RESUMO
Matrix protein 2 ectodomain (M2e) is considered an attractive component of a broadly protective, universal influenza A vaccine. Here we challenge the canonical view that antibodies against M2e are the prime effectors of protection. Intranasal immunizations of Balb/c mice with CTA1-3M2e-DD-generated M2e-specific memory CD4 T cells that were I-Ad restricted and critically protected against infection, even in the complete absence of antibodies, as observed in JhD mice. Whereas some M2e-tetramer-specific memory CD4 T cells resided in spleen and lymph nodes, the majority were lung-resident Th17 cells, that rapidly expanded upon a viral challenge infection. Indeed, immunized IL-17A-/- mice were significantly less well protected compared with wild-type mice despite exhibiting comparable antibody levels. Similarly, poor protection was also observed in congenic Balb/B (H-2b) mice, which failed to develop M2e-specific CD4 T cells, but exhibited comparable antibody levels. Lung-resident CD69+ CD103low M2e-specific memory CD4 T cells were αß TCR+ and 50% were Th17 cells that were associated with an early influx of neutrophils after virus challenge. Adoptively transferred M2e memory CD4 T cells were strong helper T cells, which accelerated M2e- but more importantly also hemagglutinin-specific IgG production. Thus, for the first time we demonstrate that M2e-specific memory CD4 T cells are broadly protective.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos de Histocompatibilidade Classe II/metabolismo , Hibridomas , Memória Imunológica , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Ligação Proteica , Domínios Proteicos/genética , Vacinação , Proteínas da Matriz Viral/genéticaRESUMO
Influenza is a global health concern. Licensed influenza vaccines induce strain-specific virus-neutralizing antibodies but hamper the induction of possibly cross-protective T-cell responses upon subsequent infection.(1) In this study, we compared protection induced by a vaccine based on the conserved extracellular domain of matrix 2 protein (M2e) with that of a conventional whole inactivated virus (WIV) vaccine using single as well as consecutive homo- and heterosubtypic challenges. Both vaccines protected against a primary homologous (with respect to hemagglutinin and neuraminidase in WIV) challenge. Functional T-cell responses were induced after primary challenge of M2e-immune mice but were absent in WIV-vaccinated mice. M2e-immune mice displayed limited inducible bronchus-associated lymphoid tissue, which was absent in WIV-immune animals. Importantly, M2e- but not WIV-immune mice were protected from a primary as well as a secondary, severe heterosubtypic challenge, including challenge with pandemic H1N1 2009 virus. Our findings advocate the use of infection-permissive influenza vaccines, such as those based on M2e, in immunologically naive individuals. The combined immune response induced by M2e-vaccine and by clinically controlled influenza virus replication results in strong and broad protection against pandemic influenza. We conclude that the challenge of the M2e-immune host induces strong and broadly reactive immunity against influenza virus infection.
Assuntos
Imunidade Celular , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas da Matriz Viral/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno/imunologia , Pulmão/imunologia , Pulmão/virologia , Camundongos , Infecções por Orthomyxoviridae/virologiaRESUMO
In 1963 I started the Laboratory of Molecular Biology (LMB) at the University of Ghent. Molecular Biology was then a new scientific discipline. Nucleic acids (NA) could be sequenced, manipulated, and recombined to form genetic information which never before had existed. Cloning of DNA-segments allowed multiplying a single molecule a billion-fold. By 1975 recombinant DNA-technology had sufficiently progressed that one could start the pursuit of a medically important goal. Our choice was to go for Interferon, a mysterious substance which could protect against viral infection, and might possibly be used as an anti-cancer agent if available in unlimited quantities. Fortunately, Piet De Somer, then Rector of the KUL, was one of the pioneers in interferon research. He encouraged his young colleague Erik De Clercq to collaborate with us. Erik brought extensive interferon expertise and reagents to the collaboration. But molecular biologists work with NA and interferon is a protein. There was a missing link which was provided by Jean Content of the Brussels Pasteur Institute. Jean had developed a system to convert interferon mRNA into protein, which was send to Leuven for quantification. This close collaboration between 3 laboratories led in January 1980 to the cloning of the human fibroblast, now interferon-beta, gene, and to the primary structure of the protein. However, 2 months earlier, Tadatsugu Taniguchi had succeeded already to obtain such a clone. But we were the first to express the clone in E. coli, and this was the definite proof that the cloned gene coded for human interferon-beta.
Assuntos
Clonagem Molecular , Expressão Gênica , Interferon beta/genética , DNA Recombinante , Humanos , Biologia MolecularRESUMO
We have previously reported on a universal human influenza A vaccine, based on the external domain of the transmembrane viral M2-protein (M2e) [Nature Medicine 5 (1999) 1119]. M2-protein is scarcely present on the virus but is abundantly expressed on virus-infected cells. The external domain, M2e, is 23-amino acids long and as such weakly immunogenic. But when presented on an appropriate carrier, such as hepatitis B virus core (HBc) particles, it induces a high titer antibody response that in mice effectively protects against a potentially lethal influenza infection. The advantage of M2e as an antigen is the conservation of its sequence that has hardly changed since the first influenza virus was isolated in 1933, despite numerous epidemics and several pandemics. Various constructs, e.g. M2e fused at the N-terminus of the HBc subunit or inserted in the immuno-dominant loop, were evaluated as a vaccine. They conferred full protection when administered together with an adjuvant. Several adjuvants were tested in conjunction with intraperitoneal vaccine administration, while the non-toxic enterotoxin mutant LT(R192G) was used for intranasal vaccination. Appropriate combinations of vaccine construct and adjuvant allowed to obtain anti-M2e IgG2a serum titers above 10,000, and this provided complete protection.
Assuntos
Vírus da Influenza A/imunologia , Vacinas contra Influenza , Influenza Humana/prevenção & controle , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Sequência Conservada , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Humanos , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
The p55 tumor necrosis factor receptor (TNF-RI) is the main receptor by which TNF exerts its effects. The signaling capacity largely depends on the presence of an intact C-terminal protein-protein interaction domain, a so-called death domain (DD). Here we report the expression and purification of the human TNF-RI DD as a fusion with the Escherichia coli thioredoxin A (TRX) protein. When expressed under control of the bacteriophage T7 promoter, TRX-DD accumulates as a soluble protein in the cytoplasm of E. coli. The TRX-DD protein was released from the cells into the periplasmic fraction after osmotic shock. Due to self-association of the DD, a large part of the material appeared as multimers; it could be removed by selective precipitation and a combination of ion-exchange and size-exclusion chromatography. This purification protocol yielded 30 mg of purified, monomeric protein from 1 liter of shake-flask culture. The purified TRX-DD was found to be functional as it still bound to the TNF-RI-associated DD protein and the intracellular part of TNF-RI. We conclude that TRX-DD is correctly folded and can be used for further structure/function analysis.
Assuntos
Antígenos CD/genética , Receptores do Fator de Necrose Tumoral/genética , Antígenos CD/química , Antígenos CD/isolamento & purificação , Clonagem Molecular , Escherichia coli , Humanos , Estrutura Terciária de Proteína , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/isolamento & purificação , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiorredoxinas/genéticaRESUMO
In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Mitocôndrias/metabolismo , Necrose , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Caspases/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Humanos , Cinética , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores do Fator de Necrose Tumoral/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/efeitos dos fármacos , Receptor fas/metabolismoRESUMO
The bioactivity of tumor necrosis factor (TNF) is mediated by two TNF receptors (TNF-Rs), more particularly TNF-RI and TNF-RII. Although human TNF (hTNF) and murine TNF (mTNF) are very homologous, hTNF binds only to mTNF-RI. By measuring the binding of a panel of mTNF/hTNF chimeras to both mTNF-R, we pinpointed the TNF region that mediates the interaction with mTNF-RII. Using site-specific mutagenesis, we identified amino acids 71-73 and 89 as the main interacting residues. Mutein hTNF-S71D/T72Y/H73 Delta/T89E interacts with both types of mTNF-R and is active in CT6 cell proliferation assays mediated by mTNF-RII. Mutein mTNF-D71S/Y72T/Delta 73H/E89T binds to mTNF-RI only and is no longer active on CT6 cells. However, the L929s cytotoxicity of this mutein (an effect mediated by mTNF-RI triggering) was also 100-fold lower than that of wild-type mTNF due to enhanced dissociation during incubation at subnanomolar concentrations. The additional mutation of amino acid 102, resulting in the mutein mTNF-D71S/Y72T/Delta 73H/E89T/P102Q, restored the trimer stability, which led to an enhanced specific activity on L929s cells. Hence the specific activity of a TNF species is governed not only by its receptor binding characteristics but also by its trimer stability after incubation at subnanomolar concentrations. In conclusion, the mutation of TNF amino acids 71-73, 89, and 102 is sufficient to obtain a mTNF mutein selective for mTNF-RI and a hTNF mutein that, unlike wild-type hTNF, also acts on mTNF-RII.
Assuntos
Antígenos CD/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Cromatografia em Gel , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
Incubation of murine tumor necrosis factor (mTNF) at subnanomolar concentrations results in partial dissociation of the trimers, coinciding with a decrease in bioactivity. Using size-exclusion chromatography, we observed that the conversion of labeled mTNF to monomers is not only prevented by coincubation with an excess of unlabeled mTNF but also with unlabeled human TNF (hTNF). Moreover, after coincubation of mTNF and hTNF four different TNF complexes were revealed by native polyacrylamide gel electrophoresis, viz. homotrimeric mTNF and hTNF, as well as two complexes with an intermediate migration pattern. Analytical gel filtration in combination with native polyacrylamide gel electrophoresis and Western blot immunodetection indicated that these new complexes consisted of heterotrimeric TNF molecules. We conclude that an exchange of monomers takes place during coincubation of two different species of TNF, which results in homotrimeric and heterotrimeric TNF. To assess receptor interaction in vitro, TNF heterotrimeric molecules were used as obtained after incubation of mTNF with labeled hTNF (which only binds to mTNF receptor I) or with labeled mutein mTNF75 (specific for mTNF receptor II). These heterotrimers were retained by both mTNF receptors, which means that the mTNF subunits incorporated in heterotrimeric complexes still can bind to both types of TNF receptor. In addition, the gradual decrease in mTNF bioactivity during preincubation at subnanomolar concentrations was prevented by the presence of mutein mTNF75, which is inactive in an L929 cytotoxicity assay, indicating that heterotrimerization can influence the overall bioactivity.
Assuntos
Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Biopolímeros , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Especificidade da Espécie , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/isolamento & purificaçãoRESUMO
Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the neuraminidase gene to a tetramerizing leucine zipper sequence; the resulting product was enzymatically active, tetrameric neuraminidase. The protective immunity induced by this engineered neuraminidase, however, remained fairly strain-specific. A third influenza A virus protein, the M2 protein, has only 23 amino acids exposed on the outer membrane surface. This extracellular part, M2e, has been remarkably conserved in all human influenza A strains since 1933. By fusing the M2e sequence to hepatitis B virus core protein, we could obtain highly immunogenic particles that induced complete, strain-independent, long-lasting protection in mice against a lethal viral challenge. Native M2 is a tetrameric protein and this conformation of the M2e part can also be mimicked by fusing this sequence to a tetramerizing leucine zipper. The potential of the resulting protein as a vaccine candidate remains to be evaluated.
Assuntos
Vacinas contra Influenza/química , Vacinas contra Influenza/genética , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Sequência de Aminoácidos , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Vírus da Influenza A , Dados de Sequência Molecular , Neuraminidase , Solubilidade , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow's medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75-100 kDa). These disulfide-stabilized bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) ("tribody") heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.
Assuntos
Anticorpos Biespecíficos/biossíntese , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Sítios de Ligação de Anticorpos/genética , Linhagem Celular , Citotoxicidade Imunológica/genética , Dimerização , Estabilidade de Medicamentos , Humanos , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/metabolismo , Fragmentos Fab das Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD/química , Antígenos CD/metabolismo , Proteínas de Transporte/metabolismo , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Virais , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Proteína de Domínio de Morte Associada a Fas , Citometria de Fluxo , Humanos , Camundongos , Necrose , Receptores Tipo I de Fatores de Necrose Tumoral , Serpinas/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Hypotension and shock observed in sepsis, SIRS, and tumor necrosis factor (TNF) or cytokine-based cancer treatment are the consequence of excessive nitric oxide (NO) production and subsequent soluble guanylate cyclase (sGC)-mediated vascular smooth muscle relaxation. We demonstrate here that, while NO synthase (NOS) inhibitors exacerbated toxicity, inhibitors of sGC activation protected against TNF-induced lethality, bradycardia, and hypotension. Importantly, sGC inhibition did not interfere with the antitumor activity of TNF. Using NOS inhibitors or iNOS-deficient animals, we furthermore observed that no protection against TNF toxicity could be obtained in the absence of NO. These data imply that iNOS- (and not eNOS-) derived NO is an endogenous protective molecule indispensable to survive a TNF challenge and exerting this beneficial effect via sGC-independent mechanisms.
Assuntos
Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Choque/metabolismo , Choque/prevenção & controle , Fator de Necrose Tumoral alfa/toxicidade , Animais , Inibidores Enzimáticos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo IIRESUMO
The cytokine interleukin-10 (IL-10) has shown promise in clinical trials for treatment of inflammatory bowel disease (IBD). Using two mouse models, we show that the therapeutic dose of IL-10 can be reduced by localized delivery of a bacterium genetically engineered to secrete the cytokine. Intragastric administration of IL-10-secreting Lactococcus lactis caused a 50% reduction in colitis in mice treated with dextran sulfate sodium and prevented the onset of colitis in IL-10(-/-) mice. This approach may lead to better methods for cost-effective and long-term management of IBD in humans.
Assuntos
Doenças Inflamatórias Intestinais/terapia , Interleucina-10/administração & dosagem , Interleucina-10/biossíntese , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Probióticos/uso terapêutico , Animais , Transporte Biológico , Colite/imunologia , Colite/patologia , Colite/prevenção & controle , Colite/terapia , Colo/imunologia , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Sulfato de Dextrana , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/prevenção & controle , Interleucina-10/genética , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lactococcus lactis/imunologia , Camundongos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismoRESUMO
Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 occurs independently of caspase activation and cytochrome c release. However, it is dependent on mitochondria and is characterized by increased production of reactive oxygen intermediates that are essential to the death process. To identify signaling molecules involved in this TNF-induced, reactive oxygen intermediate-dependent cell death pathway, we performed a comparative study by two-dimensional gel electrophoresis of phosphoproteins from a mitochondria-enriched fraction derived from TNF-treated and control cells. TNF induced rapid and persistent phosphorylation of the phosphorylation-responsive regulator of the microtubule (MT) dynamics, oncoprotein 18 (Op18). By using induced overexpression of wild type Op18 and phosphorylation site-deficient mutants S25A/S38A and S16A/S63A in L929 cells, we show that TNF-induced phosphorylation on each of the four Ser residues of Op18 promotes cell death and that Ser(16) and Ser(63) are the primary sites. This hyperphosphorylation of Op18 is known to completely turn off its MT-destabilizing activity. As a result, TNF treatment of L929 cells induced elongated and extremely tangled microtubules. These TNF-induced changes to the MT network were also observed in cells overexpressing wild type Op18 and, to a lesser extent, in cells overexpressing the S25A/S38A mutant. No changes in the MT network were observed upon TNF treatment of cells overexpressing the S16A/S63A mutant, and these cells were desensitized to TNF-induced cell death. These findings indicate that TNF-induced MT stabilization is mediated by hyperphosphorylation of Op18 and that this promotes cell death. The data suggest that Op18 and the MT network play a functional role in transduction of the cell death signal to the mitochondria.
Assuntos
Proteínas dos Microtúbulos , Microtúbulos/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Imidazóis/farmacologia , Camundongos , Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosforilação , Piridinas/farmacologia , Estatmina , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Time kinetics of phosphatidyl serine (PS) exposure were compared to other apoptotic parameters following different apoptotic stimuli. Our data indicate that anti-Fas treatment of L929sAhFas cells results in rapid exposure of PS, which precedes decrease in mitochondrial transmembrane potential (DeltaPsi(m)) and release of cytochrome c, indicating that PS exposure occurs independently of these mitochondrial events. Also during TNF-, etoposide- or staurosporine-mediated apoptosis in PC60 RI/RII cells, PS-positive cells were observed before they had a decreased DeltaPsi(m). However, during growth factor depletion-induced death of 32D cells, both phenomena seemed to occur at the same time.
Assuntos
Apoptose , Grupo dos Citocromos c/metabolismo , Mitocôndrias/metabolismo , Fosfatidilserinas/metabolismo , Animais , Linhagem Celular , Etoposídeo , Substâncias de Crescimento/deficiência , Humanos , Potenciais da Membrana , Camundongos , Estaurosporina , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa , Receptor fas/genética , Receptor fas/farmacologiaRESUMO
TNF is produced during inflammation and induces, among other activities, cell death in sensitive tumour cells. We previously reported an increased generation of ROS in TNF-treated L929 fibrosarcoma cells prior to cell death. These ROS are of mitochondrial origin and participate in the cell death process. Presently, we focus on the identification of parameters that control ROS production and subsequent cytotoxicity. From the cytotoxic properties and susceptibility to scavenging of TNF-induced ROS as compared to pro-oxidant-induced ROS we conclude that TNF-mediated ROS generation and their lethal action are confined to the inner mitochondrial membrane. Oxidative substrates, electron-transport inhibitors, glutathione and thiol-reactive agents but also caspase inhibitors modulate TNF-induced ROS production and imply the existence of a negative regulator of ROS production. Inactivation of this regulator by a TNF-induced reduction of NAD(P)H levels and/or formation of intraprotein disulfides would be responsible for ROS generation.
Assuntos
Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Fibrossarcoma , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , NAD/metabolismo , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/fisiologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In general, apoptotic stimuli lead to activation of caspases. Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members. We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts. Caspase-8 processed all procaspases examined. Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6. However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases. This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway. Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor. Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade. Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases. Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated. The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis.
Assuntos
Caspases/metabolismo , Animais , Caspases/genética , Caspases/imunologia , Caspases/isolamento & purificação , Ativação Enzimática , Escherichia coli , Expressão Gênica , Camundongos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Yeast two-hybrid technology as well as mammalian reporter assays use fusions between a protein of interest and the GAL4 DNA-binding domain (GAL4DB). We demonstrate that expression of a GAL4DB/caspase-1 chimeric protein in yeast leads to autoproteolytic cleavage of GAL4DB. Moreover, recombinant GAL4DB is a good in vitro substrate for recombinant caspase-1 and several other caspases. Cleavage sites map at the C-terminus of GAL4DB and result in release of the fused protein. The finding that GAL4DB can be cleaved by caspases has important implications for the use of caspases in two-hybrid analysis and in the interpretation of mammalian assays based on GAL4-dependent reporter gene expression.