{"gene":"DCAF15","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2019,"finding":"Crystal structure of the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex at 2.3 Å resolution revealed that DCAF15 has a distinct topology that embraces the RBM39 RRM2 domain largely via non-polar interactions, with indisulam binding in a pocket between DCAF15 and RBM39(RRM2) to coordinate additional protein-protein contacts. Point mutants of RBM39 and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif recognized by DCAF15.","method":"X-ray crystallography (2.3 Å), RBM39 point mutagenesis, indisulam analog structure-activity studies","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution crystal structure plus mutagenesis validation in a single rigorous study","pmids":["31819272"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at 4.4 Å, together with crystal structures of engineered subcomplexes, showed that DCAF15 adopts a new fold stabilized by DDA1 and that aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively recruit and degrade RBM39 and RBM23. Extensive protein-protein contacts between the ligase and substrate mitigate low-affinity interactions between the drug and DCAF15.","method":"Cryo-EM (4.4 Å), X-ray crystallography of subcomplexes, biochemical binding assays","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM plus crystal structures of subcomplexes, multiple orthogonal structural methods","pmids":["31686031"],"is_preprint":false},{"year":2019,"finding":"Crystal structure of DDA1-DDB1-DCAF15 in complex with E7820 and the RRM2 domain of RBM39 revealed that E7820 packs in a shallow pocket on the surface of DCAF15 and the modified interface binds RBM39 through the α1 helix of the RRM2 domain. Kinetic studies demonstrated that aryl sulfonamide and RBM39 bind DCAF15 in a synergistic (cooperative) manner, confirming aryl sulfonamides act as molecular glues.","method":"X-ray crystallography, kinetic binding analysis","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus kinetic mechanistic validation, independent of other structural studies","pmids":["31693911"],"is_preprint":false},{"year":2019,"finding":"Domain mapping and random mutagenesis identified DCAF15 residues Q232 and D475 as necessary for indisulam-dependent RBM39 recruitment. RBM39 is recruited to DCAF15 via its RRM2 domain and is ubiquitinated on its N terminus. RBM23, a paralog of RBM39, is also recruited to CRL4-DCAF15 through its RRM2 domain and undergoes sulfonamide-dependent degradation. Indisulam-induced splicing changes (intron retention, exon skipping) are attributable solely to RBM39 degradation.","method":"Domain mapping, random mutagenesis, ubiquitination assays, RNA-seq","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal mutagenesis in both DCAF15 and RBM39 with functional ubiquitination readouts, replicated across multiple groups","pmids":["31693891"],"is_preprint":false},{"year":2019,"finding":"Using pSILAC proteomics combined with two complementary digestion approaches, PRPF39 was identified as a novel substrate of E7070/DCAF15. PRPF39 undergoes DCAF15-dependent ubiquitination and proteasomal degradation upon E7070 treatment, establishing it as a bona fide neo-substrate of the CUL4-DCAF15 E3 ubiquitin ligase.","method":"pSILAC mass spectrometry, ubiquitination assay, proteasome inhibitor rescue, DCAF15 knockdown","journal":"Journal of proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteome-wide discovery plus targeted biological validation in a single lab with multiple orthogonal methods","pmids":["31626998"],"is_preprint":false},{"year":2020,"finding":"ARNT (aryl hydrocarbon receptor nuclear translocator) interacts with DCAF15 and undergoes proteasomal degradation induced by aryl sulfonamides indisulam and E7820 through CRL4-DCAF15, independently of the known neo-substrates RBM39 and RBM23. This degradation inhibited transcriptional activities of HIFs and AhR associated with ARNT.","method":"Co-immunoprecipitation, proteasome inhibitor rescue, RBM39/RBM23 knockdown controls, transcriptional reporter assays","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus functional degradation assays with appropriate controls, single lab","pmids":["33168788"],"is_preprint":false},{"year":2021,"finding":"ZEB1 was identified as an endogenous substrate of the CRL4-DCAF15 E3 ubiquitin ligase complex. DCAF15 acts as an adaptor that specifically recognizes the N-terminal zinc finger domain of ZEB1 and triggers its ubiquitin-proteasome-mediated degradation. DCAF15 knockdown led to ZEB1 upregulation and EMT activation, while DCAF15 overexpression suppressed ZEB1 and inhibited EMT in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, domain mapping, ubiquitination assay, DCAF15 knockdown/overexpression with EMT phenotypic readout","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP with domain mapping and functional rescue experiments, single lab","pmids":["33833131"],"is_preprint":false},{"year":2021,"finding":"DCAF15/RBM39 pathway controls KRAS4A alternative splicing. Pharmacological inhibition of RBM39 using indisulam reduces KRAS4A isoform levels and inhibits cancer stem cells, placing DCAF15 upstream of KRAS4A isoform production through RBM39-dependent splicing regulation.","method":"Indisulam treatment, isoform-specific RT-PCR/RNA analysis, cancer stem cell functional assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional pharmacological and genetic evidence linking DCAF15-RBM39 to specific splicing outcome, single lab","pmids":["34257283"],"is_preprint":false},{"year":2024,"finding":"DCAF15 directly interacts with the SMC1A subunit of the cohesin complex and destabilizes cohesin regulatory factors PDS5A and CDCA5. Loss of DCAF15 causes loss of PDS5A and CDCA5, precluding cohesin acetylation on chromatin, resulting in uncontrolled chromatin loop extrusion, defective DNA replication fork integrity, accumulation of DNA damage, and apoptosis in acute myeloid leukemia (AML) cells.","method":"Domain-focused genetic screen, DCAF15 loss-of-function (knockout), co-immunoprecipitation (DCAF15-SMC1A), cohesin acetylation assays, chromatin loop extrusion analysis, replication fork assays, DNA damage markers","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic screen plus reciprocal Co-IP plus multiple orthogonal functional assays establishing endogenous DCAF15 function with defined mechanistic pathway","pmids":["38961054"],"is_preprint":false},{"year":2025,"finding":"DCAF15 interacts with EphA2 receptor and promotes its K48- and K63-linked ubiquitination at lysine residues K646, K649, and K754, leading to EphA2 degradation. S. suis STK phosphorylates SIRT1 at S48, triggering SIRT1-mediated deacetylation of DCAF15 at K552 and K581, which enhances DCAF15-EphA2 interaction and EphA2 ubiquitination. This DCAF15-mediated EphA2 degradation disrupts blood-brain barrier integrity during S. suis meningitis.","method":"Co-immunoprecipitation, ubiquitination site mapping (K646/K649/K754), acetylation site mapping (K552/K581), SIRT1 deacetylase assay, in vitro and in vivo BBB disruption models","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with site-specific mutagenesis and PTM mapping, single lab, novel mechanistic context","pmids":["41276610"],"is_preprint":false},{"year":2024,"finding":"Network-based proteomics analysis identified RBM5 as a novel indisulam neo-substrate degraded in a proteasome-dependent manner via DCAF15, expanding the list of CRL4-DCAF15 substrates beyond RBM39 and RBM23.","method":"Network-based omics prioritization (Random Walk with Restart), proteomics, proteasome inhibitor rescue","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single method for validation (proteasome-dependent degradation), no direct DCAF15 binding shown","pmids":["bio_10.1101_2024.09.16.613231"],"is_preprint":true}],"current_model":"DCAF15 is the substrate receptor of the CRL4 (CUL4-RBX1-DDB1-DDA1-DCAF15) E3 ubiquitin ligase complex; it adopts a novel fold stabilized by DDA1 and, in the presence of aryl-sulfonamide molecular glues (indisulam, E7820, tasisulam), neo-functionalizes a shallow surface pocket to recruit and ubiquitinate splicing factors RBM39 and RBM23 (via their RRM2 α-helical degron), as well as additional substrates including PRPF39, ARNT, RBM5, ZEB1, and EphA2; endogenously, DCAF15 interacts with SMC1A of the cohesin complex and controls cohesin dynamics by destabilizing PDS5A and CDCA5, thereby regulating cohesin acetylation, chromatin loop extrusion, and DNA replication fork integrity."},"narrative":{"mechanistic_narrative":"DCAF15 is the substrate-receptor subunit of a CRL4 (CUL4-RBX1-DDB1) E3 ubiquitin ligase, where it adopts a distinct, non-canonical fold stabilized by the small subunit DDA1 [PMID:31686031]. Its defining biochemical property is that it harbors a shallow, non-conserved surface pocket that is neo-functionalized by aryl-sulfonamide molecular glues (indisulam, E7820): the drug binds cooperatively at the interface between DCAF15 and the RRM2 domain of the splicing factor RBM39, coordinating extensive protein-protein contacts that mitigate the low intrinsic affinity of the glue and recruit RBM39 for ubiquitination on its N-terminus [PMID:31819272, PMID:31693911, PMID:31693891]. RBM39 is engaged through an α-helical degron in its RRM2 domain, and the paralog RBM23 is recruited and degraded by the same mechanism [PMID:31819272, PMID:31693891]. Sulfonamide-induced degradation of RBM39 reprograms alternative splicing—including intron retention, exon skipping, and KRAS4A isoform production—accounting for the drugs' downstream effects [PMID:31693891, PMID:34257283]. Beyond RBM39/RBM23, drug-dependent neo-substrates include PRPF39 and ARNT [PMID:31626998, PMID:33168788]. Independently of molecular glues, DCAF15 also directs ubiquitin-proteasome degradation of endogenous substrates: ZEB1, recognized through its N-terminal zinc-finger domain, with consequences for EMT [PMID:33833131], and EphA2, ubiquitinated at defined lysines in a manner regulated by SIRT1-mediated deacetylation of DCAF15 [PMID:41276610]. Endogenously, DCAF15 interacts with the SMC1A subunit of cohesin and destabilizes the cohesin regulators PDS5A and CDCA5, thereby controlling cohesin acetylation, chromatin loop extrusion, replication fork integrity, and survival of acute myeloid leukemia cells [PMID:38961054].","teleology":[{"year":2019,"claim":"Established at atomic resolution how a substrate-receptor with no obvious binding cleft can be co-opted by a small molecule to recruit a neo-substrate, defining the molecular-glue mechanism for DCAF15.","evidence":"X-ray crystallography of DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) plus RBM39 point mutagenesis and indisulam analog SAR","pmids":["31819272","31693911"],"confidence":"High","gaps":["Did not define endogenous, drug-independent function of DCAF15","Limited to RBM39 RRM2; full-length substrate engagement geometry not resolved"]},{"year":2019,"claim":"Showed DCAF15 adopts a new DDA1-stabilized fold and that aryl-sulfonamides neo-functionalize a shallow non-conserved pocket selective for RBM39 and its paralog RBM23, explaining substrate selectivity.","evidence":"Cryo-EM (4.4 Å) of DDB1-DCAF15-DDA1-RBM39-E7820 with crystal structures of engineered subcomplexes and biochemical binding assays","pmids":["31686031"],"confidence":"High","gaps":["Lower resolution limits side-chain interpretation at the drug interface","Did not address whether other DCAF15 substrates use the same pocket"]},{"year":2019,"claim":"Genetically mapped the DCAF15 residues and RBM39 degron required for drug-induced recruitment and demonstrated the splicing phenotype is attributable solely to RBM39 loss, linking degradation to transcriptomic output.","evidence":"Domain mapping, random mutagenesis (DCAF15 Q232/D475), ubiquitination assays, and RNA-seq","pmids":["31693891"],"confidence":"High","gaps":["Did not establish endogenous physiological substrates","Mechanism of N-terminal ubiquitin site selection not resolved"]},{"year":2019,"claim":"Expanded the drug-dependent neo-substrate repertoire beyond RBM39/RBM23 by identifying PRPF39 through unbiased proteomics.","evidence":"pSILAC mass spectrometry with ubiquitination assay, proteasome-inhibitor rescue, and DCAF15 knockdown","pmids":["31626998"],"confidence":"Medium","gaps":["No structural basis for PRPF39 recognition","Single-lab validation"]},{"year":2020,"claim":"Identified ARNT as a sulfonamide-induced DCAF15 substrate degraded independently of RBM39/RBM23, linking DCAF15 activity to HIF and AhR transcriptional pathways.","evidence":"Co-IP, proteasome-inhibitor rescue, RBM39/RBM23 knockdown controls, transcriptional reporter assays","pmids":["33168788"],"confidence":"Medium","gaps":["Degron on ARNT not mapped","No reciprocal structural validation"]},{"year":2021,"claim":"Demonstrated that DCAF15 has an endogenous, drug-independent substrate (ZEB1) recognized via its N-terminal zinc-finger domain, controlling EMT.","evidence":"Co-IP, domain mapping, ubiquitination assay, and DCAF15 knockdown/overexpression with EMT readouts in hepatocellular carcinoma cells","pmids":["33833131"],"confidence":"Medium","gaps":["Single-lab, single-cancer context","No structure of the DCAF15-ZEB1 interface"]},{"year":2021,"claim":"Placed the DCAF15-RBM39 axis upstream of a specific oncogenic splicing event (KRAS4A isoform) with functional consequences for cancer stem cells.","evidence":"Indisulam treatment, isoform-specific RNA analysis, and cancer stem cell functional assays","pmids":["34257283"],"confidence":"Medium","gaps":["Indirect (mediated by RBM39 splicing activity rather than direct DCAF15 substrate)","Single-lab"]},{"year":2024,"claim":"Defined an endogenous, drug-independent function of DCAF15 in genome stability: it binds cohesin via SMC1A and destabilizes PDS5A/CDCA5 to control cohesin acetylation, loop extrusion, and replication fork integrity.","evidence":"Domain-focused genetic screen, DCAF15 knockout, reciprocal Co-IP (DCAF15-SMC1A), cohesin acetylation, loop extrusion, replication fork, and DNA damage assays in AML cells","pmids":["38961054"],"confidence":"High","gaps":["Whether PDS5A/CDCA5 are direct ubiquitination substrates of CRL4-DCAF15 not fully resolved","Generality beyond AML not established"]},{"year":2025,"claim":"Revealed PTM-dependent regulation of DCAF15 substrate engagement, showing SIRT1-mediated deacetylation of DCAF15 enhances EphA2 ubiquitination, with consequences for blood-brain barrier integrity.","evidence":"Co-IP, ubiquitination and acetylation site mapping, SIRT1 deacetylase assay, and in vitro/in vivo BBB models of S. suis meningitis","pmids":["41276610"],"confidence":"Medium","gaps":["Single-lab, infection-specific context","Direct degron on EphA2 not structurally defined"]},{"year":null,"claim":"It remains unresolved how DCAF15 selects among its diverse endogenous substrates without a molecular glue, and whether a unifying structural logic links cohesin regulation, ZEB1, and EphA2 degradation.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of DCAF15 with any endogenous substrate","Degron features of endogenous substrates undefined","Regulation of DCAF15 abundance/activity in normal physiology unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,6,8,9]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,3,6,9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[3,7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[8]}],"complexes":["CRL4-DCAF15 (CUL4-RBX1-DDB1-DDA1-DCAF15) E3 ubiquitin ligase"],"partners":["DDB1","DDA1","RBM39","RBM23","SMC1A","ZEB1","EPHA2","ARNT"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q66K64","full_name":"DDB1- and CUL4-associated factor 15","aliases":[],"length_aa":600,"mass_kda":66.5,"function":"Substrate-recognition component of the DCX(DCAF15) complex, a cullin-4-RING E3 ubiquitin-protein ligase complex that mediates ubiquitination and degradation of target proteins (PubMed:16949367, PubMed:31452512). The DCX(DCAF15) complex acts as a regulator of the natural killer (NK) cells effector functions, possibly by mediating ubiquitination and degradation of cohesin subunits SMC1A and SMC3 (PubMed:31452512). May play a role in the activation of antigen-presenting cells (APC) and their interaction with NK cells (PubMed:31452512) Binding of aryl sulfonamide anticancer drugs, such as indisulam (E7070) or E7820, change the substrate specificity of the DCX(DCAF15) complex, leading to promote ubiquitination and degradation of splicing factor RBM39 (PubMed:28302793, PubMed:28437394, PubMed:31452512, PubMed:31693891). RBM39 degradation results in splicing defects and death in cancer cell lines (PubMed:28302793, PubMed:28437394, PubMed:31693891). Aryl sulfonamide anticancer drugs change the substrate specificity of DCAF15 by acting as a molecular glue that promotes binding between DCAF15 and weak affinity interactor RBM39 (PubMed:31686031, PubMed:31819272). Aryl sulfonamide anticancer drugs also promote ubiquitination and degradation of RBM23 and PRPF39 (PubMed:31626998, PubMed:31686031, PubMed:31693891)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q66K64/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DCAF15","classification":"Not Classified","n_dependent_lines":161,"n_total_lines":1208,"dependency_fraction":0.13327814569536423},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DDB1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/DCAF15","total_profiled":1310},"omim":[{"mim_id":"621023","title":"RNA-BINDING MOTIF PROTEIN 23; RBM23","url":"https://www.omim.org/entry/621023"},{"mim_id":"620109","title":"DDB1- AND CUL4-ASSOCIATED FACTOR 15; DCAF15","url":"https://www.omim.org/entry/620109"},{"mim_id":"604739","title":"RNA-BINDING MOTIF PROTEIN 39; RBM39","url":"https://www.omim.org/entry/604739"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DCAF15"},"hgnc":{"alias_symbol":["MGC99481"],"prev_symbol":["C19orf72"]},"alphafold":{"accession":"Q66K64","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q66K64","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q66K64-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q66K64-F1-predicted_aligned_error_v6.png","plddt_mean":77.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DCAF15","jax_strain_url":"https://www.jax.org/strain/search?query=DCAF15"},"sequence":{"accession":"Q66K64","fasta_url":"https://rest.uniprot.org/uniprotkb/Q66K64.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q66K64/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q66K64"}},"corpus_meta":[{"pmid":"31819272","id":"PMC_31819272","title":"Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex.","date":"2019","source":"Nature chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/31819272","citation_count":188,"is_preprint":false},{"pmid":"31686031","id":"PMC_31686031","title":"Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15.","date":"2019","source":"Nature chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/31686031","citation_count":166,"is_preprint":false},{"pmid":"31693911","id":"PMC_31693911","title":"Structural Basis and Kinetic Pathway of RBM39 Recruitment to DCAF15 by a Sulfonamide Molecular Glue E7820.","date":"2019","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/31693911","citation_count":131,"is_preprint":false},{"pmid":"31693891","id":"PMC_31693891","title":"Aryl Sulfonamides Degrade RBM39 and RBM23 by Recruitment to CRL4-DCAF15.","date":"2019","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/31693891","citation_count":93,"is_preprint":false},{"pmid":"34257283","id":"PMC_34257283","title":"Targeting KRAS4A splicing through the RBM39/DCAF15 pathway inhibits cancer stem cells.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34257283","citation_count":43,"is_preprint":false},{"pmid":"32200025","id":"PMC_32200025","title":"Targeting the E3 ubiquitin ligases DCAF15 and cereblon for cancer therapy.","date":"2020","source":"Seminars in cancer biology","url":"https://pubmed.ncbi.nlm.nih.gov/32200025","citation_count":19,"is_preprint":false},{"pmid":"31626998","id":"PMC_31626998","title":"pSILAC method coupled with two complementary digestion approaches reveals PRPF39 as a new E7070-dependent DCAF15 substrate.","date":"2019","source":"Journal of proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/31626998","citation_count":17,"is_preprint":false},{"pmid":"33833131","id":"PMC_33833131","title":"Tumor suppressor DCAF15 inhibits epithelial-mesenchymal transition by targeting ZEB1 for proteasomal degradation in hepatocellular carcinoma.","date":"2021","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/33833131","citation_count":15,"is_preprint":false},{"pmid":"38961054","id":"PMC_38961054","title":"DCAF15 control of cohesin dynamics sustains acute myeloid leukemia.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38961054","citation_count":12,"is_preprint":false},{"pmid":"33168788","id":"PMC_33168788","title":"Aryl Sulfonamides Induce Degradation of Aryl Hydrocarbon Receptor Nuclear Translocator through CRL4DCAF15 E3 Ligase.","date":"2020","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/33168788","citation_count":8,"is_preprint":false},{"pmid":"41276610","id":"PMC_41276610","title":"Dcaf15-mediated EphA2 degradation triggers disruption of the blood-brain barrier during Streptococcus suis meningitis.","date":"2025","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/41276610","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.09.16.613231","title":"Network-driven identification of indisulam neo-substrates for targeted protein degradation","date":"2024-09-20","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.16.613231","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7470,"output_tokens":3150,"usd":0.03483,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10513,"output_tokens":3481,"usd":0.069795,"stage2_stop_reason":"end_turn"},"total_usd":0.104625,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structure of the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex at 2.3 Å resolution revealed that DCAF15 has a distinct topology that embraces the RBM39 RRM2 domain largely via non-polar interactions, with indisulam binding in a pocket between DCAF15 and RBM39(RRM2) to coordinate additional protein-protein contacts. Point mutants of RBM39 and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif recognized by DCAF15.\",\n      \"method\": \"X-ray crystallography (2.3 Å), RBM39 point mutagenesis, indisulam analog structure-activity studies\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution crystal structure plus mutagenesis validation in a single rigorous study\",\n      \"pmids\": [\"31819272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at 4.4 Å, together with crystal structures of engineered subcomplexes, showed that DCAF15 adopts a new fold stabilized by DDA1 and that aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively recruit and degrade RBM39 and RBM23. Extensive protein-protein contacts between the ligase and substrate mitigate low-affinity interactions between the drug and DCAF15.\",\n      \"method\": \"Cryo-EM (4.4 Å), X-ray crystallography of subcomplexes, biochemical binding assays\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM plus crystal structures of subcomplexes, multiple orthogonal structural methods\",\n      \"pmids\": [\"31686031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structure of DDA1-DDB1-DCAF15 in complex with E7820 and the RRM2 domain of RBM39 revealed that E7820 packs in a shallow pocket on the surface of DCAF15 and the modified interface binds RBM39 through the α1 helix of the RRM2 domain. Kinetic studies demonstrated that aryl sulfonamide and RBM39 bind DCAF15 in a synergistic (cooperative) manner, confirming aryl sulfonamides act as molecular glues.\",\n      \"method\": \"X-ray crystallography, kinetic binding analysis\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus kinetic mechanistic validation, independent of other structural studies\",\n      \"pmids\": [\"31693911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Domain mapping and random mutagenesis identified DCAF15 residues Q232 and D475 as necessary for indisulam-dependent RBM39 recruitment. RBM39 is recruited to DCAF15 via its RRM2 domain and is ubiquitinated on its N terminus. RBM23, a paralog of RBM39, is also recruited to CRL4-DCAF15 through its RRM2 domain and undergoes sulfonamide-dependent degradation. Indisulam-induced splicing changes (intron retention, exon skipping) are attributable solely to RBM39 degradation.\",\n      \"method\": \"Domain mapping, random mutagenesis, ubiquitination assays, RNA-seq\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal mutagenesis in both DCAF15 and RBM39 with functional ubiquitination readouts, replicated across multiple groups\",\n      \"pmids\": [\"31693891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Using pSILAC proteomics combined with two complementary digestion approaches, PRPF39 was identified as a novel substrate of E7070/DCAF15. PRPF39 undergoes DCAF15-dependent ubiquitination and proteasomal degradation upon E7070 treatment, establishing it as a bona fide neo-substrate of the CUL4-DCAF15 E3 ubiquitin ligase.\",\n      \"method\": \"pSILAC mass spectrometry, ubiquitination assay, proteasome inhibitor rescue, DCAF15 knockdown\",\n      \"journal\": \"Journal of proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteome-wide discovery plus targeted biological validation in a single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31626998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ARNT (aryl hydrocarbon receptor nuclear translocator) interacts with DCAF15 and undergoes proteasomal degradation induced by aryl sulfonamides indisulam and E7820 through CRL4-DCAF15, independently of the known neo-substrates RBM39 and RBM23. This degradation inhibited transcriptional activities of HIFs and AhR associated with ARNT.\",\n      \"method\": \"Co-immunoprecipitation, proteasome inhibitor rescue, RBM39/RBM23 knockdown controls, transcriptional reporter assays\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus functional degradation assays with appropriate controls, single lab\",\n      \"pmids\": [\"33168788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ZEB1 was identified as an endogenous substrate of the CRL4-DCAF15 E3 ubiquitin ligase complex. DCAF15 acts as an adaptor that specifically recognizes the N-terminal zinc finger domain of ZEB1 and triggers its ubiquitin-proteasome-mediated degradation. DCAF15 knockdown led to ZEB1 upregulation and EMT activation, while DCAF15 overexpression suppressed ZEB1 and inhibited EMT in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, ubiquitination assay, DCAF15 knockdown/overexpression with EMT phenotypic readout\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP with domain mapping and functional rescue experiments, single lab\",\n      \"pmids\": [\"33833131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DCAF15/RBM39 pathway controls KRAS4A alternative splicing. Pharmacological inhibition of RBM39 using indisulam reduces KRAS4A isoform levels and inhibits cancer stem cells, placing DCAF15 upstream of KRAS4A isoform production through RBM39-dependent splicing regulation.\",\n      \"method\": \"Indisulam treatment, isoform-specific RT-PCR/RNA analysis, cancer stem cell functional assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional pharmacological and genetic evidence linking DCAF15-RBM39 to specific splicing outcome, single lab\",\n      \"pmids\": [\"34257283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DCAF15 directly interacts with the SMC1A subunit of the cohesin complex and destabilizes cohesin regulatory factors PDS5A and CDCA5. Loss of DCAF15 causes loss of PDS5A and CDCA5, precluding cohesin acetylation on chromatin, resulting in uncontrolled chromatin loop extrusion, defective DNA replication fork integrity, accumulation of DNA damage, and apoptosis in acute myeloid leukemia (AML) cells.\",\n      \"method\": \"Domain-focused genetic screen, DCAF15 loss-of-function (knockout), co-immunoprecipitation (DCAF15-SMC1A), cohesin acetylation assays, chromatin loop extrusion analysis, replication fork assays, DNA damage markers\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic screen plus reciprocal Co-IP plus multiple orthogonal functional assays establishing endogenous DCAF15 function with defined mechanistic pathway\",\n      \"pmids\": [\"38961054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DCAF15 interacts with EphA2 receptor and promotes its K48- and K63-linked ubiquitination at lysine residues K646, K649, and K754, leading to EphA2 degradation. S. suis STK phosphorylates SIRT1 at S48, triggering SIRT1-mediated deacetylation of DCAF15 at K552 and K581, which enhances DCAF15-EphA2 interaction and EphA2 ubiquitination. This DCAF15-mediated EphA2 degradation disrupts blood-brain barrier integrity during S. suis meningitis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination site mapping (K646/K649/K754), acetylation site mapping (K552/K581), SIRT1 deacetylase assay, in vitro and in vivo BBB disruption models\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with site-specific mutagenesis and PTM mapping, single lab, novel mechanistic context\",\n      \"pmids\": [\"41276610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Network-based proteomics analysis identified RBM5 as a novel indisulam neo-substrate degraded in a proteasome-dependent manner via DCAF15, expanding the list of CRL4-DCAF15 substrates beyond RBM39 and RBM23.\",\n      \"method\": \"Network-based omics prioritization (Random Walk with Restart), proteomics, proteasome inhibitor rescue\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single method for validation (proteasome-dependent degradation), no direct DCAF15 binding shown\",\n      \"pmids\": [\"bio_10.1101_2024.09.16.613231\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"DCAF15 is the substrate receptor of the CRL4 (CUL4-RBX1-DDB1-DDA1-DCAF15) E3 ubiquitin ligase complex; it adopts a novel fold stabilized by DDA1 and, in the presence of aryl-sulfonamide molecular glues (indisulam, E7820, tasisulam), neo-functionalizes a shallow surface pocket to recruit and ubiquitinate splicing factors RBM39 and RBM23 (via their RRM2 α-helical degron), as well as additional substrates including PRPF39, ARNT, RBM5, ZEB1, and EphA2; endogenously, DCAF15 interacts with SMC1A of the cohesin complex and controls cohesin dynamics by destabilizing PDS5A and CDCA5, thereby regulating cohesin acetylation, chromatin loop extrusion, and DNA replication fork integrity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DCAF15 is the substrate-receptor subunit of a CRL4 (CUL4-RBX1-DDB1) E3 ubiquitin ligase, where it adopts a distinct, non-canonical fold stabilized by the small subunit DDA1 [#1]. Its defining biochemical property is that it harbors a shallow, non-conserved surface pocket that is neo-functionalized by aryl-sulfonamide molecular glues (indisulam, E7820): the drug binds cooperatively at the interface between DCAF15 and the RRM2 domain of the splicing factor RBM39, coordinating extensive protein-protein contacts that mitigate the low intrinsic affinity of the glue and recruit RBM39 for ubiquitination on its N-terminus [#0, #2, #3]. RBM39 is engaged through an α-helical degron in its RRM2 domain, and the paralog RBM23 is recruited and degraded by the same mechanism [#0, #3]. Sulfonamide-induced degradation of RBM39 reprograms alternative splicing—including intron retention, exon skipping, and KRAS4A isoform production—accounting for the drugs' downstream effects [#3, #7]. Beyond RBM39/RBM23, drug-dependent neo-substrates include PRPF39 and ARNT [#4, #5]. Independently of molecular glues, DCAF15 also directs ubiquitin-proteasome degradation of endogenous substrates: ZEB1, recognized through its N-terminal zinc-finger domain, with consequences for EMT [#6], and EphA2, ubiquitinated at defined lysines in a manner regulated by SIRT1-mediated deacetylation of DCAF15 [#9]. Endogenously, DCAF15 interacts with the SMC1A subunit of cohesin and destabilizes the cohesin regulators PDS5A and CDCA5, thereby controlling cohesin acetylation, chromatin loop extrusion, replication fork integrity, and survival of acute myeloid leukemia cells [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2019,\n      \"claim\": \"Established at atomic resolution how a substrate-receptor with no obvious binding cleft can be co-opted by a small molecule to recruit a neo-substrate, defining the molecular-glue mechanism for DCAF15.\",\n      \"evidence\": \"X-ray crystallography of DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) plus RBM39 point mutagenesis and indisulam analog SAR\",\n      \"pmids\": [\"31819272\", \"31693911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define endogenous, drug-independent function of DCAF15\", \"Limited to RBM39 RRM2; full-length substrate engagement geometry not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed DCAF15 adopts a new DDA1-stabilized fold and that aryl-sulfonamides neo-functionalize a shallow non-conserved pocket selective for RBM39 and its paralog RBM23, explaining substrate selectivity.\",\n      \"evidence\": \"Cryo-EM (4.4 Å) of DDB1-DCAF15-DDA1-RBM39-E7820 with crystal structures of engineered subcomplexes and biochemical binding assays\",\n      \"pmids\": [\"31686031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lower resolution limits side-chain interpretation at the drug interface\", \"Did not address whether other DCAF15 substrates use the same pocket\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetically mapped the DCAF15 residues and RBM39 degron required for drug-induced recruitment and demonstrated the splicing phenotype is attributable solely to RBM39 loss, linking degradation to transcriptomic output.\",\n      \"evidence\": \"Domain mapping, random mutagenesis (DCAF15 Q232/D475), ubiquitination assays, and RNA-seq\",\n      \"pmids\": [\"31693891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish endogenous physiological substrates\", \"Mechanism of N-terminal ubiquitin site selection not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Expanded the drug-dependent neo-substrate repertoire beyond RBM39/RBM23 by identifying PRPF39 through unbiased proteomics.\",\n      \"evidence\": \"pSILAC mass spectrometry with ubiquitination assay, proteasome-inhibitor rescue, and DCAF15 knockdown\",\n      \"pmids\": [\"31626998\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural basis for PRPF39 recognition\", \"Single-lab validation\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified ARNT as a sulfonamide-induced DCAF15 substrate degraded independently of RBM39/RBM23, linking DCAF15 activity to HIF and AhR transcriptional pathways.\",\n      \"evidence\": \"Co-IP, proteasome-inhibitor rescue, RBM39/RBM23 knockdown controls, transcriptional reporter assays\",\n      \"pmids\": [\"33168788\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degron on ARNT not mapped\", \"No reciprocal structural validation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated that DCAF15 has an endogenous, drug-independent substrate (ZEB1) recognized via its N-terminal zinc-finger domain, controlling EMT.\",\n      \"evidence\": \"Co-IP, domain mapping, ubiquitination assay, and DCAF15 knockdown/overexpression with EMT readouts in hepatocellular carcinoma cells\",\n      \"pmids\": [\"33833131\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab, single-cancer context\", \"No structure of the DCAF15-ZEB1 interface\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed the DCAF15-RBM39 axis upstream of a specific oncogenic splicing event (KRAS4A isoform) with functional consequences for cancer stem cells.\",\n      \"evidence\": \"Indisulam treatment, isoform-specific RNA analysis, and cancer stem cell functional assays\",\n      \"pmids\": [\"34257283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Indirect (mediated by RBM39 splicing activity rather than direct DCAF15 substrate)\", \"Single-lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined an endogenous, drug-independent function of DCAF15 in genome stability: it binds cohesin via SMC1A and destabilizes PDS5A/CDCA5 to control cohesin acetylation, loop extrusion, and replication fork integrity.\",\n      \"evidence\": \"Domain-focused genetic screen, DCAF15 knockout, reciprocal Co-IP (DCAF15-SMC1A), cohesin acetylation, loop extrusion, replication fork, and DNA damage assays in AML cells\",\n      \"pmids\": [\"38961054\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PDS5A/CDCA5 are direct ubiquitination substrates of CRL4-DCAF15 not fully resolved\", \"Generality beyond AML not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed PTM-dependent regulation of DCAF15 substrate engagement, showing SIRT1-mediated deacetylation of DCAF15 enhances EphA2 ubiquitination, with consequences for blood-brain barrier integrity.\",\n      \"evidence\": \"Co-IP, ubiquitination and acetylation site mapping, SIRT1 deacetylase assay, and in vitro/in vivo BBB models of S. suis meningitis\",\n      \"pmids\": [\"41276610\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab, infection-specific context\", \"Direct degron on EphA2 not structurally defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how DCAF15 selects among its diverse endogenous substrates without a molecular glue, and whether a unifying structural logic links cohesin regulation, ZEB1, and EphA2 degradation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of DCAF15 with any endogenous substrate\", \"Degron features of endogenous substrates undefined\", \"Regulation of DCAF15 abundance/activity in normal physiology unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 6, 8, 9]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 3, 6, 9]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [3, 7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"CRL4-DCAF15 (CUL4-RBX1-DDB1-DDA1-DCAF15) E3 ubiquitin ligase\"],\n    \"partners\": [\"DDB1\", \"DDA1\", \"RBM39\", \"RBM23\", \"SMC1A\", \"ZEB1\", \"EphA2\", \"ARNT\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}