RESUMO
The production of bio-active interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves binding of the caspase-1 prodomain to a caspase recruitment domain (CARD)-containing serine/threonine kinase known as RIP2/CARDIAK/RICK. We have identified a novel protein, COP (CARD only protein), which has a high degree of sequence identity to the caspase-1 prodomain. COP binds to both RIP2 and the caspase-1 prodomain and inhibits RIP2-induced caspase-1 oligomerization. COP inhibits caspase- 1-induced IL-1beta secretion as well as lipopolysaccharide-induced IL-1beta secretion in transfected cells. Our data indicate that COP can regulate IL-1beta secretion, implying that COP may play a role in down-regulating inflammatory responses analogous to the CARD protein ICEBERG.
Assuntos
Proteínas de Transporte/fisiologia , Inibidores de Caspase , Peptídeos e Proteínas de Sinalização Intracelular , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Caspase 1/metabolismo , Humanos , Interleucina-1/fisiologia , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Mensageiro/análise , Proteína Serina-Treonina Quinase 2 de Interação com ReceptorRESUMO
Proteins governing cell death form the basis of many normal processes and contribute to the pathogenesis of many diseases when dysregulated. Here we report the cloning of a novel human CED-4-like gene, CLAN, and several of its alternatively spliced isoforms. These caspase-associated recruitment domain (CARD)-containing proteins are expressed at varying degrees in normal human tissues and may contribute to a number of intracellular processes including apoptosis, cytokine processing, and NF-kappa B activation. The CARD of the CLAN proteins binds a number of other CARD-containing proteins including caspase-1, BCL10, NOD2, and NAC. Once their physiologic functions are uncovered, CLAN proteins may prove to be valuable therapeutic targets.
Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Proteínas de Helminto/genética , Sequência de Aminoácidos , Apoptose , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Ligação ao Cálcio/química , Caspases/química , Cromossomos Humanos Par 2 , Clonagem Molecular , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Modelos Genéticos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Hibridização de Ácido Nucleico , Plasmídeos/metabolismo , Testes de Precipitina , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Exposure of endothelial and many other cell types to tumor necrosis factor alpha generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-kappaB that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from tumor necrosis factor alpha-mediated apoptosis. In turn, some anti-apoptotic genes have been shown to modulate NF-kappaB activity. Here we demonstrate that XIAP, a NF-kappaB-dependent member of the IAP gene family, is a strong stimulator of NF-kappaB. Expression of XIAP leads to increased nuclear translocation of the p65 subunit of NF-kappaB via a novel signaling pathway that involves the mitogen-activated protein kinase kinase kinase TAK1. We show that TAK1 physically interacts with NIK and with IKK2, and both XIAP or active TAK1 can stimulate IKK2 kinase activity. Thus, XIAP may be part of a system of regulatory loops that balance a cell's response to environmental stimuli.
Assuntos
Apoptose , Endotélio Vascular/metabolismo , MAP Quinase Quinase Quinases , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Linhagem Celular , Endotélio Vascular/patologia , Humanos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
By differential screening of tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS)- activated endothelial cells (ECs), we have identified a cDNA clone that turned out to be a member of the inhibitor of apoptosis (iap) gene family. iap genes function to protect cells from undergoing apoptotic death in response to a variety of stimuli. These iap genes, hiap1, hiap2, and xiap were found to be strongly upregulated upon treatment of ECs with the inflammatory cytokines TNF-alpha, interleukin 1beta, and LPS, reagents that lead to activation of the nuclear transcription factor kappaB (NF-kappaB). Indeed, overexpression of IkappaBalpha, an inhibitor of NF-kappaB, suppresses the induced expression of iap genes and sensitizes ECs to TNF-alpha-induced apoptosis. Ectopic expression of one member of the human iap genes, human X-chromosome-linked iap (xiap), using recombinant adenovirus overrules the IkappaBalpha effect and protects ECs from TNF-alpha- induced apoptosis. We conclude that xiap represents one of the NF-kappaB-regulated genes that counteracts the apoptotic signals caused by TNF-alpha and thereby prevents ECs from undergoing apoptosis during inflammation.
Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica/genética , NF-kappa B/fisiologia , Proteínas de Neoplasias/genética , Fator de Necrose Tumoral alfa/farmacologia , Cromossomo X/genética , Adenoviridae/química , Células Cultivadas , DNA/análise , Fragmentação do DNA/genética , Endotélio Vascular/metabolismo , Citometria de Fluxo , Ligação Genética/genética , RNA Mensageiro/metabolismo , Proteínas Virais/fisiologiaRESUMO
The inhibitor of apoptosis (iap) proteins belong to a gene family that protect certain cell to undergo programmed cell death in response to a variety of stimuli. By differential screening we have identified a cDNA clone, designated piap, in porcine aortic endothelial cells (PAEC) that turned out by sequence comparison to be a porcine member of the iap family. The expression of piap is strongly up-regulated upon treatment of endothelial cells (EC) with inflammatory cytokines TNF-alpha, IL-1 beta, and LPS. In EC these stimuli lead to the activation of nuclear transcription factor kappa B (NF-kappa B) that plays a role in countering TNF-alpha induced apoptosis. We demonstrate that adenovirus mediated overexpression of I kappa B alpha, an inhibitor of NF-kappa B suppresses the expression of piap in response to TNF-alpha suggesting that piap is one of the NF-kappa B regulated genes that operates to prevent programmed cell death of EC in inflammation.
