RESUMO
The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCUG)16, in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG)106 displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. Our data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. Our Drosophila DM2 model might provide a convenient tool for in vivo drug screening.
Assuntos
Apoptose/efeitos dos fármacos , Expansão das Repetições de DNA/genética , Drosophila melanogaster/genética , Distrofia Miotônica/genética , RNA/toxicidade , Animais , Apoptose/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Humanos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Pentamidina/farmacologia , Células Fotorreceptoras de Invertebrados/efeitos dos fármacos , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/patologia , Inibidores de Proteínas Quinases/farmacologia , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/anormalidades , Retina/efeitos dos fármacos , Retina/patologia , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/metabolismoRESUMO
Trimethylated lysine 27 of histone H3 (H3K27me3) is an epigenetic mark for gene silencing and can be demethylated by the JmjC domain of UTX. Excessive H3K27me3 levels can cause tumorigenesis, but little is known about the mechanisms leading to those cancers. Mutants of the Drosophila H3K27me3 demethylase dUTX display some characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. We show that a disruption of the JmjC domain of dUTX results in a growth advantage for mutant cells over adjacent wild-type tissue due to increased proliferation. The growth advantage of dUTX mutant tissue is caused, at least in part, by increased Notch activity, demonstrating that dUTX is a Notch antagonist. Furthermore, the inactivation of Retinoblastoma (Rbf in Drosophila) contributes to the growth advantage of dUTX mutant tissue. The excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila melanogaster and may provide a model for UTX-dependent tumorigenesis in humans.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Neoplasias/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Ciclo Celular/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/genética , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Neoplasias/genética , Oxirredutases N-Desmetilantes/genética , Fenótipo , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/fisiologia , Pigmentação/genética , Interferência de RNA , Receptores Notch/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Serrate-JaggedRESUMO
BACKGROUND: Genetic studies in yeast have identified class E vps genes that form the ESCRT complexes required for protein sorting at the early endosome. In Drosophila, mutations of the ESCRT-II component vps25 cause endosomal defects leading to accumulation of Notch protein and increased Notch pathway activity. These endosomal and signaling defects are thought to account for several phenotypes. Depending on the developmental context, two different types of overgrowth can be detected. Tissue predominantly mutant for vps25 displays neoplastic tumor characteristics. In contrast, vps25 mutant clones in a wild-type background trigger hyperplastic overgrowth in a non-autonomous manner. In addition, vps25 mutant clones also promote apoptotic resistance in a non-autonomous manner. PRINCIPAL FINDINGS: Here, we genetically characterize the remaining ESCRT-II components vps22 and vps36. Like vps25, mutants of vps22 and vps36 display endosomal defects, accumulate Notch protein and--when the tissue is predominantly mutant--show neoplastic tumor characteristics. However, despite these common phenotypes, they have distinct non-autonomous phenotypes. While vps22 mutations cause strong non-autonomous overgrowth, they do not affect apoptotic resistance. In contrast, vps36 mutations increase apoptotic resistance, but have little effect on non-autonomous proliferation. Further characterization reveals that although all ESCRT-II mutants accumulate Notch protein, only vps22 and vps25 mutations trigger Notch activity. CONCLUSIONS/SIGNIFICANCE: The ESCRT-II components vps22, vps25 and vps36 display common and distinct genetic properties. Our data redefine the role of Notch for hyperplastic and neoplastic overgrowth in these mutants. While Notch is required for hyperplastic growth, it appears to be dispensable for neoplastic transformation.
Assuntos
Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Proteínas de Transporte Vesicular/genética , Animais , Apoptose , Proteínas de Transporte/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Fenótipo , Transporte Proteico/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Ubiquitinação/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase's proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1's E3 activity or drICE's ubiquitin-acceptor lysines, abrogates DIAP1's ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an "inactive" conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 "activation" and nondegradative polyubiquitylation.
Assuntos
Inibidores de Caspase , Ubiquitinação , Animais , Apoptose/genética , Apoptose/fisiologia , Caspases/genética , Caspases Efetoras/genética , Caspases Efetoras/metabolismo , Células Cultivadas , Drosophila/citologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Cinética , Modelos Biológicos , Peptídeo Hidrolases/metabolismo , Conformação Proteica , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.
Assuntos
Apoptose , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Enzimas Ativadoras de Ubiquitina/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Sequência Conservada , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutação/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fenótipo , Ligação Proteica , Receptores Notch/metabolismo , Alinhamento de Sequência , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
Appropriate cell-cell signaling is crucial for proper tissue homeostasis. Protein sorting of cell surface receptors at the early endosome is important for both the delivery of the signal and the inactivation of the receptor, and its alteration can cause malignancies including cancer. In a genetic screen for suppressors of the pro-apoptotic gene hid in Drosophila, we identified two alleles of vps25, a component of the ESCRT machinery required for protein sorting at the early endosome. Paradoxically, although vps25 mosaics were identified as suppressors of hid-induced apoptosis, vps25 mutant cells die. However, we provide evidence that a non-autonomous increase of Diap1 protein levels, an inhibitor of apoptosis, accounts for the suppression of hid. Furthermore, before they die, vps25 mutant clones trigger non-autonomous proliferation through a failure to downregulate Notch signaling, which activates the mitogenic JAK/STAT pathway. Hid and JNK contribute to apoptosis of vps25 mutant cells. Inhibition of cell death in vps25 clones causes dramatic overgrowth phenotypes. In addition, Hippo signaling is increased in vps25 clones, and hippo mutants block apoptosis in vps25 clones. In summary, the phenotypic analysis of vps25 mutants highlights the importance of receptor downregulation by endosomal protein sorting for appropriate tissue homeostasis, and may serve as a model for human cancer.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Mosaicismo , Alelos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Transporte/genética , Proliferação de Células , Sobrevivência Celular , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/genética , Endossomos/metabolismo , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica/genética , Genes de Insetos , Mutação , Transporte Proteico/fisiologia , Receptores Notch/fisiologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Proteínas de Homeodomínio/fisiologia , Neuropeptídeos/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Transativadores/fisiologia , Animais , Padronização Corporal , Caspases/metabolismo , Morte Celular/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Mutação , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Transativadores/genéticaRESUMO
Calmodulin (CaM) is an essential component of calcium signaling in multicellular organisms. We used null mutations of the Drosophila CaM gene (Cam) in combination with clonal analysis and immunolocalization to examine the effects of loss of Cam function in the ovarian germline and developing embryo. These studies have uncovered unexpected and striking movements of CaM protein within these tissues. In the ovary, evidence for transfer of CaM from an external source, across plasma membranes, into the germline cells was obtained. In late embryogenesis, maternally derived CaM protein relocalizes dramatically within the nervous system of both wildtype and Cam null embryos-a process that may also involve movement across cell membranes. These findings indicate dynamic, unsuspected elements to the in vivo functions of CaM in the whole organism.