A degron-mimicking molecular glue drives CRBN homo-dimerization and degradation
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): T Cell, Jurkat Cell
SUBMITTER: 
Despoina Kapsitidou
LAB HEAD: Debora Bonenfant
PROVIDER: PXD060261 | Pride | 2025-09-08
REPOSITORIES: pride
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Project description:Cereblon (CRBN) is a ubiquitin ligase widely harnessed for targeted protein degradation. We report the discovery of a molecular glue that drives CRBN homo-dimerization and subsequent fast, potent, and selective CRBN degradation by the ubiquitin proteasome system. The structure of the complex reveals a unique mechanism whereby two molecular glues assemble into a helix-like structure and drive ternary complex formation by mimicking a protein degron in trans.
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Project description:Molecular glue degraders (MGDs) are small molecule compounds that repurpose the ubiquitin-proteasome system to induce degradation of challenging therapeutic targets. Thalidomide-analogs are clinically effective MGDs that bind cereblon (CRBN), a substrate receptor of the Cullin-4/RING E3 ubiquitin ligase (CRL4CRBN), and impose a gain-of-function activity to recruit, ubiquitinate and degrade so-called neosubstrate proteins. Known neosubstrates bind the CRBN/MGD neosurface on the CRBN CULT domain through a structural G-loop recognition motif that mimics contacts of a natural CRBN degron. Here we report the binding mode of G3BP2, a CRBN neosubstrate that bypasses the G-loop requirement by engaging an unconventional binding site on the CRBN LON domain. The MGD-induced ternary complex interface does not resemble known protein-protein interactions (PPI) with CRBN. Instead, CRBN mimics an endogenous binding partner of G3BP2 and repurposes a preexisting PPI hotspot on the target protein. Our findings provide a novel generalizable concept for the rationalization of unconventional neosubstrate binding modes on CRBN, demonstrate unprecedented potential for the reprogrammability of this substrate receptor by MGDs, and offer opportunities for rational expansion of the target repertoire accessible to this modality.
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Project description:The PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over 50% of tumors showing aberrant AKT hyperactivation. Although potent small molecule AKT inhibitors have entered clinical trials, robust and durable therapeutic responses have not been observed. As an alternative strategy to target AKT, we report the development of INY-03-041, a pan-AKT degrader consisting of GDC-0068, an AKT inhibitor, conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase, Cereblon (CRBN). INY-03-041 induced potent degradation of all three AKT isoforms and displayed enhanced anti-proliferative effects relative to GDC-0068. Notably, INY-03-041 promoted sustained AKT degradation and inhibition of downstream signaling effects for up to 96 hours, even after compound washout. Overall, our findings suggest that AKT degradation may provide prolonged pharmacological effects compared to inhibition, and highlight the potential advantages of AKT-targeted degradation.
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Project description:Biogenesis of motile cilia in multiciliated cells (MCCs) is coupled to large-scale biosynthesis of force generating dynein motors. Shulin inhibits pre-assembled ODA (outer dynein arm) motors by packaging them into a closed conformation preventing aberrant off-target interactions with cellular microtubules in multiciliated Tetrahymena cells and co-localises with them in growing cilia. How packaged ODAs engage the Intraflagellar Transport machinery (IFT) to transit from the cytoplasm into growing cilia remains unclear. The mechanism of Shulin’s release for ODA activation inside cilia is also poorly resolved. Using proteomics, we establish links between DNAAF9 (human Shulin) and mammalian ODAs, the anterograde IFT-B complex and a ciliary small GTPase called ARL3. Based on our findings, we propose that Shulin/DNAAF9 enforces ODA inhibition to aid their unimpeded trafficking from the cytoplasm into growing multicilia via IFT and once inside the ciliary compartment, ARL3-GTP competes for a binding site to destabilise the ODA-Shulin complex thereby promoting ODA motor activation.
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Project description:Plasma proteome is the ultimate target for the biomarker discovery. It stores an endless amount of information on the pathophysiological status of a living organism, which however is still difficult to comprehensively access. The high structural complexity of the plasma proteome can be addressed by using a system-wide and unbiased tool such as mass spectrometry (LC-MS/MS) or highly sensitive targeted immunoassays as Olink Proximity Extension Assays. We have tested the performance of LC-MS/MS in data –dependent and -independent acquisition modes and Olink PEA to measure circulating plasma proteins in 173 human plasma samples from a southern Germany population-cohort. More than 300 proteins were measured by both LC-MS/MS approaches, mainly including high abundance functional plasma proteins. By using the Olink PEA technology we measured 728 plasma proteins, covering a broad dynamic range with high sensitivity down to pg/ml levels. We found 35 overlapping proteins in all three analytical platforms, predominantly secreted and highly abundant in plasma. The complementarity of the platforms was assessed by veryfing the reproducibility of data distributions, statistical correlation of measurements and gender-based differential expression analysis. Our work highlights the limitations and the advantages of both targeted and untargeted approaches and prove their complementarity. We demostrated that by combining the results of the three platforms we gain in depth proteome coverage as well as biological insights.
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Project description:Background and Purpose: Cardiotoxicity is a well-known adverse effect of radiation therapy. Measurable abnormalities in the heart function indicate advanced and often irreversible heart damage. Therefore, early detection of cardiac toxicity is necessary to delay and alleviate the development of the disease. The present study investigated long-term serum proteome alterations following local heart irradiation using a mouse model with the aim to detect biomarkers of radiation-induced cardiac toxicity. Materials and Methods: Serum samples from C57BL/6J mice were collected 20 weeks after local heart irradiation with 8 Gy or 16 Gy X-ray; the controls were sham-irradiated. The samples were analyzed by quantitative proteomics based on data-independent acquisition mass spectrometry. The proteomics data were further investigated using bioinformatics and ELISA. Results: The analysis showed radiation-induced changes in the level of several serum proteins involved in the acute phase response, inflammation and cholesterol metabolism. We found significantly enhanced expression of pro-inflammatory cytokines (TNF-, TGF-, IL-1 and IL-6) in the serum of the irradiated mice. The level of free fatty acids, total cholesterol, low density lipoprotein (LDL) and oxidized LDL was increased whereas that of high density lipoprotein was decreased by irradiation. Conclusions: This study provides information on systemic effects of heart irradiation. It elucidates a radiation fingerprint in the serum that may be used to elucidate adverse cardiac effects after radiation therapy.
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Project description:In the 1950s the drug thalidomide administered as a sedative to pregnant women led ot the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and ist IMiD derivatives recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4(CRBN)) ubiquitin ligase. Through an unbiased screen we identify the homeobox trranscription factor MEIS2 as an endogenous substrate of CRL4(CRBN). By definition, a specific target of CRL4(CRBN) is expected to have a very low intensity on negative control arrays (E1_E2), (E1_CRBN), (E1_E2_Cdt2), (E1_E2_Cdt2_revlimid), (E1_E2_Cdt2_CSN) or with CRL4(CRBN) in presence of inhibitor (E1_E2_CRBN_revlimid) and high intensity on arrays with CRL4(CRBN) (E1_E2_CRBN) or CRL4(CRBN) in presence of CSN (E1_E2_CRBN_CSN) Accordingly 16 protein microarrays were subjected to in vitro ubiquitylation using the following enzyme combinations: 3x Uba1+UbcH5a; 2x Uba1+UbcH5a+CRL4(DDB2); 3x Uba1+UbcH5a+CRL4(CRBN); 2x Uba1+UbcH5a+CRL4(CRBN)+lenalidomide; 2x Uba1+UbcH5a+CRL4(CRBN)+CSN; 2x Uba1+UbcH5a+CRL4(Cdt2); 1x Uba1+UbcH5a+CRL4(Cdt2)+CSN; 1x Uba1+UbcH5a+CRL4(Cdt2)+lenalidomide
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Project description:Parasitic African trypanosomes experience temperature fluctuations due to fever symptomatic of infection, during developmental life cycle transitions in insect vectors and mammalian hosts, or due to diurnal shift. Since trypanosomes exhibit almost exclusive polycistronic transcription, gene expression regulation is dominated by post-transcriptional controls mediated by mRNA 3'-untranslated regions (3'-UTRs). Heat-shock responses have not been described in detail at the transcriptomic or proteomic level, however, and mechanisms underpinning thermo-sensing remain largely uncharacterised. We quantified >9,300 transcripts, and >5,900 proteins in bloodstream form Trypanosoma brucei after growth at 34°C, 37°C or 40°C for six hours. Increased protein abundance correlated with temperature for several hundred genes, encoding both the classical heat-shock proteins, and a second cohort including developmentally regulated genes. Transcript abundance also increased for genes close to transcription initiation sites, but this had minimal impact on protein abundance. Notably, the classical heat-shock response was characterised by both increased mRNA and protein abundance, while increased protein abundance dominated for the second cohort, suggesting translation control. Analysis of 3'-UTRs from post-transcriptionally regulated genes, using motif searching and machine learning, revealed enrichment of (UUA)4 motifs associated with heat-shock proteins, and enrichment of poly-purine and poly-pyrimidine tracts associated with translationally controlled genes. To determine whether these low-complexity sequences increased RNA secondary structure, we disrupted the RNA interference machinery and repeated transcriptomic profiling, essentially using argonaute as an in cellulo probe for native double-stranded RNA (dsRNA). As predicted, thermo-sensitive mRNAs displayed signatures of thermo-sensitive dsRNA formation. Thus, we provide transcriptomic and proteomic profiles of the heat-shock and thermo-sensing response in the African trypanosome. Thermo-sensitive UTRs are enriched in low-complexity and complementary sequences, supporting a post-transcriptional ‘zipper’ hypothesis whereby sequences that regulate mRNA stability and/or translation are progressively unmasked as temperature increases.
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Project description:The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the anti-myeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to and putatively resistant to lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity, and a possible biomarker for the clinical assessment of anti-myeloma efficacy. We included 15 samples from multiple myeloma cell lines.
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