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A method for the detection and enrichment of endogenous cereblon substrates

ABSTRACT: C-Terminal cyclic imides are posttranslational modifications 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.

INSTRUMENT(S):

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

TISSUE(S): Heart, Brain, Hepatocyte, Liver, Cell Culture, Kidney

DISEASE(S): Disease Free

SUBMITTER: Christina Woo

LAB HEAD: Christina Woo

PROVIDER: PXD061728 | Pride | 2025-08-11

REPOSITORIES: Pride

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			Action	DRS
	HEK293T.msf	Msf
	HEK293T_F1.raw	Raw
	HEK293T_F2.raw	Raw
	HEK293T_F3.raw	Raw
	HEK293T_F4.raw	Raw

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A method for the detection and enrichment of endogenous cereblon substrates

Project description:C-Terminal cyclic imides are posttranslational modifications 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.

2025-08-11 | PXD064890 | Pride

The E3 ubiquitin ligase adaptor cereblon targets the C-terminal cyclic imide degron

Project description:The E3 ligase factor cereblon (CRBN) is a target of thalidomide and lenalidomide, which are therapeutic agents used in the treatment of hematopoietic malignancies and as ligands for targeted protein degradation. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN is unknown. Here, we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are degrons for CRBN. Dipeptides bearing the cyclic imide degron are substitutes for thalidomide when embedded within bifunctional small molecule degraders. Installation of the degron to the C-terminus of proteins induces CRBN-dependent ubiquitylation and degradation in vitro and in cells. C-Terminal cyclic imides are previously underappreciated post-translational modifications found throughout the human proteome that are endogenously recognized and removed by CRBN. The discovery of the cyclic imide degron defines a novel regulatory process controlled by these modifications, which may impact the development of therapeutic agents that engage CRBN.

2022-07-28 | PXD034248 | Pride

Genesis and regulation of C-terminal cyclic imides from protein damage

Project description:C-Terminal cyclic imides are post-translational modifications (PTMs) that can arise from spontaneous intramolecular cleavage of asparagine or glutamine residues resulting in a form of irreversible protein damage. These protein damage events are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN), indicating that this class of aging-related modifications requires cellular quality control mechanisms to prevent deleterious effects. However, the factors that determine protein or peptide susceptibility to C-terminal cyclic imide formation or their effect on protein stability have not been explored in detail. Here, we characterize the primary and secondary structures of peptides and proteins that promote intrinsic formation of C-terminal cyclic imides in comparison to deamidation, a related form of protein damage. Extrinsic effects from solution properties and stressors on the cellular proteome additionally promote C-terminal cyclic imide formation on proteins like glutathione synthetase (GSS) that are susceptible to aggregation if they are not removed by CRBN. This systematic investigation provides insight to the regions of the proteome that are prone to these unexpectedly frequent modifications, the effects of this form of protein damage on the protein stability, and the biological role of CRBN.

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The E3 ubiquitin ligase adaptor cereblon targets the C-terminal cyclic imide degron

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Discovery of a dual PROTAC for degrading WDR5 and Ikaros oncoproteins as therapeutics [ChIP-Seq]

2022-05-15 | GSE175544 | GEO

The Cyclimids: Degron-inspired cereblon binders for targeted protein degradation

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2024-01-26 | PXD041494 | Pride

RUNX proteins desensitize multiple myeloma to lenalidomide via protecting IKZFs from degradation

Project description:Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma.

2020-04-13 | GSE123660 | GEO

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Project description:The majority of clinical degraders utilize an immunomodulatory imide drug (IMiD)-based derivative, that directs their target to the E3 ligase receptor Cereblon (CRBN), however, identification of IMiD molecular glue substrates has remained underexplored. To tackle this, we design human CRBN constructs which retain all features for ternary complex formation, whilst allowing generation of homogenous and cost-efficient expression in E.coli. Extensive profiling of the construct, shows it to be the “best of both worlds” in terms of binding activity and ease of production. We next designed the ‘Enamine focused IMiD library’ and demonstrated applicability of the construct to high-throughput screening, identifying binders with high potency, ligand efficiency and specificity. Finally, we adapt our construct for proof of principle glue screening approaches enabling IMiD cellular interactome determination. Coupled with our IMiD binding landscape, the methods described here should serve as valuable tools to assist discovery of next generation CRBN glues.

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