RESUMO
MDM2, a ubiquitin E3-ligase of the RING family, has a key role in regulating p53 abundance. During normal non-stress conditions p53 is targeted for degradation by MDM2. MDM2 can also target itself and MDMX for degradation. MDMX is closely related to MDM2 but the RING domain of MDMX does not possess intrinsic E3-ligase activity. Instead, MDMX regulates p53 abundance by modulating the levels and activity of MDM2. Dimerization, mediated by the conserved C-terminal RING domains of both MDM2 and MDMX, is critical to this activity. Here we report the crystal structure of the MDM2/MDMX RING domain heterodimer and map residues required for functional interaction with the E2 (UbcH5b). In both MDM2 and MDMX residues C-terminal to the RING domain have a key role in dimer formation. In addition we show that these residues are part of an extended surface that is essential for ubiquitylation in trans. This study provides a molecular basis for understanding how heterodimer formation leads to stabilization of MDM2, yet degradation of p53, and suggests novel targets for therapeutic intervention.
Assuntos
Estrutura Quaternária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/química , Ubiquitina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Alinhamento de Sequência , Proteína Supressora de Tumor p53/metabolismoRESUMO
MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. Here, we show that after UV radiation, MIHA prevented apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functioned after release of cytochrome c and DIABLO from the mitochondria and was able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Mitocondriais , Proteínas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose , Fator Apoptótico 1 Ativador de Proteases , Western Blotting , Caspase 3 , Caspase 9 , Linhagem Celular , Meios de Cultura Livres de Soro , Ativação Enzimática , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Testes de Precipitina , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transfecção , Raios Ultravioleta , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Programmed cell death is a physiological process that eliminates unwanted cells. The bcl-2 gene regulates programmed cell death in mammalian cells, but the way it functions is not known. Expression of the human bcl-2 gene in the nematode Caenorhabditis elegans reduced the number of programmed cell deaths, suggesting that the mechanism of programmed cell death controlled by bcl-2 in humans is the same as that in nematodes.
Assuntos
Caenorhabditis elegans/fisiologia , Morte Celular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Genótipo , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
Caspases, key mediators of apoptosis, are a structurally related family of cysteine proteases that cleave their substrates at aspartic acid residues either to cause cell death or to activate cytokines as part of an immune response. They can be controlled upstream by the regulation of signals that lead to zymogen activation, or downstream by inhibitors that prevent them from reaching their substrates. This review specifically looks at caspase inhibitors as distinct from caspase regulators: those produced by the cell itself; those whose genes are carried by viruses; and artificial caspase inhibitors used for research and potentially as therapeutics.
Assuntos
Inibidores de Caspase , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas Virais/metabolismo , Animais , Caspases/química , Caspases/metabolismo , Inibidores de Cisteína Proteinase/química , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Serpinas/metabolismoRESUMO
Direct IAP binding protein with low pI/second mitochondrial activator of caspases, HtrA2/Omi and GstPT/eRF3 are mammalian proteins that bind via N-terminal inhibitor of apoptosis protein (IAP) binding motifs (IBMs) to the baculoviral IAP repeat (BIR) domains of IAPs. These interactions can prevent IAPs from inhibiting caspases, or displace active caspases, thereby promoting cell death. We have identified several additional potential IAP antagonists, including glutamate dehydrogenase (GdH), Nipsnap 3 and 4, CLPX, leucine-rich pentatricopeptide repeat motif-containing protein and 3-hydroxyisobutyrate dehydrogenase. All are mitochondrial proteins from which N-terminal import sequences are removed generating N-terminal IBMs. Whereas most of these proteins have alanine at the N-terminal position, as observed for previously described antagonists, GdH has an N-terminal serine residue that is essential for X-linked IAP (XIAP) interaction. These newly described IAP binding proteins interact with XIAP mainly via BIR2, with binding eliminated or significantly reduced by a single point mutation (D214S) within this domain. Through this interaction, many are able to antagonise XIAP inhibition of caspase 3 in vitro.
Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Mamíferos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Alanina , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Inibidores de Caspase , Inibidores Enzimáticos/farmacologia , Glutamato Desidrogenase/química , Glutamato Desidrogenase/metabolismo , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas de Repetições Ricas em Leucina , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/metabolismo , Proteômica , Serina , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Many proteins that resemble Bcl-2 or bind to it have been found using techniques that reflect interactions in vitro or depend on DNA homology, but we still do not know how this master regulator of apoptosis works.
Assuntos
Apoptose/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Modelos Biológicos , Família Multigênica , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Bcl-2, one of a family of key regulators of apoptosis, was the first cell-death machinery component to be identified, but how the family functions is still not clear. Mammalian Bax, a pro-apoptotic family member, can cause yeast cells to die, and two recent yeast genetic screens shed light on how Bax might function.
Assuntos
Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Proteína X Associada a bcl-2RESUMO
BACKGROUND: Survivin is a mammalian protein that carries a motif typical of the inhibitor of apoptosis (IAP)proteins, first identified in baculoviruses. Although baculoviral IAP proteins regulate cell death, the yeast Survivin homolog Bir1 is involved in cell division. To determine the function of Survivin in mammals, we analyzed the pattern of localization of Survivin protein during the cell cycle, and deleted its gene by homologous recombination in mice. RESULTS: In human cells, Survivin appeared first on centromeres bound to a novel para-polar axis during prophase/metaphase, relocated to the spindle midzone during anaphase/telophase, and disappeared at the end of telophase. In the mouse, Survivin was required for mitosis during development. Null embryos showed disrupted microtubule formation, became polyploid, and failed to survive beyond 4.5days post coitum. This phenotype, and the cell-cycle localization of Survivin, resembled closely those of INCENP. Because the yeast homolog of INCENP, Sli15, regulates the Aurora kinase homolog Ipl1p, and the yeast Survivin homolog Bir1 binds to Ndc10p, a substrate of Ipl1p, yeast Survivin, INCENP and Aurora homologs function in concert during cell division. CONCLUSIONS: In vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis. Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase.
Assuntos
Autoantígenos , Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Associadas aos Microtúbulos , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Centrômero/metabolismo , Proteína B de Centrômero , Cromossomos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Corantes Fluorescentes , Deleção de Genes , Genótipo , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose , Camundongos , Microscopia de Fluorescência , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose , Dados de Sequência Molecular , Proteínas de Neoplasias , Alinhamento de Sequência , Survivina , Tubulina (Proteína)/metabolismoRESUMO
To try to understand autoimmunity, attention has often fallen on the process of cell death. After all, apoptosis is used during selection of immunocytes, cells in the target organs end up dying and mutations to cell death genes have been found in some autoimmune diseases. Furthermore, some autoimmune-prone mice fail to develop disease when certain cell death genes are deleted, and transgenic mice expressing other cell death genes develop autoimmunity. However, only a tiny proportion of human autoimmune disease is associated with mutations to individual genes and even in these rare cases the genetic background has a major influence on the severity of disease. An understanding of the pathophysiology of common autoimmune diseases will require elucidation of many different systems that interact in complex ways, of which the process of apoptosis is just one.
Assuntos
Apoptose/genética , Doenças Autoimunes/genética , Autoimunidade/genética , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/fisiopatologia , Doenças Autoimunes/prevenção & controle , Modelos Animais de Doenças , Deleção de Genes , Expressão Gênica , Humanos , Sistema Imunitário/imunologia , Mutação , TransgenesRESUMO
Expression of c-myc and macromolecular synthesis have been associated with physiological cell death. We have studied their requirement for the death of factor (interleukin-3)-dependent cells (FDC-P1) bearing an inducible bcl-2 expression construct. FDC-P1 cells expressing bcl-2 turned off expression of c-myc when deprived of interleukin-3 but remained viable as long as bcl-2 was maintained. A subsequent decline in Bcl-2 allowed the cells to undergo apoptosis directly from G0, in the absence of detectable c-myc expression. Thus c-myc expression may lead to apoptosis in some cases but is not directly involved in the mechanism of physiological cell death that can be controlled by Bcl-2. The macromolecular synthesis inhibitors actinomycin D and cycloheximide triggered rapid cell death of FDC-P1 cells in the presence of interleukin-3, but the cells could be protected by Bcl-2. Thus, the cell death machinery can exist in a quiescent state and can be activated by mechanisms that do not require synthesis of RNA or protein.
Assuntos
Apoptose/fisiologia , Morte Celular/fisiologia , Genes myc , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proto-Oncogenes , Animais , Apoptose/efeitos dos fármacos , Northern Blotting , Linhagem Celular , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Expressão Gênica , Células-Tronco Hematopoéticas , Humanos , Cinética , Poli A/biossíntese , Poli A/isolamento & purificação , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2 , RNA/biossíntese , RNA/isolamento & purificação , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Fase de Repouso do Ciclo Celular , TransfecçãoRESUMO
The key effector proteins of apoptosis are a family of cysteine proteases termed caspases. Following activation of caspases, biochemical events occur that lead to DNA degradation and the characteristic morphological changes associated with apoptosis. Here we show that cytoplasmic extracts activated in vitro by proteinase K were able to cleave the caspase substrate DEVD-7-amino-4-methylcoumarin, while neither proteinase K nor nonactivated extracts were able to do so alone. Caspase-like activity was inhibited by the specific caspase inhibitor DEVD-aldehyde and by the protease inhibitor iodoacetamide, but not by N-ethylmaleimide. When added to isolated nuclei, the activated extracts caused internucleosomal DNA degradation and morphological changes typical of apoptosis. As DNA cleavage and morphological changes could be inhibited by N-ethylmaleimide but not by iodoacetamide, we conclude that during apoptosis, caspase activation causes activation of another cytoplasmic enzyme that can be inhibited by N-ethylmaleimide. Activity of this enzyme is necessary for activation of endonucleases, DNA cleavage, and changes in nuclear morphology.
Assuntos
Apoptose/fisiologia , Cisteína Endopeptidases/metabolismo , Citoplasma/enzimologia , Endonucleases/metabolismo , Animais , Compartimento Celular , Núcleo Celular/patologia , Sistema Livre de Células , Cumarínicos/metabolismo , Citoplasma/efeitos dos fármacos , Endopeptidase K/farmacologia , Ativação Enzimática , Camundongos , Modelos Biológicos , Oligopeptídeos/metabolismo , Inibidores de Proteases/farmacologia , Linfócitos T , Células Tumorais CultivadasRESUMO
The chromosome translocations typifying Burkitt's lymphoma and follicular lymphoma deregulate very different oncogenes, myc and bcl-2. Transgenic mouse models have illuminated how each contributes to lymphomagenesis. Constitutive myc expression provokes sustained cell proliferation and retards differentiation. However, the resulting expansion in cell number is self-limiting, because the cells remain dependent on cytokines and undergo apoptosis when these become limiting. In contrast, bcl-2 is the prototype of a new class of oncogene that enhances cell survival but does not promote proliferation. Coexpression of these genes leads to the rapid transformation of lymphocytes, probably because each can counter an antioncogenic aspect of the other. Several close homologues of Bcl-2 also enhance cell survival and are thus potential oncogenes; each is essential for maintenance of particular major organs. More distant Bcl-2 relatives instead promote apoptosis and can be regarded as tumor suppressors. For many but not all apoptic signals, the balance between these competing activities determines cell survival. Learning how to adjust the apoptotic threshold in cancer cells should promote development of more effective therapeutic strategies.
Assuntos
Apoptose/genética , Linfoma de Burkitt/genética , Genes bcl-2 , Genes myc , Linfoma Folicular/genética , Animais , Ciclo Celular/genética , Divisão Celular/genética , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Espermatogênese/fisiologiaRESUMO
Caspase-1 cleaves and activates the pro-inflammatory cytokine interleukin-1 beta (IL-1ß), yet the mechanism of IL-1ß release and its dependence on cell death remains controversial. To address this issue, we generated a novel inflammasome independent system in which we directly activate caspase-1 by dimerization. In this system, caspase-1 dimerization induced the cleavage and secretion of IL-1ß, which did not require processing of caspase-1 into its p20 and p10 subunits. Moreover, direct caspase-1 dimerization allowed caspase-1 activation of IL-1ß to be separated from cell death. Specifically, we demonstrate at the single cell level that IL-1ß can be released from live, metabolically active, cells following caspase-1 activation. In addition, we show that dimerized or endogenous caspase-8 can also directly cleave IL-1ß into its biologically active form, in the absence of canonical inflammasome components. Therefore, cell death is not obligatory for the robust secretion of bioactive IL-1ß.
Assuntos
Caspase 1/metabolismo , Interleucina-1beta/metabolismo , Animais , Caspase 8/metabolismo , Morte Celular , Sobrevivência Celular , DNA Girase/metabolismo , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Inflamassomos/metabolismo , Camundongos , Multimerização Proteica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The pseudokinase, MLKL (mixed-lineage kinase domain-like), is the most terminal obligatory component of the necroptosis cell death pathway known. Phosphorylation of the MLKL pseudokinase domain by the protein kinase, receptor interacting protein kinase-3 (RIPK3), is known to be the key step in MLKL activation. This phosphorylation event is believed to trigger a molecular switch, leading to exposure of the N-terminal four-helix bundle (4HB) domain of MLKL, its oligomerization, membrane translocation and ultimately cell death. To examine how well this process is evolutionarily conserved, we analysed the function of MLKL orthologues. Surprisingly, and unlike their mouse, horse and frog counterparts, human, chicken and stickleback 4HB domains were unable to induce cell death when expressed in murine fibroblasts. Forced dimerization of the human MLKL 4HB domain overcame this defect and triggered cell death in human and mouse cell lines. Furthermore, recombinant proteins from mouse, frog, human and chicken MLKL, all of which contained a 4HB domain, permeabilized liposomes, and were most effective on those designed to mimic plasma membrane composition. These studies demonstrate that the membrane-permeabilization function of the 4HB domain is evolutionarily conserved, but reveal that execution of necroptotic death by it relies on additional factors that are poorly conserved even among closely related species.
Assuntos
Apoptose , Evolução Molecular , Proteínas Quinases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Galinhas , Células HT29 , Células HeLa , Cavalos , Humanos , Lipossomos/metabolismo , Camundongos , Necrose/genética , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
Cell production within an organ is determined by the rate of immigration, proliferation, differentiation, emigration and death of cells. Abnormalities in any one of these processes will disturb normal control of cell production, thereby eliciting hyperplasia can be an early event in neoplasia. Cell death, apoptosis, is a physiological process responsible for removing unwanted cells. It is used in multi-cellular organisms for tissue remodelling during embryogenesis, regulation of cell turnover and as a defence strategy against invading pathogens. In this review article we describe the role of the bcl-2/ced-9 gene family in cancer and discuss the general implications of defects in the apoptosis program for tumourigenesis and resistance of cancer cells to chemotherapy in light of current knowledge of the molecular mechanisms of cell death.
Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Helminto/genética , Neoplasias/etiologia , Proteínas Proto-Oncogênicas/genética , Apoptose , Proteínas Reguladoras de Apoptose , Transformação Celular Neoplásica , Ciclina D1/fisiologia , Resistencia a Medicamentos Antineoplásicos , Genes bcl-2 , Genes p53 , Humanos , Mutação , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
Caspases are the key effector molecules of the physiological death process known as apoptosis, although some are involved in activation of cytokines, rather than cell death. They exist in most of our cells as inactive precursors (zymogens) that kill the cell once activated. Caspases can be controlled in two ways. The processing and activation of a caspase can be regulated by molecules such as FADD, APAF-1, Bcl-2 family members, FLIP and IAPs. Active caspases can be controlled by a variety of inhibitors that directly interact with the protease. This review describes the later direct caspase inhibitors that have been identified, products of both viral and cellular genes, and artificial caspase inhibitors that have been developed both as research tools and as pharmaceutical agents to inhibit cell death in vivo.
Assuntos
Inibidores de Caspase , Inibidores de Cisteína Proteinase , Animais , Vírus da Varíola Bovina , Humanos , Proteínas Inibidoras de Apoptose , Serpinas/metabolismo , Proteínas Virais/metabolismoRESUMO
Diablo/Smac is a mammalian pro-apoptotic protein that can antagonize the inhibitor of apoptosis proteins (IAPs). We have produced monoclonal antibodies specific for Diablo and have used these to examine its tissue distribution and subcellular localization in healthy and apoptotic cells. Diablo could be detected in a wide range of mouse tissues including liver, kidney, lung, intestine, pancreas and testes by Western blot analysis. Immunohistochemical analysis found Diablo to be most abundant in the germinal cells of the testes, the parenchymal cells of the liver and the tubule cells of the kidney. In support of previous subcellular localization analysis, Diablo was present within the mitochondria of healthy cells, but released into the cytosol following the induction of apoptosis by UV.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Mitocondriais/metabolismo , Células 3T3 , Animais , Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular Transformada , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
IAPs were identified as baculoviral proteins that could inhibit the apoptotic response of insect cells to infection. Of the viral IAPs, OpIAP and CpIAP can inhibit apoptosis, whereas AcIAP cannot. OpIAP and some mammalian homologues can inhibit mammalian cell death. Two mammalian IAPs bind to TNFRII associated factors (TRAFs), but the significance of this is unclear. Here we show that Drosophila cellular IAPs and two baculoviral IAPs (OpIAP and CpIAP) can inhibit mammalian cell death induced by overexpression of Caspases 1 and 2. IAPs must act on conserved components of the apoptotic mechanism, but as none of these IAPs could bind TRAF proteins, TRAFs are not likely to be important for IAP mediated apoptosis inhibition. As OpIAP protected against death induced by ligation of TNF receptor family members, but not by factor nor serum withdrawal from dependent cells, it can inhibit certain apoptotic pathways without affecting others.