RESUMEN
Chromosomal abnormalities are implicated in a substantial number of human developmental syndromes, but for many such disorders little is known about the causative genes. The recently described 1q41q42 microdeletion syndrome is characterized by characteristic dysmorphic features, intellectual disability and brain morphological abnormalities, but the precise genetic basis for these abnormalities remains unknown. Here, our detailed analysis of the genetic abnormalities of 1q41q42 microdeletion cases identified TP53BP2, which encodes apoptosis-stimulating protein of p53 2 (ASPP2), as a candidate gene for brain abnormalities. Consistent with this, Trp53bp2-deficient mice show dilation of lateral ventricles resembling the phenotype of 1q41q42 microdeletion patients. Trp53bp2 deficiency causes 100% neonatal lethality in the C57BL/6 background associated with a high incidence of neural tube defects and a range of developmental abnormalities such as congenital heart defects, coloboma, microphthalmia, urogenital and craniofacial abnormalities. Interestingly, abnormalities show a high degree of overlap with 1q41q42 microdeletion-associated abnormalities. These findings identify TP53BP2 as a strong candidate causative gene for central nervous system (CNS) defects in 1q41q42 microdeletion syndrome, and open new avenues for investigation of the mechanisms underlying CNS abnormalities.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/deficiencia , Deleción Cromosómica , Proteínas Supresoras de Tumor/deficiencia , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Encéfalo/anomalías , Encéfalo/patología , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/patología , Femenino , Eliminación de Gen , Ventrículos Cardíacos/anomalías , Ventrículos Cardíacos/patología , Imagen por Resonancia Magnética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Defectos del Tubo Neural/patología , Fenotipo , Síndrome , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Apoptosis is orchestrated by a family of cysteine proteases known as the caspases. Fourteen mammalian caspases have been identified, three of which (caspase-3, -6, and -7) are thought to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the relative contributions that the "executioner" caspases make to the demolition of the cell remains speculative. Here we have used cell-free extracts immuno-depleted of either caspase-3, -6, or -7 to examine the caspase requirements for apoptosis-associated proteolysis of 14 caspase substrates as well as nuclear condensation, chromatin margination, and DNA fragmentation. We show that caspase-3 is the primary executioner caspase in this system, necessary for cytochrome c/dATP-inducible cleavage of fodrin, gelsolin, U1 small nuclear ribonucleoprotein, DNA fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD), receptor-interacting protein (RIP), X-linked inhibitor of apoptosis protein (X-IAP), signal transducer and activator of transcription-1 (STAT1), topoisomerase I, vimentin, Rb, and lamin B but not for cleavage of poly(ADP-ribose) polymerase (PARP) or lamin A. In addition, caspase-3 was also essential for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse in this system. Surprisingly, although caspase-6 and -7 are considered to be important downstream effector caspases, depletion of either caspase had minimal impact on any of the parameters investigated, calling into question their precise role during the execution phase of apoptosis.
Asunto(s)
Apoptosis , Caspasas/fisiología , Adenosina Trifosfato/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/metabolismo , Caspasa 3 , Caspasa 6 , Caspasa 7 , Núcleo Celular/metabolismo , Sistema Libre de Células , Cromatina/metabolismo , Grupo Citocromo c/metabolismo , Fragmentación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/metabolismo , Gelsolina/metabolismo , Humanos , Células Jurkat , Lamina Tipo A , Lamina Tipo B , Laminas , Proteínas de Microfilamentos/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Proteína de Retinoblastoma/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Factor de Transcripción STAT1 , Factores de Tiempo , Transactivadores/metabolismo , Vimentina/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
BID, a pro-apoptotic Bcl-2 family member, promotes cytochrome c release during apoptosis initiated by CD95L or TNF. Activation of caspase-8 in the latter pathways results in cleavage of BID, translocation of activated BID to mitochondria, followed by redistribution of cytochrome c to the cytosol. However, it is unclear whether BID participates in cytochrome c release in other (non-death receptor) cell death pathways. Here, we show that BID is cleaved in response to multiple death-inducing stimuli (staurosporine, UV radiation, cycloheximide, etoposide). However BID cleavage in these contexts was blocked by Bcl-2, suggesting that proteolysis of BID occurred distal to cytochrome c release. Furthermore, addition of cytochrome c to Jurkat post-nuclear extracts triggered breakdown of BID at Asp-59 which was catalysed by caspase-3 rather than caspase-8. We provide evidence that caspase-3 catalysed cleavage of BID represents a feedback loop for the amplification of mitochondrial cytochrome c release during cytotoxic drug and UV radiation-induced apoptosis.
Asunto(s)
Apoptosis , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ácido Aspártico/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Caspasa 3 , Catálisis , Sistema Libre de Células , Cicloheximida/farmacología , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Humanos , Células Jurkat , Ratones , Mitocondrias Hepáticas/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Estaurosporina/farmacología , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas de la Nucleocápside , Nucleocápside/metabolismo , Virus de la Gastroenteritis Transmisible/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Antígenos CD13/genética , Antígenos CD13/metabolismo , Caspasa 3 , Caspasa 6 , Caspasa 7 , Caspasa 8 , Caspasa 9 , Extractos Celulares , Proteínas de la Nucleocápside de Coronavirus , Inhibidores de Cisteína Proteinasa/farmacología , Grupo Citocromo c/metabolismo , Citosol/metabolismo , Activación Enzimática , Humanos , Mitocondrias/metabolismo , Nucleocápside/genética , Oligopéptidos/farmacología , Receptores Virales/genética , Receptores Virales/metabolismo , Virus de la Gastroenteritis Transmisible/fisiología , Células Tumorales CultivadasRESUMEN
Caspases participate in the molecular control of apoptosis in several guises; as triggers of the death machinery, as regulatory elements within it, and ultimately as a subset of the effector elements of the machinery itself. The mammalian caspase family is steadily growing and currently contains 14 members. At present, it is unclear whether all of these proteases participate in apoptosis. Thus, current research in this area is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of cell death. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation. As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling (upstream) and effector (downstream) roles based upon their prodomain lengths. In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme. Here, we discuss the apoptosis-associated caspase cascade and the hierarchy of caspase activation events within it.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Animales , Activación Enzimática , Predicción , Mitocondrias , Especificidad por SustratoRESUMEN
Apoptotic protease activating factor-1 (Apaf-1) has been identified as a proximal activator of caspase-9 in cell death pathways that trigger mitochondrial damage and cytochrome c release. The mechanism of Apaf-1 action is unclear but has been proposed to involve the clustering of caspase-9 molecules, thereby facilitating autoprocessing of adjacent zymogens. Here we show that Apaf-1 can dimerize via the CED-4 homologous and linker domains of the molecule providing a means by which Apaf-1 can promote the clustering of caspase-9 and facilitate its activation. Apaf-1 dimerization was repressed by the C-terminal half of the molecule, which contains multiple WD-40 repeats, but this repression was overcome in the presence of cytochrome c and dATP. Removal of the WD-40 repeat region resulted in a constitutively active Apaf-1 that exhibited greater cytotoxicity in transient transfection assays when compared with full-length Apaf-1. These data suggest a mechanism for Apaf-1 function and reveal an important regulatory role for the WD-40 repeat region.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas/metabolismo , Factor Apoptótico 1 Activador de Proteasas , Sitios de Unión , Caspasa 9 , Línea Celular , Dimerización , Activación Enzimática , Humanos , Proteínas/genética , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiaeRESUMEN
It is now well established that the caspases, a family of cysteine proteases, play a key role in apoptosis. Although overexpressing each of the caspases in cells triggered apoptosis, the precise role and contribution of individual caspases are still unclear. Caspase-1, the first caspase discovered, was initially implicated in mammalian apoptosis because of its similarity to the gene product ced-3. Using whole cells as well as an in vitro system to study apoptosis, the role of caspase-1 in Fas-mediated apoptosis in Jurkat T cells was examined in greater detail. Using various peptide-based caspase inhibitors, our results showed that N-acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone efficiently blocked Fas-mediated apoptosis in Jurkat T cells, whereas N-acetyl-Tyr-Val-Ala-Asp aldehyde, which is more specific for caspase-1, had little effect. Cell lysates derived from anti-Fas-stimulated cells, which readily induced apoptotic nuclei morphology and DNA fragmentation in isolated thymocyte nuclei, had no caspase-1 activity using proIL-1beta as a substrate. Time-course studies showed no caspase-1 activity during the activation of apoptosis in Jurkat cells by agonistic Fas antibodies. Furthermore, no pro-caspase-1 protein nor activated form of the protein was detected in normal or apoptotic Jurkat cells. In contrast, both caspase-2 and caspase-3 were readily detected as proenzymes in control cells and their activated forms were detected in apoptotic cells. Incubation of recombinant active caspase-1 with control cell lysates did not activate the apoptotic cascade as shown by the lack of detectable apoptotic nuclei promoting activity using isolated nuclei as substrate. However, under similar conditions proIL-1beta was readily processed into the mature cytokine, indicating that the recombinant caspase-1 remained active in the presence of control cell lysates. Taken together our results demonstrate that caspase-1 is not required for the induction of apoptosis in Jurkat T cells mediated by the Fas antigen.
Asunto(s)
Apoptosis , Caspasa 1/metabolismo , Receptor fas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Caspasa 2 , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Humanos , Interleucina-1/metabolismo , Células Jurkat , Oligopéptidos/farmacología , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.
Asunto(s)
Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Animales , Apoptosis , Factor Apoptótico 1 Activador de Proteasas , Caspasa 10 , Caspasa 2 , Caspasa 3 , Caspasa 6 , Caspasa 7 , Caspasa 8 , Caspasa 9 , Extractos Celulares , Activación Enzimática , Humanos , Células Jurkat , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , ConejosRESUMEN
Recent developments in the apoptosis field have uncovered a family of cysteine proteases, the Caspases, that act as signalling components as well as effectors of the cell death machinery. Caspases are constitutively present as inactive precursors within most cells and undergo proteolytic processing in response to diverse death-inducing stimuli to initiate the death programme. Active caspases can process other caspases of the same type as well as process caspases further downstream in the pathway that ultimately leads to collapse of the cell. This cellular collapse is thought to occur as a consequence of caspase-mediated cleavage of a diverse array of cellular substrates. Regulation of entry into the death programme is controlled at a number of levels by members of the Bcl-2 family, as well as by other cell death regulatory proteins. Recent data has shed light upon the mechanism of action of these regulatory molecules and suggests that the point of caspase activation is a major checkpoint in the cell death programme. Because many transformed cell populations possess derangements in cell death-regulatory genes, such as bcl-2, such cells frequently exhibit elevated resistance to cytotoxic chemotherapy. Thus, a deeper understanding of how apoptosis is normally regulated has therapeutic implications for disease states where the normal controls on the cell death machinery have been subverted.
RESUMEN
Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.