Development of compounds for targeted degradation of mammalian cryptochrome proteins
Jack Munns1, Andrew D Beale1, Iacovos N Michaelides2
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Abstract:
The mammalian cryptochrome proteins (CRY1 and CRY2) are transcriptional repressors most notable for their role in circadian transcriptional feedback. Not all circadian rhythms depend on CRY proteins, however, and the CRY proteins are promiscuous interactors that also regulate many other processes. In cells with chronic CRY deficiency, protein homeostasis is highly perturbed, with a basal increase in cellular stress and activation of key inflammatory signalling pathways. Here, we developed tools to delineate the specific effects of CRY reduction, rather than chronic deficiency, to better understand the direct functions of CRY proteins. Performing a bioluminescence screen and immunoblot validation, we identified compounds that resulted in CRY reduction. Using these compounds, we found that circadian PERIOD2 (PER2) protein rhythms persisted under CRY-depleted conditions. By quantitative mass spectrometry, we found that CRY-depleted cells partially phenocopied the proteomic dysregulation of CRY-deficient cells, but showed minimal circadian phenotypes. We did, however, also observe substantial off-target effects of these compounds on luciferase activity and could not ascertain a specific mechanism of action. This work therefore highlights both the utility and the challenges of targeted protein degradation and bioluminescence reporter approaches in disentangling the contribution of CRY proteins to circadian rhythmicity, homeostasis and innate immune regulation.This article is part of the Theo Murphy meeting issue 'Circadian rhythms in infection and immunity'.
Insights
Mammalian cryptochrome proteins (CRY1 and CRY2) regulate circadian rhythms and cellular homeostasis. Reducing CRY levels affects protein homeostasis but not circadian rhythms, highlighting challenges in studying protein function.
Area of Science:
- Molecular Biology
- Chronobiology
- Immunology
Background:
- Mammalian cryptochromes (CRY1 and CRY2) are transcriptional repressors crucial for circadian feedback loops.
- CRY proteins also regulate non-circadian processes and are involved in protein homeostasis and inflammatory signaling.
- Chronic CRY deficiency leads to perturbed protein homeostasis and activated inflammatory pathways.
Purpose of the Study:
- To develop tools to specifically reduce CRY protein levels, differentiating effects from chronic deficiency.
- To understand the direct functions of CRY proteins in circadian rhythmicity, protein homeostasis, and innate immunity.
Main Methods:
- Bioluminescence screening and immunoblot validation to identify compounds reducing CRY levels.
- Quantitative mass spectrometry to analyze proteomic changes in CRY-depleted cells.
- Assessment of circadian PERIOD2 (PER2) protein rhythms under CRY-depleted conditions.
Main Results:
- Circadian PER2 protein rhythms persisted despite CRY depletion.
- CRY-depleted cells showed partial proteomic dysregulation similar to CRY-deficient cells but minimal circadian phenotypes.
- Identified compounds had off-target effects on luciferase activity, complicating mechanism of action determination.
Conclusions:
- Targeted protein reduction and bioluminescence reporters are useful but challenging tools for studying CRY protein functions.
- CRY proteins contribute to protein homeostasis and innate immunity, with less direct impact on circadian rhythmicity than previously thought.
- Further research is needed to overcome challenges in targeted protein degradation studies and fully elucidate CRY protein roles.
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