RESUMO
BACKGROUND: Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-ß family growth factors. RESULTS: To overcome the limitations of traditional baculovirus expression systems, we engineered "FlexiBAC". This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC can be used to produce high levels of mature, active forms of TGF-ß family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains. CONCLUSIONS: FlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle vector system reduces cloning steps and simplifies baculovirus production.
Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Ativinas/biossíntese , Ativinas/genética , Animais , Expressão Gênica , Proteínas Recombinantes/metabolismo , Spodoptera/genética , Transfecção/métodos , Cultura de Vírus/métodosRESUMO
Nutrients and growth hormones promote insulin production and the proliferation of pancreatic beta-cells. An imbalance between ever-increasing metabolic demands and insulin output causes diabetes. Recent evidence indicates that beta-cells enhance insulin gene expression depending on their secretory activity. This signalling pathway involves a catalytically inactive receptor tyrosine phosphatase, ICA512, whose cytoplasmic tail is cleaved on glucose-stimulated exocytosis of insulin secretory granules and then moves into the nucleus, where it upregulates insulin transcription. Here, we show that the cleaved cytosolic fragment of ICA512 enhances the transcription of secretory granule genes (including its own gene) by binding to tyrosine phosphorylated signal transducers and activators of transcription (STAT) 5 and preventing its dephosphorylation. Sumoylation of ICA512 by the E3 SUMO ligase PIASy, in turn, may reverse this process by decreasing the binding of ICA512 to STAT5. These findings illustrate how the exocytosis of secretory granules, through a retrograde pathway that sustains STAT activity, converges with growth hormone signalling to induce adaptive changes in beta-cells in response to metabolic demands.
Assuntos
Autoantígenos/metabolismo , Glucose/farmacologia , Hormônio do Crescimento/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Autoantígenos/genética , Núcleo Celular/metabolismo , Células Cultivadas , Ilhotas Pancreáticas/citologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Vesículas Secretórias/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição GênicaRESUMO
Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150(Glued) are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH(2) terminus of p150(Glued) binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150(Glued) and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH(2)-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH(2) and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.