RESUMO
The conserved bazooka (baz/par3) gene acts as a key regulator of asymmetrical cell divisions across the animal kingdom. Associated Par3/Baz-Par6-aPKC protein complexes are also well known for their role in the establishment of apical/basal cell polarity in epithelial cells. Here we define a novel, positive function of Baz/Par3 in the Notch pathway. Using Drosophila wing and eye development, we demonstrate that Baz is required for Notch signaling activity and optimal transcriptional activation of Notch target genes. Baz appears to act independently of aPKC in these contexts, as knockdown of aPKC does not cause Notch loss-of-function phenotypes. Using transgenic Notch constructs, our data positions Baz activity downstream of activating Notch cleavage steps and upstream of Su(H)/CSL transcription factor complex activity on Notch target genes. We demonstrate a biochemical interaction between NICD and Baz, suggesting that Baz is required for NICD activity before NICD binds to Su(H). Taken together, our data define a novel role of the polarity protein Baz/Par3, as a positive and direct regulator of Notch signaling through its interaction with NICD.
Assuntos
Proteínas de Drosophila , Receptores Notch , Transdução de Sinais , Asas de Animais , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Receptores Notch/metabolismo , Asas de Animais/metabolismo , Asas de Animais/embriologia , Asas de Animais/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Ligação Proteica , Drosophila melanogaster/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Olho/embriologia , Olho/metabolismo , Olho/crescimento & desenvolvimento , Drosophila/metabolismo , Drosophila/embriologia , Polaridade Celular , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Cancer and normal cells use multiple antiapoptotic BCL2 proteins to prevent cell death. Therapeutic targeting of multiple BCL2 family proteins enhances tumor killing but is also associated with increased systemic toxicity. Here, we demonstrate that the dual targeting of MCL1 and BCL2 proteins using the small molecules S63845 and venetoclax induces durable remissions in mice that harbor human diffuse large B-cell lymphoma (DLBCL) tumors but is accompanied by hematologic toxicity and weight loss. To mitigate these toxicities, we encapsulated S63845 or venetoclax into nanoparticles that target P-selectin, which is enriched in tumor endothelial cells. In vivo and ex vivo imaging demonstrated preferential targeting of the nanoparticles to lymphoma tumors over vital organs. Mass spectrometry analyses after administration of nanoparticle drugs confirmed tumor enrichment of the drug while reducing plasma levels. Furthermore, nanoparticle encapsulation allowed 3.5- to 6.5-fold reduction in drug dose, induced sustained remissions, and minimized toxicity. Our results support the development of nanoparticles to deliver BH3 mimetic combinations in lymphoma and in general for toxic drugs in cancer therapy.
Assuntos
Antineoplásicos/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirimidinas/administração & dosagem , Sulfonamidas/administração & dosagem , Tiofenos/administração & dosagem , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Nanopartículas/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirimidinas/efeitos adversos , Pirimidinas/uso terapêutico , Sulfonamidas/efeitos adversos , Sulfonamidas/uso terapêutico , Índice Terapêutico , Tiofenos/efeitos adversos , Tiofenos/uso terapêuticoRESUMO
AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA) in multiple signaling contexts. Here, we demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF) mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, we demonstrate that AKAP200 interacts positively with Notch in both the eye and the thorax. We further show that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, our genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, we demonstrate that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, our data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Lisossomos/metabolismo , Proteínas de Membrana/fisiologia , Proteólise , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores Notch/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Larva , Proteínas de Membrana/genética , Estabilidade Proteica , Transdução de Sinais/genética , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
X chromosome inactivation (XCI) is an epigenetic mechanism that silences the majority of genes on one X chromosome in females. Previous studies have suggested that the spread of XCI might be facilitated in part by common repeats such as long interspersed nuclear elements (LINEs). However, owing to the unusual sequence content of the X and the nonrandom distribution of genes that escape XCI, it has been unclear whether the correlation between repeat elements and XCI is a functional one. To test the hypothesis that the spread of XCI shows sequence specificity, we have analyzed the pattern of XCI in autosomal chromatin by performing DNA methylation profiling in six unbalanced X;autosome translocations. Using promoter hypermethylation as an epigenetic signature of XCI, we have determined the inactivation status of 1050 autosomal genes after translocation onto an inactive derivative X. By performing a comparative sequence analysis of autosomal genes that are either subject to or escape the X inactivation signal, we identified a number of common repetitive elements, including L1 and L2 LINEs, and DNA motifs that are significantly enriched around inactive autosomal genes. We show that these same motifs predominantly map to L1P repeat elements, are significantly enriched on the X chromosome versus the autosomes and also occur at higher densities around X-linked genes that are subject to X inactivation compared with those that escape X inactivation. These results are consistent with a potential causal relationship between DNA sequence features such as L1s and the spread of XCI, lending strong support to Mary Lyon's 'repeat hypothesis'.