RESUMO
Activation of Bax and Bak by BH3-only molecules triggers mitochondrial apoptosis. In a recent issue of Molecular Cell, Fu et al. (2009) identify a constitutively active isoform of Bax, Baxbeta, whose activity is tightly controlled by the ubiquitin-proteasome system.
Assuntos
Processamento Alternativo/fisiologia , Proteína X Associada a bcl-2/metabolismo , Apoptose , Mitocôndrias/metabolismo , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/metabolismo , Ubiquitinas/metabolismoRESUMO
While activation of BAX/BAK by BH3-only molecules (BH3s) is essential for mitochondrial apoptosis, the underlying mechanisms remain unsettled. Here we demonstrate that BAX undergoes stepwise structural reorganization leading to mitochondrial targeting and homo-oligomerization. The alpha1 helix of BAX keeps the alpha9 helix engaged in the dimerization pocket, rendering BAX as a monomer in cytosol. The activator BH3s, tBID/BIM/PUMA, attack and expose the alpha1 helix of BAX, resulting in secondary disengagement of the alpha9 helix and thereby mitochondrial insertion. Activator BH3s remain associated with the N-terminally exposed BAX through the BH1 domain to drive homo-oligomerization. BAK, an integral mitochondrial membrane protein, has bypassed the first activation step, explaining why its killing kinetics are faster than those of BAX. Furthermore, death signals initiated at ER induce BIM and PUMA to activate mitochondrial apoptosis. Accordingly, deficiency of Bim/Puma impedes ER stress-induced BAX/BAK activation and apoptosis. Our study provides mechanistic insights regarding the spatiotemporal execution of BAX/BAK-governed cell death.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína 11 Semelhante a Bcl-2 , Células Cultivadas , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Imunoprecipitação , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Modelos Biológicos , Mutação , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Proteínas Proto-Oncogênicas/genética , Estaurosporina/farmacologia , Tapsigargina/farmacologia , Proteínas Supressoras de Tumor/genética , Tunicamicina/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genéticaRESUMO
Cell cycle checkpoints are implemented to safeguard the genome, avoiding the accumulation of genetic errors. Checkpoint loss results in genomic instability and contributes to the evolution of cancer. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the role of an intact S-phase checkpoint in maintaining genome integrity. Although the basic framework of the S-phase checkpoint in multicellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome-11 band-q23 translocations disrupting the MLL gene lead to poor prognostic leukaemias. Here we assign MLL as a novel effector in the mammalian S-phase checkpoint network and identify checkpoint dysfunction as an underlying mechanism of MLL leukaemias. MLL is phosphorylated at serine 516 by ATR in response to genotoxic stress in the S phase, which disrupts its interaction with, and hence its degradation by, the SCF(Skp2) E3 ligase, leading to its accumulation. Stabilized MLL protein accumulates on chromatin, methylates histone H3 lysine 4 at late replication origins and inhibits the loading of CDC45 to delay DNA replication. Cells deficient in MLL showed radioresistant DNA synthesis and chromatid-type genomic abnormalities, indicative of S-phase checkpoint dysfunction. Reconstitution of Mll(-/-) (Mll also known as Mll1) mouse embryonic fibroblasts with wild-type but not S516A or ΔSET mutant MLL rescues the S-phase checkpoint defects. Moreover, murine myeloid progenitor cells carrying an Mll-CBP knock-in allele that mimics human t(11;16) leukaemia show a severe radioresistant DNA synthesis phenotype. MLL fusions function as dominant negative mutants that abrogate the ATR-mediated phosphorylation/stabilization of wild-type MLL on damage to DNA, and thus compromise the S-phase checkpoint. Together, our results identify MLL as a key constituent of the mammalian DNA damage response pathway and show that deregulation of the S-phase checkpoint incurred by MLL translocations probably contributes to the pathogenesis of human MLL leukaemias.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Cromatina/metabolismo , Dano ao DNA , Replicação do DNA/fisiologia , Genes Dominantes/genética , Instabilidade Genômica/fisiologia , Histona-Lisina N-Metiltransferase , Histonas/química , Histonas/metabolismo , Humanos , Leucemia/genética , Lisina/metabolismo , Metilação , Camundongos , Células Progenitoras Mieloides/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/deficiência , Proteína de Leucina Linfoide-Mieloide/genética , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Translocação Genética/genéticaRESUMO
The mixed-lineage leukemia (MLL) H3K4 methyltransferase protein, and the heterodimeric RUNX1/CBFß transcription factor complex, are critical for definitive and adult hematopoiesis, and both are frequently targeted in human acute leukemia. We identified a physical and functional interaction between RUNX1 (AML1) and MLL and show that both are required to maintain the histone lysine 4 trimethyl mark (H3K4me3) at 2 critical regulatory regions of the AML1 target gene PU.1. Similar to CBFß, we show that MLL binds to AML1 abrogating its proteasome-dependent degradation. Furthermore, a subset of previously uncharacterized frame-shift and missense mutations at the N terminus of AML1, found in MDS and AML patients, impairs its interaction with MLL, resulting in loss of the H3K4me3 mark within PU.1 regulatory regions, and decreased PU.1 expression. The interaction between MLL and AML1 provides a mechanism for the sequence-specific binding of MLL to DNA, and identifies RUNX1 target genes as potential effectors of MLL function.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Histonas/metabolismo , Mutação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/genética , Doença Aguda , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , Expressão Gênica , Células HEK293 , Histona-Lisina N-Metiltransferase , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Lisina/metabolismo , Metilação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismoRESUMO
Although the BCL-2 family constitutes a crucial checkpoint in apoptosis, the intricate interplay between these family members remains elusive. Here, we demonstrate that BIM and PUMA, similar to truncated BID (tBID), directly activate BAX-BAK to release cytochrome c. Conversely, anti-apoptotic BCL-2-BCL-X(L)-MCL-1 sequesters these 'activator' BH3-only molecules into stable complexes, thus preventing the activation of BAX-BAK. Extensive mutagenesis of BAX-BAK indicates that their activity is not kept in check by BCL-2-BCL-X(L)-MCL-1. Anti-apoptotic BCL-2 members are differentially inactivated by the remaining 'inactivator' BH3-only molecules including BAD, NOXA, BMF, BIK/BLK and HRK/DP5. BAD displaces tBID, BIM or PUMA from BCL-2-BCL-X(L) to activate BAX-BAK, whereas NOXA specifically antagonizes MCL-1. Coexpression of BAD and NOXA killed wild-type but not Bax, Bak doubly deficient cells or Puma deficient cells with Bim knockdown, indicating that activator BH3-only molecules function downstream of inactivator BH3-only molecules to activate BAX-BAK. Our data establish a hierarchical regulation of mitochondrion-dependent apoptosis by various BCL-2 subfamilies.
Assuntos
Apoptose , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína 11 Semelhante a Bcl-2 , Citocromos c/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Three forms of cell death have been described: apoptosis, autophagic cell death, and necrosis. Although genetic and biochemical studies have formulated a detailed blueprint concerning the apoptotic network, necrosis is generally perceived as a passive cellular demise resulted from unmanageable physical damages. Here, we conclude an active de novo genetic program underlying DNA damage-induced necrosis, thus assigning necrotic cell death as a form of "programmed cell death." Cells deficient of the essential mitochondrial apoptotic effectors, BAX and BAK, ultimately succumbed to DNA damage, exhibiting signature necrotic characteristics. Importantly, this genotoxic stress-triggered necrosis was abrogated when either transcription or translation was inhibited. We pinpointed the p53-cathepsin axis as the quintessential framework underlying necrotic cell death. p53 induces cathepsin Q that cooperates with reactive oxygen species (ROS) to execute necrosis. Moreover, we presented the in vivo evidence of p53-activated necrosis in tumor allografts. Current study lays the foundation for future experimental and therapeutic discoveries aimed at "programmed necrotic death."
Assuntos
Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Dano ao DNA , Necrose/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Catepsinas/genética , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias/patologia , Neoplasias/ultraestrutura , Ativação Transcricional/genética , Transplante Homólogo , Proteína Killer-Antagonista Homóloga a bcl-2/deficiência , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/metabolismoRESUMO
MLL5 is an MLL family protein and a candidate tumor suppressor located within the human chromosome band 7q22 that is frequently deleted in myeloid malignancies. In this issue of Blood, 3 independent studies report the first genetic analysis of MLL5 deficiency in mice. All 3 strains of MLL5 knockout mice exhibited defects in hematopoiesis, highlighting the critical role of MLL5 in hematopoietic stem cell functions.
RESUMO
Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Endopeptidases/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endopeptidases/genética , Estabilidade Enzimática , Evolução Molecular , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide/genética , Inibidores de Proteases/metabolismo , Precursores de Proteínas/genética , Interferência de RNA , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIAalphabeta gene and posttranslationally cleaved into alpha and beta subunits. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1-/- MEF cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset of genes during differentiation and development.
Assuntos
Endopeptidases/metabolismo , Processamento de Proteína Pós-Traducional , Fator de Transcrição TFIIA/metabolismo , Transcrição Gênica/genética , Sequência de Aminoácidos , Animais , Extratos Celulares , Núcleo Celular/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação/genética , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fator de Transcrição TFIIA/química , XenopusRESUMO
Taspase1 is a threonine protease responsible for cleaving MLL (Mixed-Lineage Leukemia) to achieve proper HOX gene expression. Subsequent studies identified additional Taspase1 substrates including Transcription Factor IIA (TFIIA) and Drosophila HCF. Taspase1 is essential for cell proliferation and is overexpressed in many cancer cell lines. Currently no small molecule inhibitors of this enzyme have been described. Here, we report the synthesis and evaluation of vinyl sulfone, vinyl ketone, epoxy ketone, and boronic acid inhibitors designed based on the preferred Taspase1 cleavage site (Ac-Ile-Ser-Gln-Leu-Asp). Specifically, we evaluated compounds in which the reactive warhead is positioned in place of the P1 aspartic acid side chain as well as at the C-terminus of the peptide. Interestingly, both classes of inhibitors were effective and vinyl ketones and vinyl sulfones showed the greatest potency for the target protease. These results suggest that Taspase1 has unique substrate recognition properties that could potentially be exploited in the design of potent and selective inhibitors of this enzyme.
Assuntos
Ácidos Borônicos/síntese química , Endopeptidases/química , Cetonas/síntese química , Inibidores de Proteases/síntese química , Sulfonas/síntese química , Sequência de Aminoácidos , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Desenho de Fármacos , Endopeptidases/metabolismo , Humanos , Cetonas/química , Cetonas/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Sulfonas/química , Sulfonas/farmacologiaRESUMO
The mixed-lineage leukemia gene (MLL, ALL1, HRX) encodes a 3,969-amino-acid nuclear protein homologous to Drosophila trithorax and is required to maintain proper Hox gene expression. Chromosome translocations in human leukemia disrupt MLL (11q23), generating chimeric proteins between the N terminus of MLL and multiple translocation partners. Here we report that MLL is normally cleaved at two conserved sites (D/GADD and D/GVDD) and that mutation of these sites abolishes the proteolysis. MLL cleavage generates N-terminal p320 (N320) and C-terminal p180 (C180) fragments, which form a stable complex that localizes to a subnuclear compartment. The FYRN domain of N320 directly interacts with the FYRC and SET domains of C180. Disrupting the interaction between N320 and C180 leads to a marked decrease in the level of N320 and a redistribution of C180 to a diffuse nuclear pattern. These data suggest a model in which a dynamic post-cleavage association confers stability to N320 and correct nuclear sublocalization of the complex, to control the availability of N320 for target genes. This predicts that MLL fusion proteins of leukemia which would lose the ability to complex with C180 have their stability conferred instead by the fusion partners, thus providing one mechanism for altered target gene expression.
Assuntos
Estruturas do Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proto-Oncogenes , Fatores de Transcrição , Motivos de Aminoácidos , Sequência de Aminoácidos , Células Cultivadas , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Mutação , Proteína de Leucina Linfoide-Mieloide , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The clinical efficacy of tyrosine kinase inhibitors supports the dependence of distinct subsets of cancers on specific driver mutations for survival, a phenomenon called "oncogene addiction." We demonstrate that PUMA and BIM are the key apoptotic effectors of tyrosine kinase inhibitors in breast cancers with amplification of the gene encoding human epidermal growth factor receptor 2 (HER2) and lung cancers with epidermal growth factor receptor (EGFR) mutants. The BH3 domain containing proteins BIM and PUMA can directly activate the proapoptotic proteins BAX and BAK to permeabilize mitochondria, leading to caspase activation and apoptosis. We delineated the signal transduction pathways leading to the induction of BIM and PUMA by tyrosine kinase inhibitors. Inhibition of the mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway caused increased abundance of BIM, whereas antagonizing the phosphoinositide 3-kinase (PI3K)-AKT pathway triggered nuclear translocation of the FOXO transcription factors, which directly activated the PUMA promoter. In a mouse breast tumor model, the abundance of PUMA and BIM was increased after inactivation of HER2. Moreover, deficiency of Bim or Puma impaired caspase activation and reduced tumor regression caused by inactivation of HER2. Similarly, deficiency of Puma impeded the regression of EGFR(L858R)-driven mouse lung tumors upon inactivation of the EGFR-activating mutant. Overall, our study identified PUMA and BIM as the sentinels that interconnect kinase signaling networks and the mitochondrion-dependent apoptotic program, which offers therapeutic insights for designing novel cell death mechanism-based anticancer strategies.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/genética , Neoplasias da Mama/metabolismo , Inativação Gênica/fisiologia , Proteínas de Membrana/metabolismo , Oncogenes/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Compostos de Bifenilo/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Receptores ErbB/metabolismo , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Lapatinib , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/genética , Camundongos , Nitrofenóis/farmacologia , Oncogenes/genética , Piperazinas/farmacologia , Plasmídeos/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Quinazolinas , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Sulfonamidas/farmacologiaRESUMO
Inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, is an archetypical tumor-initiating event in clear cell renal carcinoma (ccRCC) that leads to the activation of hypoxia-inducible transcription factors (HIFs). However, VHL mutation status in ccRCC is not correlated with clinical outcome. Here we show that during ccRCC progression, cancer cells exploit diverse epigenetic alterations to empower a branch of the VHL-HIF pathway for metastasis, and the strength of this activation is associated with poor clinical outcome. By analyzing metastatic subpopulations of VHL-deficient ccRCC cells, we discovered an epigenetically altered VHL-HIF response that is specific to metastatic ccRCC. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, we show that loss of Polycomb repressive complex 2 (PRC2)-dependent histone H3 Lys27 trimethylation (H3K27me3) activates HIF-driven chemokine (C-X-C motif) receptor 4 (CXCR4) expression in support of chemotactic cell invasion, whereas loss of DNA methylation enables HIF-driven cytohesin 1 interacting protein (CYTIP) expression to protect cancer cells from death cytokine signals. Thus, metastasis in ccRCC is based on an epigenetically expanded output of the tumor-initiating pathway.
Assuntos
Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/secundário , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Receptores CXCR4/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Sequência de Bases , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Neoplasias Renais/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Complexo Repressor Polycomb 2/genética , Receptores CXCR4/genética , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 µmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy.
Assuntos
Arsenicais/farmacologia , Neoplasias Encefálicas/prevenção & controle , Neoplasias da Mama/prevenção & controle , Endopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Endopeptidases/genética , Células HEK293 , Humanos , Cinética , Masculino , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Bibliotecas de Moléculas Pequenas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Taspase1, the mixed lineage leukemia and TFIIAalpha-beta cleaving protease, enables cell proliferation and permits oncogenic initiation. Here, we show its critical role in cancer maintenance and thus offer a new anticancer target. Taspase1 is overexpressed in primary human cancers, and deficiency of Taspase1 in cancer cells not only disrupts proliferation but also enhances apoptosis. Mechanistically, loss of Taspase1 induces the levels of CDK inhibitors (CDKI: p16, p21, and p27) and reduces the level of antiapoptotic MCL-1. Therapeutically, deficiency of Taspase1 synergizes with chemotherapeutic agents and ABT-737, an inhibitor of BCL-2/BCL-X(L), to kill cancer cells. Taspase1 alone or in conjunction with MYC, RAS, or E1A fails to transform NIH/3T3 cells or primary mouse embryonic fibroblasts, respectively, but plays critical roles in cancer initiation and maintenance. Therefore, Taspase1 is better classified as a "non-oncogene addiction" protease, the inhibition of which may offer a novel anticancer therapeutic strategy. The reliance of oncogenes on subordinate non-oncogenes during tumorigenesis underscores the non-oncogene addiction hypothesis in which a large class of non-oncogenes functions to maintain cancer phenotypes and presents attractive anticancer therapeutic targets. The emergence of successful cancer therapeutics targeting non-oncogenes to which cancers are addicted supports the future development and potential application of small-molecule Taspase1 inhibitors for cancer therapy.
Assuntos
Endopeptidases/genética , Glioblastoma/genética , Melanoma/genética , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Compostos de Bifenilo/farmacologia , Processos de Crescimento Celular/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Endopeptidases/deficiência , Endopeptidases/metabolismo , Genes myc , Genes ras , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides , Células NIH 3T3 , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Transdução Genética , TransfecçãoRESUMO
Although the proteins BAX and BAK are required for initiation of apoptosis at the mitochondria, how BAX and BAK are activated remains unsettled. We provide in vivo evidence demonstrating an essential role of the proteins BID, BIM, and PUMA in activating BAX and BAK. Bid, Bim, and Puma triple-knockout mice showed the same developmental defects that are associated with deficiency of Bax and Bak, including persistent interdigital webs and imperforate vaginas. Genetic deletion of Bid, Bim, and Puma prevented the homo-oligomerization of BAX and BAK, and thereby cytochrome c-mediated activation of caspases in response to diverse death signals in neurons and T lymphocytes, despite the presence of other BH3-only molecules. Thus, many forms of apoptosis require direct activation of BAX and BAK at the mitochondria by a member of the BID, BIM, or PUMA family of proteins.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/deficiência , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína 11 Semelhante a Bcl-2 , Caspases/metabolismo , Células Cultivadas , Cerebelo/citologia , Citocromos c/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Modelos Biológicos , Permeabilidade , Multimerização Proteica , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Estresse Fisiológico , Linfócitos T/fisiologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genéticaRESUMO
Human leukemias with chromosomal band 11q23 aberrations that disrupt the MLL/HRX/ALL-1 gene portend poor prognosis. MLL associated leukemias account for the majority of infant leukemia, approximately 10% of adult de novo leukemia and approximately 33% of therapy related acute leukemia with a balanced chromosome translocation. The 500 kD MLL precursor is processed by Taspase1 to generate mature MLL(N320/C180), which orchestrates many aspects of biology such as embryogenesis, cell cycle, cell fate and stem cell maintenance. Leukemogenic MLL translocations fuse the common MLL N-terminus (approximately 1,400 aa) in frame with more than 60 translocation partner genes (TPGs). Recent studies on MLL and MLL leukemia have greatly advanced our knowledge concerning the normal function of MLL and its deregulation in leukemogenesis. Here, we summarize the critical biological and pathological activities of MLL and MLL fusions, and discuss available models and potential therapeutic targets of MLL associated leukemias.
Assuntos
Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Animais , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Leucemia/patologia , Modelos Biológicos , Transdução de Sinais/genéticaRESUMO
The proapoptotic proteins BAX and BAK constitute the mitochondrial apoptotic gateway that executes cellular demise after integrating death signals. The lethal BAK is kept in check by voltage-dependent anion channel 2 (VDAC2), a mammalian-restricted VDAC isoform. Here, we provide evidence showing a critical role for the VADC2-BAK complex in determining thymocyte survival in vivo. Genetic depletion of Vdac2 in the thymus resulted in excessive cell death and hypersensitivity to diverse death stimuli including engagement of the T cell receptor. These phenotypes were completely rescued by the concurrent deletion of Bak but not that of Bax. Thus, the VDAC2-BAK axis provides a mechanism that governs the homeostasis of thymocytes. Our study reveals a sophisticated built-in rheostat that likely fine-tunes immune competence to balance autoimmunity and immunodeficiency.
Assuntos
Deleção Clonal/fisiologia , Linfócitos T/citologia , Canal de Ânion 2 Dependente de Voltagem/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Apoptose/genética , Apoptose/fisiologia , Autoimunidade/genética , Autoimunidade/fisiologia , Complexo CD3/imunologia , Deleção Clonal/genética , Dimerização , Feminino , Técnicas de Inativação de Genes , Genótipo , Transporte de Íons/genética , Transporte de Íons/fisiologia , Masculino , Camundongos , Camundongos Knockout , Membranas Mitocondriais/fisiologia , Timo/citologia , Canal de Ânion 2 Dependente de Voltagem/deficiência , Canal de Ânion 2 Dependente de Voltagem/genética , Proteína Killer-Antagonista Homóloga a bcl-2/deficiência , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/fisiologiaRESUMO
Discovered in 1992 from cloning the gene involved in human leukemias carrying chromosome band 11q23 translocations, the MLL/HRX/ALL-1 gene has since attracted scientists from various disciplines by its diverse functions in normal physiological and pathological processes. MLL is the human orthologue of Drosophila trithorax (trx)-the founding member of trithorax group proteins, Trx-G. Leukemogenic11q23 translocations fuse the common MLL N-terminal 1400aa in-frame with a wide variety of fusion partners that share no structural or functional homology. The 500 kD precursor MLL undergoes evolutionarily conserved site-specific cleavage mediated by Taspase1, generating the mature MLL(N320/C180) heterodimer which methylates histone H3 at lysine 4 with its carboxy-terminal SET domain. Extensive biochemical and genetic studies on MLL/trx have established its critical role in maintaining the expression of Hox/homeotic genes. By contrast, the involvement of MLL in many other essential cellular processes remains unclear. Recent reports including ours began to elucidate the intricate interplay between MLL and the cell cycle machinery, which ensures proper cell cycle phase transitions. Thus, this review will focus on this novel activity of MLL and discuss the implications of its deregulation in MLL leukemias.
Assuntos
Ciclo Celular/fisiologia , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Leucemia/metabolismo , Modelos Biológicos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Transdução de Sinais/fisiologia , Humanos , Proteína de Leucina Linfoide-Mieloide/biossínteseRESUMO
Human chromosome 11q23 translocations disrupting MLL result in poor prognostic leukemias. It fuses the common MLL N-terminal approximately 1400 amino acids in-frame with >60 different partners without shared characteristics. In addition to the well-characterized activity of MLL in maintaining Hox gene expression, our recent studies established an MLL-E2F axis in orchestrating core cell cycle gene expression including Cyclins. Here, we demonstrate a biphasic expression of MLL conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases. Specifically, SCF(Skp2) and APC(Cdc20) mark MLL for degradation at S phase and late M phase, respectively. Abolished peak expression of MLL incurs corresponding defects in G1/S transition and M-phase progression. Conversely, overexpression of MLL blocks S-phase progression. Remarkably, MLL degradation initiates at its N-terminal approximately 1400 amino acids, and tested prevalent MLL fusions are resistant to degradation. Thus, impaired degradation of MLL fusions likely constitutes the universal mechanism underlying all MLL leukemias. Our data conclude an essential post-translational regulation of MLL by the cell cycle ubiquitin/proteasome system (UPS) assures the temporal necessity of MLL in coordinating cell cycle progression.