A method for the detection and enrichment of endogenous cereblon substrates

TY - JOUR

T1 - A method for the detection and enrichment of endogenous cereblon substrates

AU - Lloyd, Hannah C.

AU - Li, Yuli

AU - Payne, N. Connor

AU - Zhao, Zhenguang

AU - Xu, Wenqing

AU - Kroupova, Alena

AU - Zollman, David

AU - Long, Tengfang

AU - Kabir, Farah

AU - Chen, Mei

AU - Freeman, Rebecca

AU - Feng, Ethan Yang

AU - Xi, Sarah Y.

AU - Hsu, Ya Chieh

AU - Ciulli, Alessio

AU - Mazitschek, Ralph

AU - Woo, Christina M.

N1 - Publisher Copyright: © 2025 Elsevier Ltd

PY - 2025/8/21

Y1 - 2025/8/21

N2 - 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.

AB - 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.

KW - cereblon

KW - cyclic imide

KW - degron

KW - E3 ligase

KW - posttranslational modification

KW - protein degradation

KW - protein engineering

UR - https://www.scopus.com/pages/publications/105012776529

U2 - 10.1016/j.chembiol.2025.07.002

DO - 10.1016/j.chembiol.2025.07.002

M3 - Article

C2 - 40782806

AN - SCOPUS:105012776529

SN - 2451-9456

VL - 32

SP - 1028-1041.e13

JO - Cell Chemical Biology

JF - Cell Chemical Biology

IS - 8

ER -