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Abstract Target validation is key to the development of protein degrading molecules such as proteolysis‐targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin‐proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3‐catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN‐pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP‐HaloTag fusion with higher activities than VHL‐engaging HaloPROTACs at sub‐micromolar concentrations of the compound. The CRBN‐engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.
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The E3 Ligases Spsb1 and Spsb4 Regulate RevErbα Degradation and Circadian Period
The time-dependent degradation of core circadian clock proteins is essential for the proper functioning of circadian timekeeping mechanisms that drive daily rhythms in gene expression and, ultimately, an organism’s physiology. The ubiquitin proteasome system plays a critical role in regulating the stability of most proteins, including the core clock components. Our laboratory developed a cell-based functional screen to identify ubiquitin ligases that degrade any protein of interest and have started screening for those ligases that degrade circadian clock proteins. This screen identified Spsb4 as a putative novel E3 ligase for RevErbα. In this article, we further investigate the role of Spsb4 and its paralogs in RevErbα stability and circadian rhythmicity. Our results indicate that the paralogs Spsb1 and Spsb4, but not Spsb2 and Spsb3, can interact with and facilitate RevErbα ubiquitination and degradation and regulate circadian clock periodicity.
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- Award ID(s):
- 1832069
- PAR ID:
- 10547893
- Publisher / Repository:
- SAGE Publications
- Date Published:
- Journal Name:
- Journal of Biological Rhythms
- Volume:
- 34
- Issue:
- 6
- ISSN:
- 0748-7304
- Format(s):
- Medium: X Size: p. 610-621
- Size(s):
- p. 610-621
- Sponsoring Org:
- National Science Foundation
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