Proteomics

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Development of compounds for targeted degradation of mammalian cryptochrome proteins


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 in order to better understand the direct functions of CRY proteins. Through 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 that are required to disentangle the contribution of CRY to circadian rhythmicity, homeostasis and innate immune regulation.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Sew Peak-Chew  

LAB HEAD: John S O'Neill

PROVIDER: PXD058309 | Pride | 2025-01-22

REPOSITORIES: pride

Dataset's files

Source:
Action DRS
CRM6010_TMT18_Jack_300322.xlsx Xlsx
CRM6010_TMT18_MS3RTS_F1.raw Raw
CRM6010_TMT18_MS3RTS_F10.raw Raw
CRM6010_TMT18_MS3RTS_F11.raw Raw
CRM6010_TMT18_MS3RTS_F12.raw Raw
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