated that vCLAP activates NF-kappaB through an IkappaB kinase (IKK)-dependent pathway, but the underlying mechanism remains unknown. In this report, we demonstrate that vCLAP associates stably with the IKK complex through direct binding to the C-terminal region of IKKgamma. Consistent with this finding, IKKgamma was found to be essential for vCLAP-induced NF-kappaB activation, and the association between vCLAP and the IKK complex induced persistent activation of the IKKs. Moreover, enforced oligomerization of the isolated C-terminal region of vCLAP, which interacts with IKKgamma, can trigger NF-kappaB activation. Finally, substitution of the C-terminal region of IKKgamma, which interacts with vCLAP, with the CARD of vCLAP or Bcl10 produced a molecule that was able to activate NF-kappaB when ectopically expressed in IKKgamma-deficient cells. These data suggest that vCLAP-induced oligomerization of IKKgamma, which is mediated by the CARD of vCLAP, could be the mechanism by which vCLAP induces activation of NF-kappaB.

Asunto(s)

Proteínas Portadoras/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Animales , Proteínas Portadoras/química , Caspasas/metabolismo , Células Cultivadas , Glicina/metabolismo , Quinasa I-kappa B , Estructura Terciaria de Proteína , Ratas , Proteínas Virales/química

RESUMEN

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.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Caspasas/metabolismo , FN-kappa B/metabolismo , Proteínas/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteína 10 de la LLC-Linfoma de Células B , Ratones , Datos de Secuencia Molecular , Proteínas/química

RESUMEN

To understand the mechanism of activation of the IkappaB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFalpha stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKgamma, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-kappaB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKgamma or truncated IKKalpha or IKKbeta lacking their serine-cluster domains can also induce IKK and NF-kappaB activation. These data suggest that IKKgamma functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.

Asunto(s)

Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Animales , Biopolímeros , Línea Celular , Activación Enzimática , Humanos , Quinasa I-kappa B , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores

RESUMEN

Molecules that regulate NF-kappaB activation play critical roles in apoptosis and inflammation. We describe the cloning of the cellular homolog of the equine herpesvirus-2 protein E10 and show that both proteins regulate apoptosis and NF-kappaB activation. These proteins were found to contain N-terminal caspase-recruitment domains (CARDs) and novel C-terminal domains (CTDs) and were therefore named CLAPs (CARD-like apoptotic proteins). The cellular and viral CLAPs induce apoptosis downstream of caspase-8 by activating the Apaf-1-caspase-9 pathway and activate NF-kappaB by acting upstream of the NF-kappaB-inducing kinase, NIK, and the IkB kinase, IKKalpha. Deletion of either the CARD or the CTD domain inhibits both activities. The CARD domain was found to be important for homo- and heterodimerization of CLAPs. Substitution of the CARD domain with an inducible FKBP12 oligomerization domain produced a molecule that can induce NF-kappaB activation, suggesting that the CARD domain functions as an oligomerization domain, whereas the CTD domain functions as the effector domain in the NF-kappaB activation pathway. Expression of the CARD domain of human CLAP abrogates tumor necrosis factor-alpha-induced NF-kappaB activation, suggesting that cellular CLAP plays an essential role in this pathway of NF-kappaB activation.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Apoptosis , FN-kappa B/metabolismo , Proteínas/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Secuencia de Aminoácidos , Proteína 10 de la LLC-Linfoma de Células B , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Clonación Molecular , Genes Reporteros , Humanos , Quinasa I-kappa B , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/genética , Proteínas/química , ARN Mensajero/metabolismo , Alineación de Secuencia , Transducción de Señal , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Virales/genética , Quinasa de Factor Nuclear kappa B

RESUMEN

The summer leaf isoform of the pokeweed (Phytolacca americana) antiviral protein, PAP II, was produced in high yields from inclusion bodies in recombinant E. coli. On the basis of its sequence similarity with the spring leaf isoform (PAP I) and with the A chain of ricin, a three-dimensional model of the protein was constructed as an aid in the design of active site mutants. PAP II variants mutated in residues Asp 88 (D88N), Tyr 117 (Y117S), Glu 172 (E172Q), Arg 175 (R175H) and a combination of Asp 88 and Arg 175 (D88N/R175H) were produced in E. coli and assayed for their ability to inhibit protein synthesis in a rabbit reticulocyte lysate. All of these mutations had effects deleterious to the enzymatic activity of PAP II. The results were interpreted in the light of three reaction mechanisms proposed for ribosome-inactivating proteins (RIPs). We conclude that none of the proposed mechanisms is entirely consistent with the data presented here.

Asunto(s)

Antivirales/metabolismo , N-Glicosil Hidrolasas/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/química , Dominio Catalítico/genética , Sistema Libre de Células , Relación Dosis-Respuesta a Droga , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Conejos , Reticulocitos , Proteínas Inactivadoras de Ribosomas Tipo 1 , Estaciones del Año , Homología de Secuencia de Aminoácido

RESUMEN

Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA. The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant. Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract. In this complex, the enzymatic activity of the toxin was strongly inhibited. This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis. PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80 degrees C or by treatment by alkaline or acidic pH or by 7 M urea. The other components involved in the complex remain unknown.

Asunto(s)

Antivirales/análisis , N-Glicosil Hidrolasas , Proteínas de Plantas/análisis , Animales , Antivirales/farmacología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Ratones , Peso Molecular , Proteínas de Plantas/farmacología , Plantas/química , Desnaturalización Proteica , Proteínas Inactivadoras de Ribosomas Tipo 1

RESUMEN

Pokeweed antiviral proteins (PAP) represent a family of protein toxins isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). We isolated, sequenced and characterized for the first time a complete cDNA encoding a pokeweed antiviral protein expressed in seeds. The cDNA of PAP-S consists of 1249 nucleotides and encodes a mature 262 amino acid protein. Its predicted amino acid sequence is more similar to PAP (76%) than to PAP II (31%). It is known from literature that PAP-S is more active in inhibiting protein synthesis than other members of the PAP family. Therefore, the cDNA of PAP-S was expressed in Escherichia coli and the biological activity of the recombinant protein was compared with that of PAP purified from spring leaves. In a rabbit translation system, the median inhibitory concentrations (IC50) of recombinant PAP-S and native PAP were determined as 0.07 and 0.29 nM, respectively. Although the PAP-S protein in seeds is glycosylated, PAP-S can be expressed in Escherichia coli in a very active form, indicating that post-translational modification in pokeweed does not seem to alter its ability to inhibit protein synthesis.

Asunto(s)

N-Glicosil Hidrolasas , Proteínas de Plantas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Semillas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistema Libre de Células , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/farmacología , Conejos , Proteínas Inactivadoras de Ribosomas Tipo 1 , Homología de Secuencia de Aminoácido

RESUMEN

Monoclonal antibodies specific to pokeweed antiviral protein (PAP), a ribosome-inactivating protein (RIP), were obtained after six unsuccessful fusions. A special procedure including injections of low doses of purified PAP from spring leaves in a short period was adopted. Some clones highly specific to PAP react with recombinant PAP. One clone cross-reacts with PAP-S isolated from seeds but none cross-reacts with the isoform PAP II isolated from summer leaves. These antibodies represent a useful tool to investigate the mechanisms of PAP biosynthesis and plant protection involving RIPs.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , N-Glicosil Hidrolasas , Proteínas de Plantas/inmunología , Inhibidores de la Síntesis de la Proteína/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Hibridomas/química , Hibridomas/metabolismo , Inmunización , Ratones , Ratones Endogámicos BALB C , Proteínas de Plantas/aislamiento & purificación , Inhibidores de la Síntesis de la Proteína/aislamiento & purificación , Proteínas Inactivadoras de Ribosomas Tipo 1

RESUMEN

Three distinct ribosome-inactivating proteins (RIPs) were isolated from pokeweed (Phytolacca americana). We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokeweed. The cDNA of PAP II consists of 1,187 nucleotides and encodes a mature protein of 285 amino acids. Its predicted amino acid sequence is only 33% similar to PAP and PAP-S. The NH2 terminal extrapeptide (25 amino acid residues) was similar but not identical to that of PAP's extrapeptide. The cDNA of PAP II was expressed in E. coli. The growth of the transformants was strongly inhibited after induction of the gene. Furthermore, PAP II, which was produced in E. coli, inhibited protein synthesis in a rabbit reticulocyte translation system. Thus, recombinant PAP II would appear to be as functional as native PAP in inhibiting protein synthesis in both prokaryotes and eukaryotes.

Asunto(s)

ADN Complementario/genética , Escherichia coli/genética , Expresión Génica , N-Glicosil Hidrolasas , Proteínas de Plantas/genética , Plantas/química , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Plantas/genética , Poli A/metabolismo , Inhibidores de la Síntesis de la Proteína , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/efectos de los fármacos , Alineación de Secuencia