RESUMO
A series of compounds was designed and prepared as inhibitors of interleukin-1beta converting enzyme (ICE), also known as caspase-1. These inhibitors, which employ a diphenyl ether sulfonamide, were designed to improve potency by forming favorable interactions between the diphenyl ether rings and the prime side hydrophobic region. An X-ray crystal structure of a representative member of the diphenyl ether sulfonamide series bound to the active site of caspase-1 was obtained.
Assuntos
Inibidores de Caspase , Inibidores Enzimáticos/síntese química , Éteres/síntese química , Sulfonamidas/síntese química , Caspase 1/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Éteres/química , Éteres/farmacologia , Modelos Moleculares , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Max is the central component of the Myc/Max/Mad network of transcription factors that regulate growth, differentiation and apoptosis. Whereas the Myc and Mad genes and proteins are highly regulated, Max expression is constitutive and no post-translational regulation is known. We have found that Max is targeted during Fas-induced apoptosis. Max is first dephosphorylated and subsequently cleaved by caspases. Two specific cleavage sites for caspases in Max were identified, one at IEVE(10) decreasing S and one at SAFD(135) decreasing G near the C-terminus, which are cleaved in vitro by caspase-5 and caspase-7 respectively. Mutational analysis indicates that both sites are also used in vivo. Thus Max represents the first caspase-5 substrate. The unusual cleavage after a glutamic acid residue is observed only with full-length, DNA-binding competent Max protein but not with corresponding peptides, suggesting that structural determinants might be important for this activity. Furthermore, cleavage by caspase-5 is inhibited by the protein kinase CK2-mediated phosphorylation of Max at Ser-11, a previously mapped phosphorylation site in vivo. These findings suggest that Fas-mediated dephosphorylation of Max is required for cleavage by caspase-5. The modifications that occur on Max in response to Fas signalling affect the DNA-binding activity of Max/Max homodimers. Taken together, our findings uncover three distinct processes, namely dephosphorylation and cleavage by caspase-5 and caspase-7, that target Max during Fas-mediated apoptosis, suggesting the regulation of the Myc/Max/Mad network through its central component.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ácido Glutâmico , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Células COS , Caspase 7 , Chlorocebus aethiops , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Ligação a DNA/química , Dimerização , Humanos , Imunoglobulina M/farmacologia , Células Jurkat , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transfecção , Receptor fas/fisiologiaRESUMO
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a TNF family member and potent apoptosis inducer. In contrast to TNF-alpha or Fas ligand, relatively little is known about the signaling events activated by TRAIL. In particular, the initial caspase(s) required for TRAIL-induced apoptosis remains to be determined Caspase-3-like protease but not caspase-1-like protease (YVADase) activity rapidly increased in HeLa cells in response to TRAIL treatment. The increase in protease activity correlated with the profile of apoptotic cell death that was inhibited by the pan-caspase inhibitor Z-VAD-fmk. In response to TRAIL, caspase-8, an initiator caspase in death receptor-mediated apoptosis, was activated within 1 h in association with Bid cleavage, cytochrome c release, caspase-3 activation, and DNA fragmentation factor 45 cleavage. Z-IETD-fmk, a caspase-8 inhibitor, completely blocked caspase-8 activation and resulted in inhibition of caspase-3 (a caspase-3-like protease) activation and apoptotic cell death. Overexpression of a caspase-8 dominant negative mutant inhibited apoptosis induced by TRAIL. Caspase-8-deficient Jurkat cells were resistant to both TRAIL and Fas-induced apoptosis, whereas wild-type Jurkat cells were susceptible to both TRAIL- and Fas-induced apoptosis. The caspase-8-reintro duced caspase-8-deficient Jurkat cells acquired normal susceptibility to both TRAIL and agonistic Fas antibody. Reverse transcription-PCR and sequence analyses have revealed that these caspase-8-deficient Jurkat cell express wild-type caspase-10. Therefore, our data indicate that caspase-8 is required for TRAIL-induced apoptosis and suggest that caspase-10 may play a minor role, if any, in TRAIL-induced apoptosis.
Assuntos
Apoptose/fisiologia , Caspases/fisiologia , Glicoproteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Reguladoras de Apoptose , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Células HeLa , Humanos , Células Jurkat , Glicoproteínas de Membrana/farmacologia , Oligopeptídeos/farmacologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Atypical protein kinase C zeta (PKCzeta) is known to transduce signals that influence cell proliferation and survival. Here we show that recombinant human caspases can process PKCzeta at three sites in the hinge region between the regulatory and catalytic domains. Caspase-3, -6, -7, and -8 chiefly cleaved human PKCzeta at EETD downward arrowG, and caspase-3 and -7 also cleaved PKCzeta at DGMD downward arrowG and DSED downward arrowL, respectively. Processing of PKCzeta expressed in transfected cells occurred chiefly at EETD downward arrowG and DGMD downward arrowG and produced carboxyl-terminal polypeptides that contained the catalytic domain. Epitope-tagged PKCzeta that lacked the regulatory domain was catalytically active following expression in HeLa cells. Induction of apoptosis in HeLa cells by tumor necrosis factor alpha plus cycloheximide evoked the conversion of full-length epitope-tagged PKCzeta to two catalytic domain polypeptides and increased PKCzeta activity. A caspase inhibitor, zVAD-fmk, prevented epitope-tagged PKCzeta processing and activation following the induction of apoptosis. Induction of apoptosis in rat parotid C5 cells produced catalytic domain polypeptides of endogenous PKCzeta and increased PKCzeta activity. Caspase inhibitors prevented the increase in PKCzeta activity and production of the catalytic domain polypeptides. Treatment with lactacystin, a selective inhibitor of the proteasome, caused polyubiquitin-PKCzeta conjugates to accumulate in cells transfected with the catalytic domain or full-length PKCzeta, or with a PKCzeta mutant that was resistant to caspase processing. We conclude that caspases process PKCzeta to carboxyl-terminal fragments that are catalytically active and that are degraded by the ubiquitin-proteasome pathway.
Assuntos
Caspases/metabolismo , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteína Quinase C/metabolismo , Ubiquitinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Domínio Catalítico , Linhagem Celular , Cricetinae , Cicloeximida/farmacologia , Primers do DNA , Ativação Enzimática , Células HeLa , Humanos , Hidrólise , Complexo de Endopeptidases do Proteassoma , Proteína Quinase C/química , Processamento de Proteína Pós-Traducional , Ratos , Radioisótopos de Enxofre , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Mitochondrial cytochrome c release plays a critical role in apoptotic signal cascade after the activation of cell surface death receptors. We investigated the role played by nitric oxide (NO) in mitochondrial apoptotic signaling in tumor necrosis factor alpha (TNF-alpha) plus actinomycin D (TNF-alpha/ActD)-induced apoptosis. NO produced either by S-nitroso-N-acetyl-DL-penicillamine (SNAP) or inducible NO synthase (iNOS) prevented TNF-alpha/ActD-induced apoptosis in hepatocytes and also inhibited both caspase-8-like (IETDase) and caspase-3-like protease (DEVDase) activity as well as mitochondrial cytochrome c release. Recombinant human (rh) caspase-8 induced the cleavage of the cytochrome c-effluxing factor Bid and cytochrome c release from purified mitochondria in the reconstitution system with Bid(+/+) cytosol, but not with Bid(-/-) cytosol. The addition of SNAP and the caspase-8 inhibitor Ac-IETD-fmk inhibited caspase-8-dependent Bid cleavage and cytochrome c release. The inhibitory effect of NO on caspase-8 was reversed by dithiothreitol (DTT). Furthermore, rh-caspase-8 was found to be modified by S-nitrosylation with 1.7 moles of NO bound per mole of enzyme. Treatment of hepatocytes with interleukin 1beta (IL-1beta) plus interferon gamma (IFN-gamma), which induced iNOS expression and NO production, suppressed TNF-alpha/ActD-induced Bid cleavage and mitochondrial cytochrome c release. The NOS inhibitor N(G)-monomethyl-L-arginine (NMA) inhibited the protective effects of IL-1beta and IFN-gamma. The liver-specific NO donor V-PYRRO/NO also inhibited in vivo elevation of IETDase activity, Bid cleavage, and mitochondrial cytochrome c release in the livers of rats injected with TNF-alpha plus D-galactosamine. Our results indicate that one mechanism by which NO protects hepatocytes from TNF-alpha/ActD-induced apoptosis is via the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Óxido Nítrico/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/metabolismo , Caspase 3 , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/fisiologia , Células Cultivadas , Grupo dos Citocromos c/antagonistas & inibidores , Grupo dos Citocromos c/fisiologia , Dactinomicina/farmacologia , Masculino , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Ratos , Ratos Sprague-DawleyAssuntos
Anti-Inflamatórios/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Inibidores de Cisteína Proteinase/química , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Caspase 1/química , Caspase 1/genética , Caspase 1/metabolismo , Caspases/química , Caspases/deficiência , Caspases/genética , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/farmacologia , Citocinas/metabolismo , Desenho de Fármacos , Humanos , Transdução de Sinais , Especificidade por SubstratoRESUMO
The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1beta-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7x10(2) M(-1).s(-1). The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.
Assuntos
Inibidores de Caspase , Serpinas/farmacologia , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Caspases Iniciadoras , Humanos , Hidrólise , Modelos Químicos , Ligação Proteica , Proteínas Recombinantes/antagonistas & inibidoresRESUMO
Degradation of chromosomal DNA into nucleosome-sized fragments is one of the characteristics of apoptotic cell death. Here, we examined whether caspase-activated DNase (CAD) is responsible for the DNA fragmentation that occurs upon exposure to various apoptotic stimuli. When human Jurkat cells were exposed to etoposide, or UV or gamma radiation, a caspase-3-like protease was activated, and nuclear DNA was fragmented. Human TF-1 cells, which are dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF), also underwent apoptosis accompanied by the activation of caspase-3-like protease and DNA fragmentation, when cultured without the cytokine. Both Jurkat and TF-1 cells expressed two forms of ICAD, ICAD-L and ICAD-S, which were cleaved upon exposure to these apoptotic stimuli. Among eight different caspases examined, recombinant caspases 3 and 7 specifically cleaved ICAD synthesized in a cell-free system. An expression plasmid containing mouse ICAD-L mutated at the caspase-3-recognition sites was then introduced into Jurkat and TF-1 cells. When the transformants were induced to undergo apoptosis (by treatment with etoposide, UV or gamma radiation for Jurkat cells, or factor withdrawal for TF-1 cells) they did not show DNA fragmentation, although they still died as a result of these stimuli. These results indicated that CAD, released from ICAD by caspase activation, is involved in the nuclear DNA fragmentation induced by these apoptotic stimuli.
Assuntos
Apoptose , Caspases/metabolismo , DNA/metabolismo , Desoxirribonucleases/metabolismo , Raios gama , Nucleossomos/metabolismo , Raios Ultravioleta , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Caspase 3 , Linhagem Celular , Fragmentação do DNA , Desoxirribonucleases/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Etoposídeo/toxicidade , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , Cinética , Camundongos , Nucleossomos/efeitos dos fármacos , Plasmídeos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We have evaluated 619 aspartic ketones with 9 different types of prime-side groups (acyloxymethyl, aryloxymethyl, arylthiomethyl, alkylthiomethyl, acylamino-oxymethyl, sulfonylaminomethyl, alpha-ketoamide, alpha-(1-phenyl-3-trifluoromethyl-pyrazol-5-yl)oxymethyl (PTP), and aliphatic ketones) as inhibitors of caspase-1. The inhibitory behaviors could be classified as reversible, inactivating, or bimodal (i.e. reversible inhibition followed by slow inactivation) based on the kinetically observed formation of reversible thiohemiketal complexes and conversion to an irreversible thioether adduct, and the mechanism of any given ketone was only poorly predictable on the basis of leaving group structure and chemistry. Among 201 bimodal inhibitors, the rate of conversion of the reversible thiohemiketal complex to the inactive thioether (k(i)) was strictly first-order, consistent with direct conversion of the thiohemiketal to the thioether with no intermediate collapse to free ketone and thiolate. We have examined 22 crystallographic structures of caspase-1 complexed as a thiohemiketal with the inhibitors from 8 different ketone classes, and found the Cys285S-C-C(alpha)-leaving group dihedral angle to be near either to 60 degrees or to 180 degrees. Only the 180 degrees conformation was permissive for SN2 displacement of the leaving group and, furthermore, positioned His237Ndelta to stabilize developing charge on the leaving group. Among these structures and 19 additional complexes, all showed a strong interaction between His237Ndelta and the ketone or thiohemiketal oxygen. We therefore propose a proteolytic mechanism for caspase-1 involving polarization of the scissile carbonyl by the His237 imidazolium group. During thiohemiketal/thioether conversion (but probably not during peptide hydrolysis), the leaving group is stabilized by the His237 imidazolium.
Assuntos
Caspase 1/química , Inibidores de Caspase , Cetonas/farmacologia , Cristalografia por Raios X , Concentração Inibidora 50 , Cinética , Modelos Químicos , Modelos Moleculares , Piridinas/farmacologia , Fatores de TempoRESUMO
We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Animais , Caspase 3 , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose , Oligopeptídeos/farmacologia , Rad51 Recombinase , Radiação Ionizante , Serpinas/farmacologia , Fator de Necrose Tumoral alfa , Células U937 , Proteínas Virais/farmacologiaRESUMO
It is now known that caspase-3-like protease activation can promote Bcl-2 cleavage and mitochondrial cytochrome c release and that these events can lead to further downstream caspase activation. NO has been proposed as a potent, endogenous inhibitor of caspase-3-like protease activity. Experiments were carried out to determine whether NO could interrupt Bcl-2 cleavage or cytochrome c release by the inhibition of caspase activity linking these events. NO inhibited the capacity of purified caspase-3 to cleave recombinant Bcl-2. Both Bcl-2 cleavage and cytochrome c release were inhibited in tumor necrosis factor alpha- and actinomycin D-treated MCF-7 cells exposed to NO donors. The NO-mediated inhibition of Bcl-2 cleavage and cytochrome c release occurred in association with an inhibition of apoptosis and caspase-3-like activation. Thus, NO suppresses a key step in the positive feedback amplification of apoptotic signaling by preventing Bcl-2 cleavage and cytochrome c release.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Caspase , Grupo dos Citocromos c/metabolismo , Óxido Nítrico/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Caspase 3 , Dactinomicina/farmacologia , Ativação Enzimática , Fígado/citologia , Fígado/enzimologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Procytokine processing by caspase-1 is required for the maturation and release of IL-1beta and IFN-gamma-inducing factor (IGIF) (or IL-18) from activated macrophages (Mphi). Nitric oxide (NO) has emerged as a potent inhibitor of cysteine proteases. Here, we tested the hypothesis that NO regulates cytokine release by inhibiting IL-1beta-converting enzyme (ICE) or caspase-1 activity. Activated RAW264.7 cells released four to five times more IL-1beta, but not TNF-alpha, in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine. Stimulated peritoneal Mphi from wild-type mice (inducible NO synthase (iNOS)+/+) also released more IL-1beta if exposed to N(G)-monomethyl-L-arginine, whereas Mphi from iNOS knockout mice (iNOS-/-) did not. Inhibition of NO synthesis in stimulated RAW264.7 cells also resulted in a threefold increase in intracellular caspase-1 activity. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited caspase-1 activity in cells as well as the activity of purified recombinant caspase-1 and also prevented the cleavage of pro-IL-1beta and pro-IGIF by recombinant caspase-1. The inhibition of caspase-1 by NO was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. An in vivo role for the regulation of caspase-1 by NO was established in iNOS knockout animals, which exhibited significantly higher plasma levels of IL-1beta and IFN-gamma than their wild-type counterparts at 10 h following LPS injection. Taken together, these data indicate that NO suppresses IL-1beta and IGIF processing by inhibiting caspase-1 activity, providing evidence for a unique role for induced NO in regulating IL-1beta and IGIF release.
Assuntos
Caspase 1/metabolismo , Interleucina-18/metabolismo , Interleucina-1/metabolismo , Macrófagos Peritoneais/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/imunologia , Animais , Células COS , Inibidores de Caspase , Inibidores Enzimáticos/farmacologia , Interleucina-1/imunologia , Interleucina-18/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Camundongos , Óxido Nítrico Sintase/antagonistas & inibidores , ômega-N-Metilarginina/farmacologiaRESUMO
Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreactive fragments of Mr 75 K and 30 K In apoptotic SH-SY5Y human neuroblastoma cells, HMW calpastatin was degraded to a major immunoreactive fragment of 75 K. In both cell types, fragmentation of HMW calpastatin was blocked by a caspase-specific inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene. In vitro translated HMW calpastatin was sensitive to proteolysis by recombinant caspase-1, -3, and -7. By contrast, in vitro translated LMW calpastatin (which lacks domains L and I) was cleaved into multiple fragments only by caspase-1 and was relatively resistant to caspase-3, -7, and other caspases tested. Consistently with that, purified erythroid LMW calpastatin was also highly susceptible to caspase-1 digestion. Recombinant human calpastatin spanning domain I through III (CAST(DI-III)) was found cleaved by caspase-1 at at least three sites, located in either the A or the C helix of domains I and III (ALDD137*L, LSSD203*F and ALAD404*S), while only a single site (ALDD137*L) was cleaved by caspase-3. These findings suggest that both HMW and LMW calpastatins are more vulnerable to caspase-1 than to caspase-3. Surprisingly, both erythroid LMW calpastatin and recombinant CAST(DI-III) fragmented by caspase-1 suffered only a less than twofold reduction of inhibitory activity toward calpain. We propose that the proteolysis of calpastatin in early apoptosis might have yet unidentified effects on the cross-talk between the two protease systems.
Assuntos
Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/antagonistas & inibidores , Caspases , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/fisiologia , Inibidores de Cisteína Proteinase/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sítios de Ligação/efeitos dos fármacos , Calpaína/metabolismo , Caspase 1 , Caspase 3 , Humanos , Hidrólise , Células Jurkat , Peso Molecular , Neuroblastoma , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
The degradation of alphaII- and betaII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both alphaII- and betaII-subunits. Major C-terminal alphaII-spectrin BDPs were detected at approximately 150, 145, and 120 kDa (alphaII-BDP150, alphaII-BDP145, and alphaII-BDP120, respectively); major C-terminal betaII-spectrin BDPs were at approximately 110 and 85 kDa (betaII-BDP110 and betaII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that alphaII-BDP150 and alphaII-BDP120 were generated by cleavages at DETD1185*S1186 and DSLD1478*S1479, respectively. For betaII-spectrin, a major caspase site was detected at DEVD1457*S1458, and both betaII-BDP110 and betaII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146*S2147, was found to account for betaII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both alphaII- and betaII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.
Assuntos
Apoptose , Caspases , Cisteína Endopeptidases/metabolismo , Espectrina/metabolismo , Sequência de Aminoácidos , Caspase 3 , Humanos , Hidrólise , Cinética , Espectrina/química , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
BACKGROUND: Fas is a member of the tumour necrosis factor (TNF) receptor family. Activation of Fas by its ligand or an agonistic anti-Fas antibody causes apoptosis in Fas-bearing cells, by activating various members of the caspase family. RESULTS: Specific fluorogenic substrates (MCA-DEVDAPK[dnp] and MCA-VEVDAPK[dnp]) for caspases 3 and 6 were prepared. Using these substrates, a gradual increase of the caspase 3-and 6-like proteases were detected during the Fas engagement in human Jurkat. This activation of caspases correlated well with the cleavage of poly(ADP-ribose) polymerase and lamin B1, as well as with DNA fragmentation. When the recombinant caspases were added to the extracts from Jurkat cells, caspase 3 produced active caspase 6-like protease, while caspase 6 activated the caspase 3 protease, suggesting that these proteases can activate each other. The caspase-treated cell extracts, as well as the extracts from the Fas-activated cells, caused the proteolysis of nuclear proteins and DNA degradation. The cleavage of nuclear proteins was inhibited by caspase inhibitors, while the same inhibitors had no effect on DNA degradation. CONCLUSIONS: At one stage of the caspase cascade, caspases activate each other, and amplify the apoptotic signal. Caspases downstream of the cascade then cause the proteolysis of nuclear proteins and DNA degradation.
Assuntos
Caspases , Cisteína Endopeptidases/fisiologia , Fragmentação do DNA/fisiologia , Lamina Tipo B , Proteínas Nucleares/metabolismo , Receptor fas/fisiologia , Apoptose/fisiologia , Western Blotting , Caspase 1 , Caspase 3 , Caspase 6 , Extratos Celulares , DNA Topoisomerases Tipo I/metabolismo , Ativação Enzimática , Corantes Fluorescentes , Humanos , Células Jurkat , Laminas , Oligopeptídeos/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases , Proteínas/metabolismo , Especificidade por Substrato , Fatores de TempoRESUMO
The caspases are a family of at least 10 human cysteine proteases that participate in cytokine maturation and in apoptotic signal transduction and execution mechanisms. Peptidic inhibitors of these enzymes are capable of blocking cytokine maturation and apoptosis, demonstrating their crucial roles in these processes. We have recently discovered that nitric oxide (NO), produced either extracellularly by NO donors or intracellularly by the inducible nitric oxide synthase, prevented apoptosis in hepatocytes. Caspase-3-like activity was found to be inhibited under these conditions. To investigate further the interaction between NO and caspases, we utilized purified human recombinant caspases and examined the effect of NO on enzymatic activities of different caspases. We report here that of the seven caspases studied, all were reversibly inhibited by NO. Dithiothreitol was able to reverse the NO inhibition, indicating direct S-nitrosylation of caspase catalytic cysteine residue by NO. Our results support the concept that NO is an endogenous regulator of caspase activity.
Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Fígado/enzimologia , Óxido Nítrico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Boratos/farmacologia , Células Cultivadas , Citosol/enzimologia , Dactinomicina/farmacologia , Ditiotreitol/farmacologia , Humanos , Cinética , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Cloreto de Mercúrio/farmacologia , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Compostos Nitrosos , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Ratos , Ratos Sprague-Dawley , S-Nitroso-N-Acetilpenicilamina , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We report that the serine protease granzyme B (GrB), which is crucial for granule-mediated cell killing, initiates apoptosis in target cells by first maturing caspase-10. In addition, GrB has a limited capacity to mature other caspases and to cause cell death independently of the caspases. Compared with other members, GrB in vitro most efficiently processes caspase-7 and -10. In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present. Furthermore, GrB matured caspase-3 with less efficiency than caspase-7 or caspase-10. With the caspases fully inactivated by peptidic inhibitors, GrB induced in Jurkat cells growth arrest and, over a delayed time period, cell death. Thus, the primary mechanism by which GrB initiates cell death is activation of the caspases through caspase-10. However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates. The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.
Assuntos
Apoptose/imunologia , Caspases , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/imunologia , Citotoxicidade Imunológica , Serina Endopeptidases/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama , Caspase 3 , Caspase 7 , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Cisteína Endopeptidases/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Granzimas , Humanos , Células Jurkat , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/metabolismo , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1beta converting enzyme (ICE) family proteases, we have examined in detail the specificities beyond the P1 position of caspase-1, -2, -3, -4, -6, and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors.
Assuntos
Caspases , Cisteína Endopeptidases/metabolismo , Caspase 1 , Caspase 3 , Caspase 6 , Caspase 7 , Cromatografia Líquida de Alta Pressão , Cisteína Endopeptidases/química , Corantes Fluorescentes/metabolismo , Cinética , Oligopeptídeos/metabolismo , Transdução de Sinais , Especificidade por SubstratoRESUMO
FADD/MORT1 is a death domain (DD)-containing adaptor/signaling molecule that interacts with the intracellular DD of FAS/APO-I (CD95) and tumor necrosis factor receptor 1 and the prodomain of caspase-8 (Mch5/MACH/FLICE). FADD engagement of caspase-8 presumably activates this caspase and leads to apoptosis. Another DD-containing adaptor/signaling molecule, CRADD, was identified and was shown to induce apoptosis. CRADD has a dual-domain structure similar to that of FADD. It has an NH2-terminal caspase homology domain that interacts with caspase-2 and a COOH-terminal DD that interacts with RIP. CRADD is constitutively expressed in many tissues and thus could play a role in regulating apoptosis in mammalian cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose/fisiologia , Proteínas de Transporte/genética , Proteínas/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Proteína Adaptadora de Sinalização CRADD , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteína Ligante Fas , Proteína de Domínio de Morte Associada a Fas , Feto , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Homologia de Sequência de Aminoácidos , Receptor fas/genéticaRESUMO
The response of eukaryotic cells to ionizing radiation (IR) includes induction of apoptosis. However, the signals that regulate this response are unknown. The present studies demonstrate that IR treatment of U-937 cells is associated with: (i) internucleosomal DNA fragmentation; (ii) cleavage of poly(ADP-ribose) polymerase; (iii) cleavage of protein kinase C delta; and (iv) induction of an Ac-DEVD-p-nitroanilide cleaving activity. Overexpression of the cowpox protein CrmA blocked tumor necrosis factor (TNF)-induced apoptosis but had no effect on IR-induced DNA fragmentation or cleavage of poly(ADP-ribose) polymerase and protein kinase C delta. By contrast, overexpression of the baculovirus p35 protein blocked both IR- and TNF-induced apoptosis. The results further demonstrate that the IR-induced proteolytic activity is directly inhibited by the addition of purified recombinant p35, but not by CrmA. We show that the CPP32 protease is sensitive to p35 and not CrmA. We also show that IR induces activation of CPP32 and that this event, like induction of apoptosis, is sensitive to overexpression of p35 and not CrmA. These findings indicate that IR-induced apoptosis involves activation of CPP32 and that this CrmA-insensitive apoptotic pathway is distinct from those induced by TNF and certain other stimuli.