RESUMO
Heat shock proteins HSPA4L and HSPA4 are closely related members of the HSP110 family and act as cochaperones. We generated Hspa4l(-/-)Hspa4(-/-) mice to investigate a functional complementarity between HSPA4L and HSPA4 during embryonic development. Hspa4l(-/-)Hspa4(-/-) embryos exhibited marked pulmonary hypoplasia and neonatal death. Compared with lungs of wild-type, Hspa4l(-/-), and Hspa4(-/-) embryos, Hspa4l(-/-)Hspa4(-/-) lungs were characterized by diminished saccular spaces and increased mesenchymal septa. Mesenchymal hypercellularity was determined to be due to an increased cell proliferation index and decreased cell death. A significant increase in expression levels of prosurvival protein B cell leukemia/lymphoma 2 may be the cause for inhibition of apoptotic process in lungs of Hspa4(-/-)Hspa4l(-/-) embryos. Accumulation of glycogen and diminished expression of surfactant protein B, prosurfactant protein C, and aquaporin 5 in saccular epithelium suggested impaired maturation of type II and type I pneumocytes in the Hspa4l(-/-)Hspa4(-/-) lungs. Further experiments showed a significant accumulation of ubiquitinated proteins in the lungs of Hspa4l(-/-)Hspa4(-/-) embryos, indicating an impaired chaperone activity. Our study demonstrates that HSPA4L and HSPA4 collaborate in embryonic lung maturation, which is necessary for adaptation to air breathing at birth.
Assuntos
Proteínas de Choque Térmico HSP110/deficiência , Proteínas de Choque Térmico HSP70/deficiência , Pulmão/metabolismo , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/fisiopatologia , Animais , Apoptose , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP70/genética , Pulmão/anormalidades , Pulmão/fisiopatologia , Pneumopatias/genética , Pneumopatias/metabolismo , Pneumopatias/fisiopatologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Fenótipo , Respiração , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/fisiopatologia , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo , UbiquitinaçãoRESUMO
The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Junções Íntimas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Animais , Células Cultivadas , Cães , Células Epiteliais/citologia , Fase G1/fisiologia , Proteínas de Choque Térmico HSP110/química , Proteínas de Choque Térmico HSP110/deficiência , Hipertermia Induzida , Ligação Proteica , Fase S/fisiologia , Transdução de Sinais , Proteína da Zônula de Oclusão-1RESUMO
Protein aggregates are a common pathological feature of most neurodegenerative diseases (NDs). Understanding their formation and regulation will help clarify their controversial roles in disease pathogenesis. To date, there have been few systematic studies of aggregates formation in Drosophila, a model organism that has been applied extensively in modeling NDs and screening for toxicity modifiers. We generated transgenic fly lines that express enhanced-GFP-tagged mutant Huntingtin (Htt) fragments with different lengths of polyglutamine (polyQ) tract and showed that these Htt mutants develop protein aggregates in a polyQ-length- and age-dependent manner in Drosophila. To identify central regulators of protein aggregation, we further generated stable Drosophila cell lines expressing these Htt mutants and also established a cell-based quantitative assay that allows automated measurement of aggregates within cells. We then performed a genomewide RNA interference screen for regulators of mutant Htt aggregation and isolated 126 genes involved in diverse cellular processes. Interestingly, although our screen focused only on mutant Htt aggregation, several of the identified candidates were known previously as toxicity modifiers of NDs. Moreover, modulating the in vivo activity of hsp110 (CG6603) or tra1, two hits from the screen, affects neurodegeneration in a dose-dependent manner in a Drosophila model of Huntington's disease. Thus, other aggregates regulators isolated in our screen may identify additional genes involved in the protein-folding pathway and neurotoxicity.