RESUMO
Tumour necrosis factor (tumour necrosis factor-alpha/cachectin) plays a critical role in certain physiological defensive responses but causes severe damage to the host organism when produced in excess. There are two forms of tumour necrosis factor, a type II membrane protein of relative molecular mass 26,000 (26K) and a soluble, 17K form generated from the cell-bound protein by proteolytic cleavage. The two forms of tumour necrosis factor and lymphotoxin-alpha (tumour necrosis factor-beta/lymphotoxin), a related protein, have similar but apparently not identical biological activities. A therapeutic agent which inhibited the release of tumour necrosis factor, but did not reduce the cell-associated activity or the level of lymphotoxin-alpha, might preserve the benefits of these cytokines while preventing tumour necrosis factor-induced damage. Here we describe a potent inhibitor of tumour necrosis factor processing and report that it protects mice from a lethal dose of endotoxin.
Assuntos
Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Ácidos Hidroxâmicos/farmacologia , Linfotoxina-alfa/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
We have purified the IL-1 beta converting enzyme from the THP-1 cell line using standard chromatographic techniques and obtained the N-terminal amino acid sequence of this novel protein. After stimulation of THP-1 cells with lipopolysaccharide, hydroxyurea, and silica, the protease was solubilized by multiple freeze/thawing. The protein was purified by ion-exchange chromatography, affinity chromatography on blue agarose, gel filtration, and chromatofocusing. The molecular weight of the protein is approximately 22,000 Da and the pI is between 7.1 and 6.8. The overall yield for this procedure was 16% of the activity found in the initial cell lysates. An antiserum raised against a peptide based on the N-terminus was used to precipitate the protease, confirming our identification of the 22,000-Da protein as the IL-1 beta converting enzyme.
Assuntos
Interleucina-1/metabolismo , Metaloendopeptidases/isolamento & purificação , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Western Blotting , Caspase 1 , Linhagem Celular , Cromatografia , Hidroxiureia/farmacologia , Técnicas de Imunoadsorção , Ponto Isoelétrico , Lipopolissacarídeos , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Peso Molecular , Dióxido de Silício/farmacologiaRESUMO
Cowpox virus effectively inhibits inflammatory responses against viral infection in the chick embryo. This study demonstrates that one of the viral genes necessary for this inhibition, the crmA gene (a cytokine response modifier gene), encodes a serpin that is a specific inhibitor of the interleukin-1 beta converting enzyme. This serpin can prevent the proteolytic activation of interleukin-1 beta, thereby suppressing an interleukin-1 beta response to infection. However, the modification of this single cytokine response is not sufficient to inhibit inflammatory responses. This suggests that cowpox virus encodes several cytokine response modifiers that act together to inhibit the release of pro-inflammatory cytokines in response to infection. These viral countermeasures to host defenses against infection may contribute significantly to the pathology associated with poxvirus infections.
Assuntos
Vírus da Varíola Bovina/enzimologia , Interleucina-1/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Serina Endopeptidases/metabolismo , Serpinas/genética , Proteínas Virais , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caspase 1 , Embrião de Galinha , Vírus da Varíola Bovina/genética , Vírus da Varíola Bovina/imunologia , Genes Virais , Inflamação/enzimologia , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas Estruturais Virais/genéticaRESUMO
Interleukin-1 beta (IL-1 beta) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor. A complementary DNA encoding a protease that carries out this cleavage has been cloned. Recombinant expression in COS-7 cells enabled the cells to process precursor IL-1 beta to the mature form. Sequence analysis indicated that the enzyme itself may undergo proteolytic processing. The gene encoding the protease was mapped to chromosomal band 11q23, a site frequently involved in rearrangement in human cancers.
Assuntos
Cromossomos Humanos Par 11 , Precursores Enzimáticos/genética , Metaloendopeptidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Caspase 1 , Linhagem Celular , Bandeamento Cromossômico , Clonagem Molecular , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/isolamento & purificação , Humanos , Metaloendopeptidases/biossíntese , Metaloendopeptidases/isolamento & purificação , Dados de Sequência Molecular , Neutrófilos/enzimologia , Oligodesoxirribonucleotídeos , Poli A/genética , Poli A/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , TransfecçãoRESUMO
Interleukin-1 beta (IL-1 beta) is released from an inactive precursor by a proteolytic cleavage. A monocytic protease has been identified that appears to be involved in the physiological activation of this cytokine. Two situations have been found in which precursor IL-1 beta exists without the monocytic processing enzyme, and in these cases other proteases, such as neutrophil elastase, cathepsin G and cathepsin L, may be involved in generating the active cytokine.
