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1.
Haemophilia ; 18(6): 941-7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22812621

RESUMO

The objective of the present study was to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the new recombinant FVIII compound turoctocog alfa and a Glyco-PEGylated FVIII derivative thereof (N8-GP) in Haemophilia A dogs. Six haemophilic dogs divided into two groups were included in the study. Each dog was administered a dose of 125 U kg(-1) , blood samples were collected at predetermined time points for both pharmacokinetic (FVIII measured by one-stage aPTT assay) and pharmacodynamic [whole blood clotting time (WBCT)] evaluations. After intravenous administration to haemophilic dogs, the plasma concentration at the first sampling point was comparable for turoctocog alfa and N8-GP, and the clearance was estimated to be 6.5 and 3.9 mL h(-1) kg(-1) for turoctocog alfa and N8-GP respectively. Both turoctocog alfa and N8-GP were able to reduce the WBCT time to normal levels (<20 min), however, the reduced clearance was reflected in the WBCT, which returned to baseline at a later time point for N8-GP as compared with dogs dosed with turoctocog alfa. The clearance was 40% reduced for N8-GP as compared with turoctocog alfa. Simulations of a multiple dosing regimen in dogs, suggest that to maintain WBCT <20 min N8-GP can be dosed at reduced intervals, e.g. with 4 days between doses, whereas turoctocog alfa will have to be dosed with 2½ day between doses. Data thereby supports N8-GP as an alternative to standard rFVIII replacement therapy, with a more convenient dosing regimen.


Assuntos
Fator VIII/farmacocinética , Hemofilia A/tratamento farmacológico , Polietilenoglicóis/farmacocinética , Animais , Cães , Fator VIII/análise , Fator VIII/uso terapêutico , Feminino , Meia-Vida , Masculino , Tempo de Tromboplastina Parcial , Polietilenoglicóis/análise , Polietilenoglicóis/química , Polietilenoglicóis/uso terapêutico , Tromboelastografia , Tempo de Coagulação do Sangue Total
2.
Mol Oral Microbiol ; 27(1): 45-56, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22230465

RESUMO

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.


Assuntos
Periodontite Agressiva/enzimologia , Periodontite Crônica/enzimologia , Líquido do Sulco Gengival/enzimologia , Porphyromonas gingivalis/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Adesinas Bacterianas/metabolismo , Adulto , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Aggregatibacter actinomycetemcomitans/metabolismo , Periodontite Agressiva/microbiologia , Bacteroides/isolamento & purificação , Bacteroides/metabolismo , Estudos de Casos e Controles , Periodontite Crônica/microbiologia , Cisteína Endopeptidases/metabolismo , Elafina/análise , Elafina/metabolismo , Feminino , Cisteína Endopeptidases Gingipaínas , Gengivite/enzimologia , Gengivite/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Porphyromonas gingivalis/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/análise , Inibidor Secretado de Peptidases Leucocitárias/análise , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Serina Proteases/análise , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/análise , Inibidores de Serina Proteinase/metabolismo , Estatísticas não Paramétricas , Treponema denticola/isolamento & purificação , Treponema denticola/metabolismo
3.
J Thromb Haemost ; 8(8): 1763-72, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20524980

RESUMO

BACKGROUND: Canine models have been good predictors of efficacy of hemophilia treatments, including recombinant human coagulation factor (F)VIIa (hFVIIa). However, canine FVIIa and tissue factor (TF) have remained incompletely characterized. OBJECTIVE: To explore canine-human cross-species FVIIa-TF compatibility in order to strengthen the predictive value of canine models in research on FVIIa and TF. METHODS: Canine FVIIa (cFVIIa) and canine TF((1-217)) [cTF((1-217))] were produced by recombinant techniques, and canine-human cross-species FVIIa-TF interactions were characterized in vitro. RESULTS: Recombinant cFVIIa and soluble cTF((1-217)) were produced and purified to homogeneity. hFVIIa and cFVIIa bound with comparably high affinities to cTF((1-217)) (K(D)=6.0±0.7 nm and K(D)=6.0±0.3 nm, respectively) and to cell surface-expressed cTF (K(D)=8.4±0.4 nm and K(D)=7.2±1.2 nm, for (125) I-labeled hFVIIa and cFVII, respectively). In contrast, cFVIIa bound to human TF (hTF) with decreased affinity, both in solution and on cell surfaces. The decreased binding resulted in reduced activity of cFVIIa in functional assays with hTF((1-209)) . In direct comparison, cFVIIa was more active than hFVIIa, both in the absence and the presence of cognate TF. CONCLUSION: The present finding that hFVIIa binds to cTF essentially as it does to hTF substantiates the hypothesis that human FVIIa-TF biology can be reliably recapitulated in canine models on administration of hFVIIa to dogs.


Assuntos
Fator VII/metabolismo , Tromboplastina/metabolismo , Animais , Coagulação Sanguínea , Membrana Celular/metabolismo , Clonagem Molecular , Modelos Animais de Doenças , Cães , Fator VIIa/química , Fibroblastos/metabolismo , Humanos , Cinética , Ligação Proteica , Proteínas Recombinantes/química , Especificidade da Espécie
4.
Cell Death Differ ; 15(5): 938-45, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18309328

RESUMO

Drosophila Nedd2-like caspase (DRONC), an initiator caspase in Drosophila melanogaster and ortholog of human caspase-9, is cleaved during its activation in vitro and in vivo. We show that, in contrast to conclusions from previous studies, cleavage is neither necessary nor sufficient for DRONC activation. Instead, our data suggest that DRONC is activated by dimerization, a mechanism used by its counterparts in humans. Subsequent cleavage at Glu352 stabilizes the active dimer. Since cleavage is at a Glu residue, it has been proposed that DRONC is a dual Asp- and Glu-specific caspase. We used positional-scanning peptide libraries to define the P1-P4 peptide sequence preferences of DRONC, and show that it is indeed equally active on optimized tetrapeptides containing either Asp or Glu in P1. Furthermore, mutagenesis reveals that Asp and Glu residues are equally tolerated at the primary autoprocessing site of DRONC itself. However, when its specificity is tested on a natural substrate, the Drosophila executioner caspase DRICE, a clear preference for Asp emerges. The formerly proposed Glu preference is thus incorrect. DRONC does not differentiate between Asp and Glu in poor substrates, but prefers Asp when tested on a good substrate.


Assuntos
Caspases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Conformação Proteica , Animais , Caspase 9/genética , Caspase 9/metabolismo , Caspases/química , Caspases/genética , Dimerização , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Ativação Enzimática , Humanos , Especificidade por Substrato
5.
Proc Natl Acad Sci U S A ; 98(25): 14250-5, 2001 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-11734640

RESUMO

A critical step in the induction of apoptosis is the activation of the apoptotic initiator caspase 9. We show that at its normal physiological concentration, caspase 9 is primarily an inactive monomer (zymogen), and that activity is associated with a dimeric species. At the high concentrations used for crystal formation, caspase 9 is dimeric, and the structure reveals two very different active-site conformations within each dimer. One site closely resembles the catalytically competent sites of other caspases, whereas in the second, expulsion of the "activation loop" disrupts the catalytic machinery. We propose that the inactive domain resembles monomeric caspase 9. Activation is induced by dimerization, with interactions at the dimer interface promoting reorientation of the activation loop. These observations support a model in which recruitment by Apaf-1 creates high local concentrations of caspase 9 to provide a pathway for dimer-induced activation.


Assuntos
Caspases/química , Caspases/metabolismo , Apoptose , Caspase 9 , Domínio Catalítico , Dimerização , Ativação Enzimática , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
6.
Biochem J ; 357(Pt 2): 575-80, 2001 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11439111

RESUMO

Caspases play an important role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, two of which, baculovirus p35 and members of the inhibitor of apoptosis (IAP) family, are thought to be caspase specific. However, caspases are members of the clan of cysteine proteases designated CD, which also includes animal and plant legumains, and the bacterial proteases clostripain, gingipain-R and gingipain-K. Since these proteases have been proposed to have a common mechanism and evolutionary origin, we hypothesized that the caspase inhibitors may also regulate these other proteases. We tested this hypothesis by examining the effect of the natural caspase inhibitors on other members of protease clan CD. The IAP family proteins were found to have only a slight inhibitory effect on gingipain-R. The cowpox viral cytokine-response modifier A (CrmA) serpin had no effect on any of the proteases tested but a single point mutation of CrmA (Asp-->Lys) resulted in strong inhibition of gingipain-K. More substantial, with respect to the hypothesis, was the strong inhibition of gingipain-K by wild-type p35. The site in p35, required for inhibition of gingipain-K, was mapped to Lys94, seven residues C-terminal to the caspase inhibitory site. Our data indicate that the virally encoded caspase inhibitors have adopted a mechanism that allows them to regulate disparate members of clan CD proteases.


Assuntos
Inibidores de Caspase , Caspases/química , Inibidores de Cisteína Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Baculoviridae/enzimologia , Clonagem Molecular , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleopoliedrovírus/enzimologia , Proteínas de Plantas/antagonistas & inibidores , Mutação Puntual , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Serpinas/farmacologia , Especificidade por Substrato , Proteínas Virais/química , Proteínas Virais/farmacologia
7.
Biochim Biophys Acta ; 1499(3): 191-8, 2001 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11341966

RESUMO

We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis.


Assuntos
Bombyx/genética , Proteínas de Drosophila , Proteínas de Insetos/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Sequência de Aminoácidos , Animais , Apoptose , Baculoviridae/genética , Sequência de Bases , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular , Clonagem Molecular , Inibidores de Cisteína Proteinase/metabolismo , Primers do DNA/genética , Proteínas Inibidoras de Apoptose , Proteínas de Insetos/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Spodoptera , Proteína X Associada a bcl-2
8.
J Biol Chem ; 276(11): 8087-93, 2001 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-11098060

RESUMO

TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-alpha- and Fas-induced apoptosis in vivo. (160)LEVD(163) was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)- and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1- and TRAF2-mediated nuclear factor-kappaB activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF- and Fas-induced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.


Assuntos
Apoptose , Caspases/metabolismo , Proteínas/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Feminino , Humanos , Ativação Linfocitária , Linfócitos/metabolismo , NF-kappa B/metabolismo , Fator 1 Associado a Receptor de TNF , Células Tumorais Cultivadas , Receptor fas/fisiologia
9.
Curr Biol ; 10(21): 1359-66, 2000 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-11084335

RESUMO

BACKGROUND: Inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All IAPs have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. IAPs physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. This allows them to function as sensors and inhibitors of death signals that emanate from a variety of pathways. RESULTS: Here we report the characterization of ML-IAP, a novel human IAP that contains a single BIR and RING finger motif. ML-IAP is a powerful inhibitor of apoptosis induced by death receptors and chemotherapeutic agents, probably functioning as a direct inhibitor of downstream effector caspases. Modeling studies of the structure of the BIR domain revealed it to closely resemble the fold determined for the BIR2 domain of X-IAP. Deletion and mutational analysis demonstrated that integrity of the BIR domain was required for anti-apoptotic function. Tissue survey analysis showed expression in a number of embryonic tissues and tumor cell lines. In particular, the majority of melanoma cell lines expressed high levels of ML-IAP in contrast to primary melanocytes, which expressed undetectable levels. These melanoma cells were significantly more resistant to drug-induced apoptosis. CONCLUSIONS: ML-IAP, a novel human IAP, inhibits apoptosis induced by death receptors and chemotherapeutic agents. The BIR of ML-IAP possesses an evolutionarily conserved fold that is necessary for anti-apoptotic activity. Elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the pathogenesis of this malignancy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos CD/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Inibidores de Caspase , Caspases/genética , Caspases/metabolismo , Linhagem Celular , Doxorrubicina/farmacologia , Genes Reporter , Humanos , Proteínas Inibidoras de Apoptose , Melanócitos/metabolismo , Melanoma/genética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Conformação Proteica , Estrutura Terciária de Proteína , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Células Tumorais Cultivadas , Receptor fas/metabolismo
10.
Biochem J ; 350 Pt 2: 563-8, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10947972

RESUMO

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.


Assuntos
Caspase 1/química , Caspases/química , Peptídeos/química , Tirosina/análogos & derivados , Alanina/química , Apoptose , Sítios de Ligação , Caspase 3 , Caspase 6 , Caspase 7 , Caspase 8 , Caspase 9 , Catálise , Escherichia coli/metabolismo , Corantes Fluorescentes/farmacologia , Glicina/química , Humanos , Cinética , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Serina/química , Especificidade por Substrato , Tirosina/farmacologia , ortoaminobenzoatos/farmacologia
12.
Structure ; 8(7): 789-97, 2000 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-10903953

RESUMO

BACKGROUND: Cowpox virus expresses the serpin CrmA (cytokine response modifier A) in order to avoid inflammatory and apoptotic responses of infected host cells. The targets of CrmA are members of the caspase family of proteases that either initiate the extrinsic pathway of apoptosis (caspases 8 and 10) or trigger activation of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 (caspase 1). RESULTS: We have determined the structure of a cleaved form of CrmA to 2.26 A resolution. CrmA has the typical fold of a cleaved serpin, even though it lacks the N-terminal half of the A helix, the entire D helix, and a portion of the E helix that are present in all other known serpins. The reactive-site loop of CrmA was mutated to contain the optimal substrate recognition sequence for caspase 3; however, the mutation only marginally increased the ability of CrmA to inhibit caspase 3. Superposition of the reactive-site loop of alpha1-proteinase inhibitor on the cleaved CrmA structure provides a model for virgin CrmA that can be docked to caspase 1, but not to caspase 3. CONCLUSIONS: CrmA exemplifies viral economy, selective pressure having resulted in a 'minimal' serpin that lacks the regions not needed for structural integrity or inhibitory activity. The docking model provides an explanation for the selectivity of CrmA. Our demonstration that engineering optimal substrate recognition sequences into the CrmA reactive-site loop fails to generate a good caspase 3 inhibitor is consistent with the docking model.


Assuntos
Apoptose/efeitos dos fármacos , Vírus da Varíola Bovina/química , Serpinas/química , Proteínas Virais/química , Sequência de Aminoácidos , Caspases/metabolismo , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Serpinas/genética , Serpinas/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato , Subtilisina/metabolismo , Proteínas Virais/genética , Proteínas Virais/farmacologia
13.
Proc Natl Acad Sci U S A ; 97(4): 1427-32, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10677478

RESUMO

We cloned a new inhibitor of apoptosis protein (IAP) homolog, SfIAP, from Spodoptera frugiperda Sf-21 cells, a host of insect baculoviruses. SfIAP contains two baculovirus IAP repeat domains followed by a RING domain. SfIAP has striking amino acid sequence similarity with baculoviral IAPs, CpIAP and OpIAP, suggesting that baculoviral IAPs may be host-derived genes. SfIAP and baculoviral CpIAP inhibit Bax but not Fas-induced apoptosis in human cells. Their apoptosis-suppressing activity in mammalian cells requires both baculovirus IAP repeat and RING domains. Further biochemical data suggest that SfIAP and CpIAP are specific inhibitors of mammalian caspase-9, the pinnacle caspase in the mitochondria/cytochrome c pathway for apoptosis, but are not inhibitors of downstream caspase-3 and caspase-7. Thus the mechanisms by which insect and baculoviral IAPs suppress apoptosis may involve inhibition of an insect caspase-9 homologue. Peptides representing the IAP-binding domain of the Drosophila cell death protein Grim abrogated human caspase suppression by SfIAP and CpIAP, implying evolutionary conservation of the functions of IAPs and their inhibitors.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Bactérias/genética , Inibidores de Caspase , Proteínas de Drosophila , Inibidores Enzimáticos/química , Proteínas de Insetos/química , Proteínas , Spodoptera/química , Sequência de Aminoácidos , Animais , Apoptose/genética , Proteínas de Bactérias/química , Baculoviridae/química , Caspase 9 , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Evolução Molecular , Humanos , Proteínas Inibidoras de Apoptose , Dados de Sequência Molecular , Neuropeptídeos/química , Fragmentos de Peptídeos/farmacologia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/farmacologia
14.
Biochim Biophys Acta ; 1477(1-2): 299-306, 2000 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-10708865

RESUMO

Animal development and homeostasis is a balance between cell proliferation and cell death. Physiologic, and sometimes pathologic, cell death - apoptosis - is driven by activation of a family of proteases known as the caspases, present in almost all nucleated animal cells. The enzymatic properties of these proteases are governed by a dominant specificity for substrates containing Asp, and by the use of a Cys side chain for catalyzing peptide bond cleavage. The primary specificity for Asp turns out to be very rare among proteases, and currently the only other known mammalian proteases with the same primary specificity is the physiological caspase activator granzyme B. Like most other proteases, the caspases are synthesized as inactive zymogens whose activation requires limited proteolysis or binding to co-factors. To transmit the apoptotic execution signal, caspase zymogens are sequentially activated through either an intrinsic or an extrinsic pathway. The activation of caspases at the apex of each pathway, the initiators, occurs by recruitment to specific adapter molecules through homophilic interaction domains, and the activated initiators directly process the executioner caspases to their catalytically active forms. In the present communication we review the different mechanisms underlying the selective activation of the caspases.


Assuntos
Caspases/metabolismo , Precursores Enzimáticos/metabolismo , Serina Endopeptidases/metabolismo , Animais , Apoptose , Sítios de Ligação , Caspases/química , Ativação Enzimática , Precursores Enzimáticos/química , Granzimas , Humanos , Conformação Proteica , Transdução de Sinais , Especificidade por Substrato
16.
Cell Death Differ ; 6(11): 1054-9, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10578173

RESUMO

Caspase stands for cysteine-dependent aspartate specific protease, and is a term coined to define proteases related to interleukin 1beta converting enzyme and CED-3.1 Thus their enzymatic properties are governed by a dominant specificity for substrates containing Asp, and by the use of a Cys side-chain for catalyzing peptide bond cleavage. The use of a Cys side chain as a nucleophile during peptide bond hydrolysis is common to several protease families. However, the primary specificity for Asp turns out to be very rare among protease families throughout biotic kingdoms. Of all known mammalian proteases only the caspase activator granzyme B, a serine protease, has the same primary specificity. In addition to this unusual primary specificity, caspases are remarkable in that certain of their zymogens have intrinsic proteolytic activity. This latter property is essential to trigger the proteolytic pathways that lead to apoptosis. Here we review the known enzymatic properties of the caspases and their zymogens within the broad context of structure:mechanism:activity relationships of proteases in general.


Assuntos
Caspases/metabolismo , Animais , Sítios de Ligação , Catálise , Ativação Enzimática , Humanos , Peptídeos/metabolismo , Especificidade por Substrato
17.
EMBO J ; 18(19): 5242-51, 1999 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-10508158

RESUMO

Several human inhibitor of apoptosis (IAP) family proteins function by directly inhibiting specific caspases in a mechanism that does not require IAP cleavage. In this study, however, we demonstrate that endogenous XIAP is cleaved into two fragments during apoptosis induced by the tumor necrosis factor family member Fas (CD95). The two fragments produced comprise the baculoviral inhibitory repeat (BIR) 1 and 2 domains (BIR1-2) and the BIR3 and RING (BIR3-Ring) domains of XIAP. Overexpression of the BIR1-2 fragment inhibits Fas-induced apoptosis, albeit at significantly reduced efficiency compared with full-length XIAP. In contrast, overexpression of the BIR3-Ring fragment results in a slight enhancement of Fas-directed apoptosis. Thus, cleavage of XIAP may be one mechanism by which cell death programs circumvent the anti-apoptotic barrier posed by XIAP. Interestingly, ectopic expression of the BIR3-Ring fragment resulted in nearly complete protection from Bax-induced apoptosis. Use of purified recombinant proteins revealed that BIR3-Ring is a specific inhibitor of caspase-9 whereas BIR1-2 is specific for caspases 3 and 7. Therefore XIAP possesses two different caspase inhibitory activities which can be attributed to distinct domains within XIAP. These data may provide an explanation for why IAPs have evolved with multiple BIR domains.


Assuntos
Caspases/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Apoptose/imunologia , Sequência de Bases , Inibidores de Caspase , Primers do DNA , Humanos , Hidrólise , Proteínas Inibidoras de Apoptose , Células Jurkat , Proteínas/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Receptor fas/imunologia
18.
Blood ; 94(5): 1683-92, 1999 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10477693

RESUMO

Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-delta. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, 'apoptotic' substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.


Assuntos
Plaquetas/fisiologia , Calpaína/fisiologia , Ativação Plaquetária , Sequência de Aminoácidos , Apoptose/fisiologia , Plaquetas/patologia , Caspase 3 , Caspase 9 , Caspases/fisiologia , Humanos , Dados de Sequência Molecular , Transdução de Sinais
19.
Methods ; 17(4): 313-9, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10196102

RESUMO

Caspases and their involvement in programmed cell death have been an area of significant interest since their initial identification in 1992. To facilitate the search for new components involved in cell death, and to aid researchers in understanding the interactions between currently known cell death proteins, we describe a number of techniques commonly used in the preparation and characterization of caspases.


Assuntos
Apoptose , Caspases/química , Caspases/biossíntese , Caspases/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica/genética , Cinética , Inibidores de Proteases/farmacologia , Espectrofotometria , Especificidade por Substrato
20.
J Biol Chem ; 274(13): 8359-62, 1999 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-10085063

RESUMO

The recombinant form of the proapoptotic caspase-9 purified following expression in Escherichia coli is processed at Asp315, but largely inactive; however, when added to cytosolic extracts of human 293 cells it is activated 2000-fold in the presence of cytochrome c and dATP. Thus, the characteristic activities of caspase-9 are context-dependent, and its activation may not recapitulate conventional caspase activation mechanisms. To explore this hypothesis we produced recombinant forms of procaspase-9 containing mutations that disabled one or both of the interdomain processing sites of the zymogen. These mutants were able to activate downstream caspases, but only in the presence of cytosolic factors. The mutant with both processing sites abolished had 10% of the activity of wild-type, and was able to support apoptosis, with equal vigor to wild-type, when transiently expressed in 293 cells. Thus caspase-9 has an unusually active zymogen that does not require proteolytic processing, but instead is dependent on cytosolic factors for expression of its activity.


Assuntos
Caspases/metabolismo , Apoptose/genética , Caspase 9 , Caspases/genética , Linhagem Celular , Cumarínicos/metabolismo , Grupo dos Citocromos c/metabolismo , Citosol/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Ativação Enzimática , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Mutação/genética , Oligopeptídeos/metabolismo , Proteínas Recombinantes/metabolismo
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