Abstract
The E3 ligase substrate adapter cereblon (CRBN), the primary target of clinical agents thalidomide and lenalidomide, recognizes endogenous substrates bearing the C-terminal cyclic imide modification. Although C-terminal cyclic imides can form spontaneously, an enzyme that regulates their formation and thereby promotes a biological pathway connecting substrates to CRBN is unknown. Here we report that protein carboxymethyltransferase (PCMT1) promotes formation of C-terminal cyclic imides on C-terminal asparagine residues of CRBN substrates. PCMT1 and CRBN coregulate the levels of metabolic enzymes including glutamine synthetase and inorganic pyrophosphatase 1 in vitro, in cells and in vivo, and this regulation is associated with the proepileptic phenotype of CRBN knockout mouse models. The discovery of an enzyme that regulates CRBN substrates through the C-terminal cyclic imide reveals a previously unknown biological pathway that is perturbed by thalidomide derivatives and provides a biochemical basis for the connection between multiple biological processes and CRBN.
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Data availability
All data are available in the main text and Supplementary Information. Proteomics data have been deposited to the PRIDE repository under the identifier PXD061730. The crystal structure of human CRBN–DDB1 in complex with GLUL-cN has been deposited to the PDB under the accession code 9NR3. Source data are provided with this paper.
Code availability
All code used in this study is commercially available or provided in a public repository.
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Acknowledgements
We thank S. Ichikawa, H. A. Flaxman, N. C. Payne, M. A. Leon-Duque and T. Long for helpful discussions, T. Wang for support with peptide synthesis, M. Chen and S. Trager from the Harvard University Mass Spectrometry and Proteomics Resource Laboratory for support with MS experiments, D. Cui and B. Tresco from the Harvard University Laukien-Purcell Instrumentation Center for support with peptide MS experiments, and J. A. Nelson and Z. T. Niziolek from the Harvard University Bauer Core for support with flow cytometry. His6-CRBN–DDB1 was a generous gift from Boehringer Ingelheim. Support from the National Institutes of Health (grant no. R01GM141406, C.M.W.), the Blavatnik Biomedical Accelerator at Harvard University (C.M.W.), Mark Foundation for Cancer Research (C.M.W.), Merkin Family Foundation (C.M.W.), the Starr Foundation (C.M.W.) and Spanish Ministerio de Ciencia, Innovación y Universidades (MICINU/FEDER; grant no. PID2021-125118OB-I00, M.G.) is gratefully acknowledged. N.Z. is supported by Howard Hughes Medical Institute.
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Z.Z., W.X. and A.K.D.P. designed and synthesized peptides. E.Y.F., S.C., W.X., H.C.L. and A.K.D.P. generated recombinant and engineered proteins. Z.Z., W.X. and E.Y.F. performed in vitro enzyme activity and TR-FRET binding studies. S.C. and N.Z. designed and conducted structural, BLI and in vitro ubiquitination studies. Z.Z., W.X. and E.Y.F. performed cellular studies. H.C.L. developed cerebody methods. A.H.-L., P.P.-V. and M.G. designed and conducted in vivo measurements. E.Y.F. and W.X. analyzed mouse tissue samples. C.M.W. conceived of the project. C.M.W., Z.Z., W.X., E.Y.F. and S.C. drafted the paper. All authors reviewed and edited the paper.
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The Woo Laboratory, under the direction of C.M.W., receives or has received sponsored research support from Amgen, Ono Pharmaceuticals and Merck. N.Z. is one of the scientific cofounders and a shareholder of SEED Therapeutics, and serves as a member of the scientific advisory boards of SyntheX, Molecular Glue Labs, Cold Start Therapeutics and Differentiated Therapeutics, with financial interests. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Formation of cyclic imides from asparagine.
(a) Scheme of protein damage events that occur through Asn residues and result in the C-terminal cyclic imide or isoaspartate post-translational modifications. (b) Chemical scheme and conversion of model peptides incubated in 20 mM NH4OAc (pH 7.4) buffer at 37 °C (n = 3). We detected two distinct hydrolysis products, which were constitutional isomers, but were unable to assign their identities. Data represent mean ± S.D. (c) Scheme of PCMT1 correction of isoaspartate modifications.
Extended Data Fig. 2 Efficiency of PCMT1-mediated C-terminal cyclic imide formation on various peptides.
(a) Time-course of peptides representing the C-termini of proteins previously linked to CRBN incubated with PCMT1 and SAM in 50 mM Tris-Cl (pH 7.4) buffer at 37 °C (n = 3). (b) Time-course incubation of Fmoc-FN and GGGFN with PCMT1 and SAM in 50 mM Tris-Cl (pH 7.4) buffer at 37 °C (n = 3). (c) Time-course incubation of synthetically methylated peptide PFQYKN(OMe) in 50 mM Tris-Cl (pH 7.4) buffer at 37 °C with or without PCMT1 and SAM. (d) Time-course incubation of Fmoc-FN and FQYKN with the catalytically inactive PCMT1 S60A mutant and SAM in 50 mM Tris-Cl (pH 7.4) buffer at 37 °C (n = 2). For all these data, note that the grey curves likely represent the sum of two species, the C-terminal Asn or Gln (that is, the starting material) or the C-terminal amide (a hydrolysis product), which are indistinguishable by mass spectrometry. Indicated sample sizes (n) represent biological replicates.
Extended Data Fig. 3 Sequence preference of PCMT1 activity and CRBN recognition.
(a) Structure of thalidomide-FITC, the TR-FRET tracer used to assess the engagement of ligands with His6-CRBN–DDB1 in this study. (b) Conversion efficiency and CRBN binding of GLUL C-terminal peptides with variable lengths after 14 h incubation with PCMT1 by mass spectrometry and TR-FRET assay, respectively (n = 3). (c) Conversion efficiency and CRBN binding of GLUL C-terminal peptides with variable –1 residue after 14 h incubation with PCMT1 by mass spectrometry and TR-FRET assay, respectively (n = 3). (d) Conversion efficiency and CRBN binding of GLUL C-terminal peptides with variable –2 residue after 14 h incubation with PCMT1 by mass spectrometry and TR-FRET assay, respectively (n = 3). Data represent mean ± S.D. in all graphs with error bars. Indicated sample sizes (n) represent biological replicates.
Extended Data Fig. 4 Full-length GLUL is recognized and regulated by PCMT1 and CRBN in cells.
(a) In vitro ubiquitination assay of Venus-GLUL with WT or catalytically inactive PCMT1 (n = 2). (b) Size exclusion chromatography of the GFP-GLUL protein before or after electroporation. The oligomerization state of the protein did not change upon electroporation. (c) Cerebody enrichment for APP from WT, PCMT1-KO, or CRBN-KO HEK293T cells after treatment with DMSO or 100 µM lenalidomide for 24 h (n = 3). Multiple bands are observed corresponding to different isoforms of APP with different degrees of PTMs. Enriched APP may stem from nonspecific binding. (d) Western blot of endogenous GLUL levels in sgCtrl, PCMT1-KO, or CRBN-KO HEK293T cells (n = 3). Quantified relative GLUL levels are shown. (e) Cycloheximide (CHX) chase assay to monitor the degradation of GLUL over 24 h in sgCtrl, PCMT1-KO, or CRBN-KO HEK293T cells (n = 2). Mean relative GLUL levels are shown. (f–g) Western blot of GLUL levels in sgCtrl, PCMT1-KO, or CRBN-KO HEK293T cells grown to low or high confluence, along with representative images of cells in culture from the same experiment (n = 3). Quantified relative GLUL levels, normalized to sgCtrl within each condition, are shown. (h) Western blot of GLUL levels in sgCtrl, PCMT1-KO, or CRBN-KO HEK293T cells treated with DMSO or 1 µM MLN4924 for 24 h (n = 3). Mean relative GLUL levels are shown. Indicated sample sizes (n) represent biological replicates.
Extended Data Fig. 5 Glutamine-triggered GLUL degradation is independent of PCMT1.
(a) Scheme of GLUL-catalyzed condensation of glutamate and ammonium into glutamine. (b) Time-course GLUL buildup in WT, sgCtrl, PCMT1-KO, or CRBN-KO HEK293T cells incubated in glutamine-free media (n = 3). (c) Time-course GLUL degradation of glutamine-starved sgCtrl, PCMT1-KO, or CRBN-KO HEK293T cells after refeeding with 4 mM glutamine (n = 3). Quantified relative GLUL levels are shown. (d) Normalized mRNA level of GLUL in HEK293T cells with or without glutamine starvation for 48 h (n = 3). Data represent mean ± S.D. Indicated sample sizes (n) represent biological replicates.
Extended Data Fig. 6 Impact of clinical agents on the GLUL-cN level in multiple cell lines.
(a) Cerebody enrichment for GLUL-cN from WT, PCMT1-KO, or CRBN-KO HEK293T cells after treatment with DMSO or 100 µM lenalidomide for 24 h (n = 3). (b) Cerebody enrichment for GLUL-cN from different human and mouse cell lines after treatment with DMSO or 100 µM lenalidomide for 24 h (n = 3). Elution for SK-N-SH and MEF was performed with 3× FLAG peptide, while elution for Neuro-2a was performed with 200 µM lenalidomide. Indicated sample sizes (n) represent biological replicates.
Extended Data Fig. 7 Time- and dose-dependence of clinical agents’ impact on GLUL-cN in cells.
(a) Cerebody enrichment for Glul-cN from Neuro-2a cells after treatment with varying doses of lenalidomide for 24 h (n = 3). (b) Cerebody enrichment for Glul-cN from Neuro-2a cells after treatment with 100 µM lenupalidomide for varying durations (n = 3). (c) Cerebody enrichment for Glul-cN from Neuro-2a cells after treatment with DMSO, 5 µM TAK-243, 1 µM MLN4924, or 10 µM MG132 for 6 h (n = 3). Indicated sample sizes (n) represent biological replicates.
Extended Data Fig. 8 Characterization of GLUL-cN binding to CRBN.
(a) Close-up view of GLUL-cN in complex with CRBN. GLUL-cN is shown as a stick model, together with its positive Fo – Fc electron density (purple mesh) calculated and contoured at 2.5σ before it was built into the CRBN-DDB1 model. The C-terminal cN and the backbones of upstream residues were resolved with continuous density, along with clear density for the side chain of the tyrosine residue at the –2 position. CRBN is shown as an orange cartoon with the tri-Trp side chains highlighted. (b) GLUL-cN–CRBN aligned with CRBN–lenalidomide (PDB: 5FQD). For GLUL-cN–CRBN, CRBN is shown as an orange cartoon with the tri-Trp side chains highlighted. GLUL-cN is shown as sticks (carbon: green, oxygen: red, nitrogen: blue). For CRBN–lenalidomide, CRBN is shown as a purple cartoon, and lenalidomide (Len) is shown as sticks (carbon: purple, oxygen: red, nitrogen: blue). (c) GLUL-cN–CRBN aligned with MsCI4–QMQcN (PDB: 8BC6). CRBN is shown as an orange cartoon with the tri-Trp side chains highlighted. GLUL-cN is shown as sticks (carbon: green, oxygen: red, nitrogen: blue). MsCI4 is shown as a cyan cartoon, and the peptide (QMQcN) is shown as sticks (carbon: cyan, oxygen: red, nitrogen: blue, sulfur: yellow). (d–h) BLI measurements of GLUL-cN binding to CRBN WT or mutants (n = 2 biological replicates). (i) The C-terminal residues of GLUL across species from different classes of vertebrates. The conserved C-terminal N residues are highlighted in red.
Extended Data Fig. 9 Genetic knockout of Crbn leads to accumulation of Glul and Ppa1 and neurological phenotypes in mouse models.
(a) Volcano plot of tryptic peptides derived from protein C-termini that bear a C-terminal N or Q identified from the hippocampi of 4 individual WT or Crbn−/− mice (two male, two female). P-values for the abundance ratios were calculated using a one-way ANOVA with TukeyHSD post-hoc test. (b) Western blot of Glul and Ppa1 levels in WT or Crbn−/− mouse hippocampus (n = 3). (c) Normalized mRNA levels of Glul, Ppa1, Pcmt1, and Crbn in WT or Crbn−/− mouse hippocampus (n = 3). Comparisons were performed using unpaired two-tailed t-tests, and p-values are indicated. (d) The C-terminal residues of PPA1 across species from different classes of vertebrates. The conserved C-terminal N residues are highlighted in red. (e) Comparison of the time from KA injection to the first observation of convulsions (stage 3 of Racine scale) in WT (n = 19) and Crbn−/− (n = 16) mice. (f) Comparison of Racine scores of WT (n = 19) and Crbn−/− (n = 16) mice during each phase of the convulsion response. Comparisons were performed using unpaired two-tailed t-tests, and p-values are indicated. Data represent mean ± S.D. in all graphs with error bars. Indicated sample sizes (n) represent biological replicates.
Extended Data Fig. 10 PPA1 is a substrate of PCMT1 and CRBN.
(a) Time-course incubation of the peptide representing the C-terminus of human PPA1 with PCMT1 and SAM in 50 mM Tris-Cl (pH 7.4) buffer at 37 °C (n = 3). (b) Time-course incubation of the peptide representing the C-terminus of mouse Ppa1 with PCMT1 and SAM in 50 mM Tris-Cl (pH 7.4) buffer at 37 °C (n = 3). (c) TR-FRET assay of the human or mouse PPA1 C-terminal peptides after 14 h incubation with or without PCMT1 (n = 3). Data represent mean ± S.D. (d) Cerebody enrichment for PPA1-cN from different human and mouse cell lines after treatment with DMSO or 100 µM lenalidomide for 24 h (n = 3). Basal levels of enriched PPA1 may stem from nonspecific binding. Elution for HEK293T and Neuro-2a was performed with 200 µM lenalidomide, while elution for SK-N-SH was performed with 3× FLAG peptide. Indicated sample sizes (n) represent biological replicates.
Supplementary information
Supplementary Information
Supplementary Tables 1–4, Figs. 1 and 2, Notes 1 (materials and instrumentation) and 2 (synthetic procedures), NMR spectra, and LC–MS traces.
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Zhao, Z., Xu, W., Feng, E.Y. et al. PCMT1 generates the C-terminal cyclic imide degron on CRBN substrates. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-02106-9
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DOI: https://doi.org/10.1038/s41589-025-02106-9
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