Highlights d Identification of 17 new ecDHFR DD stabilizers, dose responses, counter screens d Dissection of minimal chemical and molecular requirements for ecDHFR DD stabilization d HeLa cell death sensitization by concomitant hDHFR inhibition and dnHSF1 stabilization d Simultaneous repression of ocular microglia and ecDHFR DD stabilization in vivo
SUMMARY
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)
.
SUMMARY
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)
.
The Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. In the absence of TMP, a DHFR stabilizer, the DD is degraded by the ubiquitin proteasome system (UPS). To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, which may have a compromised UPS, we evaluated the DHFR DD system in aged mice (up to 24 mo), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4-/- mice). Aged, LIRD, and Abca4-/- mice all had similar proteasomal activities and high-molecular weight ubiquitin levels compared to control mice. However, rd2 mice displayed compromised chymotrypsin activity compared to control mice. Nonetheless, the DHFR DD was effectively degraded in all model systems, including rd2 mice. Moreover, TMP increased DHFR DD-dependent retinal bioluminescence in all mouse models, however the fold induction was slightly, albeit significantly, lower in Abca4-/- mice. Thus, the destabilized DHFR DD-based approach allows for efficient control of protein abundance in aged mice and retinal degeneration mouse models, laying the foundation to use this strategy in a wide variety of mice for the conditional control of gene therapies to potentially treat multiple eye diseases.
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