RESUMO
Cellular FLICE-like inhibitory protein (cFLIP) is a member of the Death Domain superfamily with pivotal roles in many cellular processes and disease states, including cancer and autoimmune disorders. In the context of the death-inducing signaling complex (DISC), cFLIP isoforms regulate extrinsic apoptosis by controlling procaspase-8 activation. The function of cFLIP is mediated through a series of protein-protein interactions, engaging the two N-terminal death effector domains (DEDs). Here, we solve the structure of an engineered DED1 domain of cFLIP using solution nuclear magnetic resonance (NMR) and we define the interaction with FADD and calmodulin, protein-protein interactions that regulate the function of cFLIP in the DISC. cFLIP DED1 assumes a canonical DED fold characterized by six α helices and is able to bind calmodulin and FADD through two separate interfaces. Our results clearly demonstrate the role of DED1 in the cFLIP/FADD association and contribute to the understanding of the assembly of DISC filaments.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Calmodulina/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Engenharia de Proteínas/métodos , Sítios de Ligação , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Mapas de Interação de Proteínas , Estrutura Secundária de ProteínaRESUMO
Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 8/química , Proteína de Domínio de Morte Associada a Fas/química , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Domínio Catalítico , Clonagem Molecular , Microscopia Crioeletrônica , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Morte Celular Regulada/genética , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Regenerative capacity in vital organs is limited by fibrosis propensity. Idiopathic pulmonary fibrosis (IPF), a progressive lung disease linked with aging, is a classic example. In this study, we show that in flow cytometry, immunoblots (IB) and in lung sections, FLIP levels can be regulated, in vivo and in vitro, through SIRT1 activity inhibition by CMH (4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide), a small molecule that, as we determined here by structural biology calculations, docked into its nonhistone substrate Ku70-binding site. Ku70 immunoprecipitations and immunoblots confirmed our theory that Ku70-deacetylation, Ku70/FLIP complex, myofibroblast resistance to apoptosis, cell survival, and lung fibrosis in bleomycin-treated mice, are reduced and regulated by CMH. Thus, small molecules associated with SIRT1-mediated regulation of Ku70 deacetylation, affecting FLIP stabilization in fibrotic-lung myofibroblasts, may be a useful strategy, enabling tissue regeneration.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Ácidos Hidroxâmicos/administração & dosagem , Fibrose Pulmonar Idiopática/tratamento farmacológico , Autoantígeno Ku/metabolismo , Pulmão/citologia , Sirtuína 1/química , Sirtuína 1/metabolismo , Acetilação/efeitos dos fármacos , Animais , Sítios de Ligação , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacosRESUMO
Pharmacological targeting via small molecule-based chemical probes has recently acquired an emerging importance as a valuable tool to delineate molecular mechanisms. Induction of apoptosis via CD95/Fas and TRAIL-R1/2 is triggered by the formation of the death-inducing signaling complex (DISC). Caspase-8 activation at the DISC is largely controlled by c-FLIP proteins. However molecular mechanisms of this control have just started to be uncovered. In this study we report the first-in-class chemical probe targeting c-FLIPL in the heterodimer caspase-8/c-FLIPL. This rationally designed small molecule was aimed to imitate the closed conformation of the caspase-8 L2' loop and thereby increase caspase-8 activity after initial processing of the heterodimer. In accordance with in silico predictions, this small molecule enhanced caspase-8 activity at the DISC, CD95L/TRAIL-induced caspase activation, and subsequent apoptosis. The generated computational model provided further evidence for the proposed effects of the small molecule on the heterodimer caspase-8/c-FLIPL. In particular, the model has demonstrated that boosting caspase-8 activity by the small molecule at the early time points after DISC assembly is crucial for promoting apoptosis induction. Taken together, our study allowed to target the heterodimer caspase-8/c-FLIPL and get new insights into molecular mechanisms of its activation.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Multimerização Proteica , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 8/química , Linhagem Celular Tumoral , Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Proteína Ligante Fas , Humanos , Modelos Moleculares , Reprodutibilidade dos Testes , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Domínio Efetor de Morte , Sequência de Aminoácidos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Células HeLa , Humanos , Células Jurkat , Modelos Moleculares , Isoformas de Proteínas/metabolismo , Transporte Proteico , Receptor fas/metabolismoRESUMO
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 8/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteína de Domínio de Morte Associada a Fas/química , Proteínas Virais/química , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Sítios de Ligação , Proteínas Adaptadoras de Sinalização CARD , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Domínio Efetor de Morte , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Expressão Gênica , Humanos , Células Jurkat , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismo , Receptor fas/farmacologiaRESUMO
The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis.
Assuntos
Apoptose , Fatores de Transcrição NFATC/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Pontos de Checagem do Ciclo Celular , Transformação Celular Neoplásica , Proteína Ligante Fas/química , Proteína Ligante Fas/metabolismo , Humanos , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/metabolismo , Fatores de Transcrição NFATC/química , Proteínas Nucleares/metabolismo , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIPL and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIPL and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Calmodulina/química , Apoptose , Sítios de Ligação , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular Tumoral , Humanos , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos/metabolismo , Linfócitos T/metabolismo , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/genética , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Immunoblotting , Interleucina-2/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas c-raf , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Linfócitos T/citologia , Fator 2 Associado a Receptor de TNF/metabolismoAssuntos
Receptor fas/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspases/metabolismo , Morte Celular , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Células HeLa , Humanos , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
The antiapoptotic protein FLIP(S) is a key suppressor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human glioblastoma multiforme (GBM) cells. We previously reported that a novel phosphatase and tensin homologue (PTEN)-Akt-atrophin-interacting protein 4 (AIP4) pathway regulates FLIP(S) ubiquitination and stability, although the means by which PTEN and Akt were linked to AIP4 activity were unclear. Here, we report that a second regulator of ubiquitin metabolism, the ubiquitin-specific protease 8 (USP8), is a downstream target of Akt, and that USP8 links Akt to AIP4 and the regulation of FLIP(S) stability and TRAIL resistance. In human GBM xenografts, levels of USP8 correlated inversely with pAkt levels, and genetic or pharmacologic manipulation of Akt regulated USP8 levels in an inverse manner. Overexpression of wild-type USP8, but not catalytically inactive USP8, increased FLIP(S) ubiquitination, decreased FLIP(S) half-life, decreased FLIP(S) steady-state levels, and decreased TRAIL resistance, whereas short interfering RNA (siRNA)-mediated suppression of USP8 levels had the opposite effect. Because high levels of the USP8 deubiquitinase correlated with high levels of FLIP(S) ubiquitination, USP8 seemed to control FLIP(S) ubiquitination through an intermediate target. Consistent with this idea, overexpression of wild-type USP8 decreased the ubiquitination of the FLIP(S) E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIP(S) interaction, whereas siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIP(S) levels by USP8 overexpression was reversed by the introduction of siRNA targeting AIP4. These results show that USP8, a downstream target of Akt, regulates the ability of AIP4 to control FLIP(S) stability and TRAIL sensitivity.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Glioblastoma/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Apoptose , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Citometria de Fluxo , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Imunoprecipitação , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Phosphatase and tensin homologue (PTEN) loss and activation of the Akt-mammalian target of rapamycin (mTOR) pathway increases mRNA translation, increases levels of the antiapoptotic protein FLIP(S), and confers resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in glioblastoma multiforme (GBM). In PTEN-deficient GBM cells, however, the FLIP(S) protein also exhibited a longer half-life than in PTEN mutant GBM cells, and this longer half-life correlated with decreased FLIP(S) polyubiquitination. FLIP(S) half-life in PTEN mutant GBM cells was reduced by exposure to an Akt inhibitor, but not to rapamycin, suggesting the existence of a previously undescribed, mTOR-independent linkage between PTEN and the ubiquitin-dependent control of protein stability. Total levels of the candidate FLIP(S) E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) were comparable in PTEN wild-type (WT) and PTEN mutant GBM cells, although in PTEN-deficient cells, AIP4 was maintained in a stable polyubiquitinated state that was less able to associate with FLIP(S) or with the FLIP(S)-containing death inducing signal complex. Small interfering RNA-mediated suppression of AIP4 levels in PTEN WT cells decreased FLIP(S) ubiquitination, prolonged FLIP(S) half-life, and increased TRAIL resistance. Similarly, the Akt activation that was previously shown to increase TRAIL resistance did not alter AIP4 levels, but increased AIP4 ubiquitination, increased FLIP(S) steady-state levels, and suppressed FLIP(S) ubiquitination. These results define the PTEN-Akt-AIP4 pathway as a key regulator of FLIP(S) ubiquitination, FLIP(S) stability, and TRAIL sensitivity and also define a novel link between PTEN and the ubiquitin-mediated control of protein stability.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Glioblastoma/metabolismo , PTEN Fosfo-Hidrolase/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Western Blotting , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/patologia , Meia-Vida , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The tumor suppressor p53 protein supports growth arrest and is able to induce apoptosis, a signaling cascade regulated by sequential activation of caspases. Mechanisms that lead from p53 to activation of individual initiator caspases are still unclear. The present model for caspase-2 activation includes PIDDosome complex formation. However, in certain experimental models, elimination of complex constituents PIDD or RAIDD did not significantly influence caspase-2 activation, suggesting the existence of an alternative activation platform for caspase-2. Here we have investigated the link between p53 and caspase-2 in further detail and report that the latter is able to utilize the CD95 DISC as an activation platform. The recruitment of caspase-8 to this complex is required for activation of caspase-2. In the experimental system used, the DISC is formed through a distinct, p53-dependent upregulation of CD95. Moreover, we show that caspase-2 and -8 cleave Bid, and that both act simultaneously upstream of mitochondrial cytochrome c release. Finally, a direct interaction between the two caspases and the ability of caspase-8 to cleave caspase-2 are demonstrated. Thus, the observed functional link between caspase-8 and -2 within the DISC represents an alternative mechanism to the PIDDosome for caspase-2 activation in response to DNA damage.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 2/metabolismo , Dano ao DNA , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 2/genética , Caspase 8/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fluoruracila/farmacologia , Genes p53 , Células HCT116 , Humanos , Cinética , RNA Interferente Pequeno/metabolismo , Ativação Transcricional , Regulação para Cima , Receptor fas/metabolismoRESUMO
We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP (c-FLIPL), but not the short form (c-FLIPS), enhanced adhesion and motility. Downregulation of c-FLIPL with siRNA decreased phosphorylation of FAK and ERK, while overexpression of c-FLIPL increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed c-FLIPL-promoted motility. Inhibition of ROCK by Y27632 suppressed the c-FLIPL-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of c-FLIPL increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced c-FLIPL- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the c-FLIPL-promoted cell motility. We conclude that c-FLIPL promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Movimento Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Transfecção , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Suppression of apoptosis is one of the hallmarks of carcinogenesis. Tumor cells endure apoptotic pressure by overexpressing several antiapoptotic proteins, and FLICE inhibitory protein (FLIP) is one of the important antiapoptotic proteins that have been shown to be overexpressed in various primary tumor cells. FLIP has two death-effector domains in tandem, mimicking the prodomain of procaspase-8. It is recruited to Fadd in death-inducing signaling complex, thereby preventing the activation of procaspase-8. To date, three isoforms of human cytosolic FLIP (c-FLIP) and six viral homologs (v-FLIP) have been identified. Recently, the crystal structure of v-FLIP MC159 was determined for the first time as an atomic-detail FLIP structure, which revealed that two death effector domains are packed tightly against each other mainly through conserved hydrophobic interactions. The overexpression of c-FLIP in tumor cells has been shown to be the determinant of the tumor's resistance to death ligands such as FasL and TRAIL. It has also been shown that the down-regulation of c-FLIP results in sensitizing resistant tumor cells. Therefore, the agents directly targeting c-FLIP at mRNA and protein levels are expected to be developed in near future and tested for the potential as a new class of anti-cancer drugs.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Inibidores de Caspase , Caspases/metabolismo , Humanos , Neoplasias/patologia , Transdução de SinaisRESUMO
Apoptosis signaling through CD95 (Fas/APO-1) involves aggregation and clustering of the receptor followed by its actin-dependent internalization. Internalization is required for efficient formation of the death-inducing signaling complex (DISC) with maximal recruitment of FADD, caspase-8/10 and c-FLIP occurring when the receptor has reached an endosomal compartment. The first detectable event during CD95 signaling is the formation of SDS-stable aggregates likely reflecting intense oligomerization of the receptor. We now demonstrate that these SDS-stable forms of CD95 correspond to very high molecular weight DISC complexes (hiDISC) and are the sites of caspase-8 activation. hiDISCs are found both inside and outside of detergent-resistant membranes. The formation of SDS-stable CD95 aggregates involves palmitoylation of the membrane proximal cysteine 199 in CD95. Cysteine 199 mutants no longer form SDS-stable aggregates, and inhibition of palmitoylation reduces internalization of CD95 and activation of caspase-8. Our data demonstrate that SDS-stable forms of CD95 are the sites of apoptosis initiation and represent an important early step in apoptosis signaling through CD95 before activation of caspases.