. 2025 Aug 21;32(8):1028-1041.e13.

doi: 10.1016/j.chembiol.2025.07.002. Epub 2025 Aug 8.

A method for the detection and enrichment of endogenous cereblon substrates

Hannah C Lloyd¹, Yuli Li¹, N Connor Payne², Zhenguang Zhao¹, Wenqing Xu¹, Alena Kroupova³, David Zollman³, Tengfang Long¹, Farah Kabir¹, Mei Chen⁴, Rebecca Freeman⁵, Ethan Yang Feng¹, Sarah Y Xi¹, Ya-Chieh Hsu⁵, Alessio Ciulli³, Ralph Mazitschek⁶, Christina M Woo⁷

Affiliations

¹ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
² Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
³ Centre for Targeted Protein Degradation, School of Life Sciences, University of Dundee, Dundee DD1 5JJ, UK.
⁴ Harvard Center for Mass Spectrometry, Division of Science, Faculty of Arts and Sciences, Harvard University, Cambridge, MA 02138, USA.
⁵ Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
⁶ Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA 02115, USA.
⁷ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: cwoo@chemistry.harvard.edu.

PMID: 40782806
PMCID: PMC12360616 (available on 2026-08-05)
DOI: 10.1016/j.chembiol.2025.07.002

A method for the detection and enrichment of endogenous cereblon substrates

Hannah C Lloyd et al. Cell Chem Biol. 2025.

. 2025 Aug 21;32(8):1028-1041.e13.

doi: 10.1016/j.chembiol.2025.07.002. Epub 2025 Aug 8.

Authors

Affiliations

¹ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
² Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
³ Centre for Targeted Protein Degradation, School of Life Sciences, University of Dundee, Dundee DD1 5JJ, UK.
⁴ Harvard Center for Mass Spectrometry, Division of Science, Faculty of Arts and Sciences, Harvard University, Cambridge, MA 02138, USA.
⁵ Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
⁶ Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA 02115, USA.
⁷ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: cwoo@chemistry.harvard.edu.

PMID: 40782806
PMCID: PMC12360616 (available on 2026-08-05)
DOI: 10.1016/j.chembiol.2025.07.002

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.

Keywords: E3 ligase; cereblon; cyclic imide; degron; posttranslational modification; protein degradation; protein engineering.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests R.M. and N.C.P. are inventors on patent applications related to the CoraFluor TR-FRET technology used in this work. The Woo Lab receives or has received sponsored research support from Amgen, Ono Pharmaceuticals, and Merck. The Ciulli laboratory receives or has received sponsored research support from Almirall, Amgen, Amphista Therapeutics, Boehringer Ingelheim, GlaxoSmithKline, Eisai, Merck KGaA, Nurix Therapeutics, Ono Pharmaceuticals, and Tocris-Biotechne. A.C. is a co-founder and shareholder of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms.

Update of

A method for the detection and enrichment of endogenous cereblon substrates.
Lloyd HC, Li Y, Payne NC, Zhao Z, Xu W, Kroupova A, Zollman D, Long T, Kabir F, Chen M, Freeman R, Feng EY, Xi S, Hsu YC, Ciulli A, Mazitschek R, Woo CM. Lloyd HC, et al. bioRxiv [Preprint]. 2025 Mar 26:2025.03.24.645063. doi: 10.1101/2025.03.24.645063. bioRxiv. 2025. Update in: Cell Chem Biol. 2025 Aug 21;32(8):1028-1041.e13. doi: 10.1016/j.chembiol.2025.07.002. PMID: 40196695 Free PMC article. Updated. Preprint.

References

1. Higgins JJ, Pucilowska J, Lombardi RQ, Rooney JP (2004). A mutation in a novel ATP-dependent Lon protease gene in a kindred with mild metal retardation. Neurology 63, 1927–1931. - PMC - PubMed
1. Ito T, Ando H, Suzuki T, Ogura T, Hotta K, Imamura Y, Yamaguchi Y, and Handa H (2010). Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350. 10.1126/science.1177319. - DOI - PubMed
1. Lupas AN, Zhu H, and Korycinski M (2015). The thalidomide-binding domain of cereblon defines the CULT domain family and is a new member of the beta-tent fold. PLoS Comput Biol 11, e1004023. 10.1371/journal.pcbi.1004023. - DOI - PMC - PubMed
1. Kronke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, Svinkina T, Heckl D, Comer E, Li X, et al. (2014). Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305. 10.1126/science.1244851. - DOI - PMC - PubMed
1. Lu G, Middleton RE, Sun H, Naniong M, Ott CJ, Mitsiades CS, Wong KK, Bradner JE, and Kaelin WG Jr. (2014). The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309. 10.1126/science.1244917. - DOI - PMC - PubMed

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

A method for the detection and enrichment of endogenous cereblon substrates

Affiliations

A method for the detection and enrichment of endogenous cereblon substrates

Authors

Affiliations

Abstract

Conflict of interest statement

Update of

Similar articles

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials

Miscellaneous

Abstract

Conflict of interest statement

Update of

Similar articles

References

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials

Miscellaneous