RESUMEN
The Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, we evaluated the DHFR DD system in the eyes of aged mice (up to 24 months), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4 -/- mice). The DHFR DD was effectively degraded in all model systems, including rd2 mice, which showed significant defects in chymotrypsin proteasomal activity. Moreover, trimethoprim (TMP) administration stabilized the DHFR DD in all mouse models. Thus, the DHFR DD-based approach allows for control of protein abundance in a variety of mouse models, laying the foundation to use this strategy for the conditional control of gene therapies to potentially treat multiple eye diseases.
RESUMEN
The use of destabilizing domains (DDs) to conditionally control the abundance of a protein of interest (POI) through a small-molecule stabilizer has gained increasing traction both in vitro and in vivo. Yet there are specific considerations for the development and accurate control of user-defined POIs via DDs, as well as the identification of novel (and potentially synergistic) small-molecule stabilizers. Here, we describe a platform for achieving these goals. For complete details on the use and execution of this protocol, please refer to Ramadurgum et al. (2020).
Asunto(s)
Clonación Molecular/métodos , Proteínas Recombinantes , Línea Celular , Escherichia coli/genética , Células HEK293 , Humanos , Dominios Proteicos/genética , Ingeniería de Proteínas , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Destabilizing domains (DDs) have been used successfully to conditionally control the abundance of proteins of interest (POIs) in a small-molecule-dependent manner in mice, worms (Caenorhabditis elegans), and Drosophila. However, development of such systems must account for delivery of the DD-POIs to the target tissue, accessibility of the target tissue to the small molecule, and quantification of stabilization. Here, we describe the considerations and steps to take in order to effectively implement a DD-POI in mouse ocular and hepatic tissue. For complete details on the use and execution of this protocol, please refer to Datta et al. (2018), Ramadurgum and Hulleman (2020), and Ramadurgum et al. (2020).
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Estabilidad Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Animales , Ojo/metabolismo , Inyecciones Intravítreas/métodos , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Dominios Proteicos/genética , Dominios Proteicos/fisiologíaRESUMEN
Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.
