RESUMEN
The Hippo pathway is an important regulator of organ growth and cell fate. The major mechanism by which Hippo is known to control transcription is by dictating the nucleo-cytoplasmic shuttling rate of Yorkie, a transcription co-activator, which promotes transcription with the DNA binding protein Scalloped. The nuclear biophysical behavior of Yorkie and Scalloped, and whether this is regulated by the Hippo pathway, remains unexplored. Using multiple live-imaging modalities on Drosophila tissues, we found that Scalloped interacts with DNA on a broad range of timescales, and enrichment of Scalloped at sites of active transcription is mediated by longer DNA dwell times. Further, Yorkie increased Scalloped's DNA dwell time, whereas the repressors Nervous fingers 1 (Nerfin-1) and Tondu-domain-containing growth inhibitor (Tgi) decreased it. Therefore, the Hippo pathway influences transcription not only by controlling nuclear abundance of Yorkie but also by modifying the DNA binding kinetics of the transcription factor Scalloped.
Asunto(s)
Cromatina , Proteínas de Drosophila , Drosophila melanogaster , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Transactivadores , Transcripción Genética , Proteínas Señalizadoras YAP , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cromatina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas Señalizadoras YAP/metabolismo , Transactivadores/metabolismo , Transactivadores/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN/metabolismo , ADN/genéticaRESUMEN
Enzymatic prospection indicated that L-asparaginase from Erwinia carotovora (ECAR-LANS) posses low glutaminase activity and much effort has been made to produce therapeutic ECAR-LANS. However, its low stability precludes its use in therapy. Herein, biochemical and biophysical assays provided data highlighting the influence of solubilization and storage into ECAR-LANS structure, stability, and activity. Moreover, innovations in recombinant expression and purification guaranteed the purification of functional tetramers. According to solubilization condition, the L-asparaginase activity and temperature of melting ranged up to 25-32%, respectively. CD spectra indicate the tendency of ECAR-LANS to instability and the influence of ß-structures in activity. These results provide relevant information to guide formulations with prolonged action in the bloodstream.