A method for the detection and enrichment of endogenous cereblon substrates.

Cell chemical biology
Authors
Keywords
Abstract

C-terminal cyclic imides are posttranslational modifications (PTMs) on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. Here, we develop an antibody-like reagent, termed "cerebody," for visualizing and enriching C-terminal cyclic imide-modified proteins. We describe the engineering of CRBN derivatives to produce cerebody and use it to identify CRBN substrates by western blot and enrichment from whole-cell and tissue lysates. CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation.

Year of Publication
2025
Journal
Cell chemical biology
Volume
32
Issue
8
Pages
1028-1041.e13
Date Published
08/2025
ISSN
2451-9448
DOI
10.1016/j.chembiol.2025.07.002
PubMed ID
40782806
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