{"gene":"UFC1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2007,"finding":"Crystal structure of human UFC1 determined at 1.6 Å resolution reveals a canonical E2 domain plus an additional N-terminal domain. The UBA5 binding site on UFC1 was mapped by structural comparison with Ubc12 and confirmed by mutational analyses. The N-terminal unique domain contributes to thermal stability.","method":"X-ray crystallography (1.6 Å resolution) + site-directed mutagenesis","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure combined with mutagenesis to define binding site and functional domain","pmids":["17825256"],"is_preprint":false},{"year":2008,"finding":"NMR and X-ray structures of human UFC1 show the catalytic core domain with active-site Cys116 in a flexible, solvent-accessible loop forming a thioester bond with UFM1. The N-terminal helix adopts different conformations in crystal vs. solution, suggesting a role in mediating specificity. A model of the UFC1–UFM1 complex positions UFM1 C-terminal Gly83 near Cys116 for thioester formation.","method":"NMR spectroscopy + X-ray crystallography + structural modeling","journal":"Journal of structural and functional genomics","confidence":"High","confidence_rationale":"Tier 1 / Strong — two orthogonal structural methods (NMR + X-ray) identifying active site residue and thioester chemistry, replicated independently from PMID:17825256","pmids":["19101823"],"is_preprint":false},{"year":2018,"finding":"Biallelic UFC1 mutations in humans impair UFM1–UFC1 thioester intermediate formation, resulting in widespread reduction of cellular ufmylation and severe early-onset encephalopathy with progressive microcephaly, establishing UFC1 as the E2-like enzyme essential for ufmylation in human brain development.","method":"Biochemical thioester intermediate formation assays + human genetics (patient-derived mutations)","journal":"Brain : a journal of neurology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct biochemical assay of thioester intermediate with patient mutations, multiple families, replicated biochemical defect matching UFM1 mutation phenotype","pmids":["29868776"],"is_preprint":false},{"year":2021,"finding":"Crystal structure of UFC1 bound to the C-terminus of UBA5 reveals that UBA5 interacts with UFC1 via a short linear sequence not found in other E1–E2 complexes. A region of UBA5 outside the adenylation domain is dispensable for UFC1 binding but critical for UFM1 transfer, and moves adjacent to UFC1's active-site Cys to compensate for a missing loop in UFC1 that exists in other E2s.","method":"X-ray crystallography of UFC1–UBA5 C-terminal peptide complex + biochemical transfer assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure plus functional biochemical assays defining mechanistic basis of UFM1 transfer","pmids":["34588452"],"is_preprint":false},{"year":2021,"finding":"The last 20 residues of UBA5's unstructured C-terminal region are pivotal for binding to UFC1 and accelerate UFM1 transfer to UFC1. The NMR structure of UFC1 bound to this 20-residue UBA5 peptide combined with ITC confirmed the interaction mechanism.","method":"NMR spectroscopy + isothermal titration calorimetry + biochemical UFM1 transfer assays","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure plus ITC and functional transfer assay, single lab but multiple orthogonal methods","pmids":["34299007"],"is_preprint":false},{"year":2014,"finding":"UBA5 residues 381–404 constitute the minimal region required for UFC1 recognition, and residues 364–404 are necessary for transthiolation of UFM1 to UFC1; the UBA5 C-terminus–UFC1 fusion complex crystallized and diffracted to 3.0 Å.","method":"Deletion mutagenesis + biochemical transthiolation assay + X-ray crystallography (preliminary)","journal":"Acta crystallographica. Section F, Structural biology communications","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — functional deletion mapping with biochemical assay confirmed minimal binding region; crystal analysis preliminary (3.0 Å, no full structure reported)","pmids":["25084390"],"is_preprint":false},{"year":2014,"finding":"UFC1 (Ufc1) interacts with the cytoplasmic domain of NCAM140, co-localizes with NCAM140 and UFM1, and Ufm1 increases endocytosis of NCAM140, suggesting possible ufmylation of NCAM140 as a cell-surface protein.","method":"Protein macroarray + co-localization imaging + endocytosis assay","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, macroarray pulldown with co-localization, ufmylation of NCAM140 not directly demonstrated","pmids":["24726913"],"is_preprint":false},{"year":2023,"finding":"UFL1 (E3) binds UFC1 (E2) via the UFL1 N-terminal helix; crystal structure of UFL1–DDRGK1 fusion and UFC1 together with NMR and biochemical assays reveals a conserved UFL1–UFC1 interface and competition between UFL1 and UBA5 for binding to UFC1 that shifts in favor of UFL1 after UFM1 charging of UFC1.","method":"X-ray crystallography (UFL1–DDRGK1 structure) + NMR + biochemical competition assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus NMR and biochemical assays with multiple orthogonal methods establishing the E3–E2 interaction and regulatory mechanism","pmids":["37988244"],"is_preprint":false},{"year":2025,"finding":"UFC1 contains a TAK motif rather than the canonical HPN motif found in other E2 enzymes. Crystal structures of UFC1 mutants including one mimicking the oxyanion intermediate, combined with in vitro ufmylation assays, demonstrate that UFC1 employs two distinct oxyanion holes: one stabilizing the oxyanion intermediate during E3-mediated trans-ufmylation and another for cis-driven auto-ufmylation, with C-alpha hydrogen bonding contributing to stabilization.","method":"X-ray crystallography of UFC1 mutants (including oxyanion-intermediate mimic) + in vitro ufmylation activity assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures of multiple mutants plus functional activity assays dissecting catalytic mechanism, single lab but multiple orthogonal methods","pmids":["40280917"],"is_preprint":false},{"year":2025,"finding":"Depletion of UFC1 (along with other ufmylation machinery components UBA5, UFL1, UFBP1, UFM1) significantly reduces infectious virion production for orthoflaviviruses (dengue, Zika, West Nile, yellow fever) but does not affect viral RNA translation or replication, placing UFC1/ufmylation at a post-replication step in viral assembly.","method":"siRNA knockdown of UFC1 + viral infectivity assay + RNA replication assay (orthoflavivirus cell biology)","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with defined phenotypic readout and epistatic placement, but preprint, single lab","pmids":["bio_10.1101_2025.01.09.632082"],"is_preprint":true},{"year":2025,"finding":"The HLD14-associated UFC1 p.Arg23Gln missense mutation causes aggregation of UFC1 primarily in lysosomes in oligodendroglial FBD-102b cells, reduces Akt kinase phosphorylation, decreases expression of differentiation and myelination marker proteins, and impairs morphological differentiation (membrane extension).","method":"Mutant UFC1 expression in differentiating oligodendroglial cell line + immunofluorescence localization + Western blotting for Akt phosphorylation and myelination markers","journal":"Medicines (Basel, Switzerland)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — direct localization with functional consequences (signaling and morphogenesis phenotype) but single lab, single cell model","pmids":["39846712"],"is_preprint":false}],"current_model":"UFC1 is the E2-like conjugating enzyme of the ufmylation pathway that accepts activated UFM1 from the E1 enzyme UBA5 via thioester bond formation at its active-site Cys116, facilitated by a short linear C-terminal sequence of UBA5 that docks onto UFC1 in a manner unique among E1–E2 pairs; UFC1 then transfers UFM1 to substrate proteins in concert with the E3 ligase UFL1, which competes with UBA5 for UFC1 binding via its N-terminal helix; the reaction proceeds through dual oxyanion holes (one for E3-driven trans-ufmylation, one for cis auto-ufmylation) defined by UFC1's atypical TAK motif, and loss-of-function mutations in UFC1 cause severe early-onset encephalopathy with progressive microcephaly in humans by globally impairing cellular ufmylation."},"narrative":{"mechanistic_narrative":"UFC1 is the E2-like conjugating enzyme of the ufmylation pathway, accepting activated UFM1 from the E1 enzyme UBA5 and relaying it to substrate proteins [PMID:29868776, PMID:34588452]. It comprises a canonical E2 catalytic core plus a stabilizing N-terminal domain, and carries out thioester chemistry through its active-site Cys116, which sits in a flexible, solvent-exposed loop positioned to capture the C-terminal Gly83 of UFM1 [PMID:17825256, PMID:19101823]. Charging of UFC1 depends on an unusual mode of E1 docking: a short linear sequence in the unstructured C-terminus of UBA5 (its last ~20 residues, within region 364–404) binds UFC1 and accelerates transthiolation, with part of UBA5 reaching toward the UFC1 active-site Cys to compensate for a loop absent in UFC1 but present in other E2s [PMID:34588452, PMID:34299007, PMID:25084390]. Hand-off to substrates is directed by the E3 ligase UFL1, whose N-terminal helix engages a conserved interface on UFC1 and competes with UBA5 for binding; this competition shifts toward UFL1 once UFC1 is UFM1-charged, coupling E1 release to E3 engagement [PMID:37988244]. Catalysis is mediated by an atypical TAK motif (in place of the canonical HPN motif) that builds two distinct oxyanion holes—one supporting E3-driven trans-ufmylation and one for cis auto-ufmylation [PMID:40280917]. Biallelic loss-of-function UFC1 mutations that impair UFM1–UFC1 thioester formation cause severe early-onset encephalopathy with progressive microcephaly by globally reducing cellular ufmylation [PMID:29868776].","teleology":[{"year":2007,"claim":"Establishing the architecture of UFC1 was the first step in defining how this enzyme could function within the ufmylation cascade, by identifying both its E2 core and where it engages the upstream E1.","evidence":"X-ray crystallography at 1.6 Å with site-directed mutagenesis mapping the UBA5 binding site by comparison to Ubc12","pmids":["17825256"],"confidence":"High","gaps":["Did not define the catalytic active-site residue or thioester chemistry","UBA5 binding mode inferred by comparison, not from a complex structure"]},{"year":2008,"claim":"Identifying Cys116 in a flexible loop poised to receive UFM1 established the catalytic basis for UFC1's E2 thioester chemistry.","evidence":"NMR and X-ray structures plus modeling of the UFC1–UFM1 complex positioning UFM1 Gly83 near Cys116","pmids":["19101823"],"confidence":"High","gaps":["The UFC1–UFM1 complex was modeled, not experimentally solved","Role of the N-terminal helix conformational change in specificity not functionally tested"]},{"year":2014,"claim":"Mapping the minimal UBA5 segment needed for UFC1 recognition versus transthiolation separated the binding determinant from the transfer-competent region, beginning to explain the unusual E1–E2 interaction.","evidence":"Deletion mutagenesis with biochemical transthiolation assays and preliminary crystallization of a UBA5 C-terminus–UFC1 fusion","pmids":["25084390"],"confidence":"Medium","gaps":["Full crystal structure of the complex not reported (preliminary 3.0 Å)","Atomic basis of why a region outside the binding determinant is needed for transfer unresolved"]},{"year":2014,"claim":"A candidate substrate link was explored by showing UFC1 interacts with and co-localizes with the NCAM140 cytoplasmic domain, raising the possibility of ufmylation of a cell-surface protein.","evidence":"Protein macroarray, co-localization imaging, and NCAM140 endocytosis assays","pmids":["24726913"],"confidence":"Low","gaps":["Ufmylation of NCAM140 was not directly demonstrated","Single lab, macroarray-based interaction without reciprocal validation","Functional relevance to ufmylation pathway unclear"]},{"year":2018,"claim":"Patient mutations that block UFM1–UFC1 thioester formation tied UFC1 enzymatic activity directly to human brain development, establishing it as the essential E2 of ufmylation in vivo.","evidence":"Biochemical thioester intermediate assays with patient-derived mutations across multiple families","pmids":["29868776"],"confidence":"High","gaps":["Substrates whose loss of ufmylation drives the neurological phenotype not identified","Tissue-specific reasons for brain vulnerability unresolved"]},{"year":2021,"claim":"Solving the UFC1–UBA5 C-terminus complex revealed a short linear docking motif unique among E1–E2 pairs and showed how UBA5 substitutes for a missing UFC1 loop to enable transfer.","evidence":"X-ray crystallography of a UFC1–UBA5 C-terminal peptide complex plus NMR, ITC, and biochemical transfer assays (two independent studies)","pmids":["34588452","34299007"],"confidence":"High","gaps":["Dynamics of the charged intermediate during transfer not visualized","How UBA5 release is triggered after charging not defined here"]},{"year":2023,"claim":"Defining the UFL1 N-terminal helix interface on UFC1 and its competition with UBA5 explained how UFM1-charging switches the enzyme from E1 acceptor to E3-directed donor.","evidence":"Crystal structure of a UFL1–DDRGK1 fusion with UFC1, plus NMR and biochemical competition assays","pmids":["37988244"],"confidence":"High","gaps":["Structure of the full E2–E3–substrate transfer complex not resolved","How charging mechanistically increases UFL1 affinity not detailed at atomic level"]},{"year":2025,"claim":"Discovery of UFC1's atypical TAK motif and dual oxyanion holes explained how a single E2 supports both E3-driven trans-ufmylation and cis auto-ufmylation.","evidence":"Crystal structures of UFC1 mutants including an oxyanion-intermediate mimic, with in vitro ufmylation assays","pmids":["40280917"],"confidence":"High","gaps":["Physiological balance between trans- and cis-ufmylation not quantified in cells","Substrate determinants selecting each pathway unknown"]},{"year":2025,"claim":"Linking UFC1 to orthoflavivirus assembly placed ufmylation at a post-replication step required for infectious virion production.","evidence":"siRNA knockdown of UFC1 (and other ufmylation components) with viral infectivity and RNA replication assays (preprint)","pmids":["bio_10.1101_2025.01.09.632082"],"confidence":"Medium","gaps":["Preprint, single lab","The ufmylated substrate mediating the assembly defect not identified","Direct evidence of ufmylation at the assembly step lacking"]},{"year":2025,"claim":"A disease-associated missense mutation was shown to mislocalize UFC1 and disrupt oligodendroglial differentiation signaling, connecting UFC1 dysfunction to myelination defects.","evidence":"Mutant UFC1 expression in a differentiating oligodendroglial cell line with immunofluorescence and Western blotting for Akt phosphorylation and myelination markers","pmids":["39846712"],"confidence":"Medium","gaps":["Single cell model, single lab","Whether mislocalization reflects loss of ufmylation activity not established","Link between Akt signaling and ufmylation not mechanistically defined"]},{"year":null,"claim":"The physiological substrates ufmylated by UFC1 and how their modification underlies neurodevelopmental phenotypes and viral assembly remain unresolved.","evidence":"No discovery in the timeline directly identifies a validated UFC1 ufmylation substrate driving disease or viral phenotypes","pmids":[],"confidence":"Low","gaps":["No validated endogenous substrate identified","Mechanism linking global ufmylation loss to tissue-specific defects unknown","Cellular localization context of ufmylation activity uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,3,8]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[3,8]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[2,8]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,3,7]}],"complexes":[],"partners":["UBA5","UFM1","UFL1","DDRGK1","NCAM140"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y3C8","full_name":"Ubiquitin-fold modifier-conjugating enzyme 1","aliases":[],"length_aa":167,"mass_kda":19.5,"function":"E2-like enzyme which specifically catalyzes the second step in ufmylation (PubMed:15071506, PubMed:29868776, PubMed:30626644, PubMed:34588452, PubMed:35394863, PubMed:36121123, PubMed:38383789). Accepts the ubiquitin-like modifier UFM1 from the E1 enzyme UBA5 and forms an intermediate with UFM1 via a thioester linkage (PubMed:15071506, PubMed:29868776, PubMed:34588452, PubMed:38383789). Ufmylation is involved in various processes, such as ribosome recycling, response to DNA damage, interferon response or reticulophagy (also called ER-phagy) (PubMed:27351204, PubMed:32160526, PubMed:35394863, PubMed:37036982, PubMed:38383789)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9Y3C8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UFC1","classification":"Not Classified","n_dependent_lines":250,"n_total_lines":1208,"dependency_fraction":0.20695364238410596},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000143222","cell_line_id":"CID001783","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3},{"compartment":"er","grade":2}],"interactors":[{"gene":"SAR1B","stoichiometry":0.2},{"gene":"UFM1","stoichiometry":0.2},{"gene":"UBA5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001783","total_profiled":1310},"omim":[{"mim_id":"618076","title":"NEURODEVELOPMENTAL DISORDER WITH SPASTICITY AND POOR GROWTH; NEDSG","url":"https://www.omim.org/entry/618076"},{"mim_id":"617899","title":"LEUKODYSTROPHY, HYPOMYELINATING, 14; HLD14","url":"https://www.omim.org/entry/617899"},{"mim_id":"617132","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 44; DEE44","url":"https://www.omim.org/entry/617132"},{"mim_id":"616177","title":"DDRGK DOMAIN-CONTAINING PROTEIN 1; DDRGK1","url":"https://www.omim.org/entry/616177"},{"mim_id":"613372","title":"UFM1-SPECIFIC LIGASE 1; UFL1","url":"https://www.omim.org/entry/613372"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nuclear speckles","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UFC1"},"hgnc":{"alias_symbol":["HSPC155"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y3C8","domains":[{"cath_id":"3.10.110.10","chopping":"3-155","consensus_level":"high","plddt":95.3318,"start":3,"end":155}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3C8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3C8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3C8-F1-predicted_aligned_error_v6.png","plddt_mean":93.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UFC1","jax_strain_url":"https://www.jax.org/strain/search?query=UFC1"},"sequence":{"accession":"Q9Y3C8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y3C8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y3C8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3C8"}},"corpus_meta":[{"pmid":"25449213","id":"PMC_25449213","title":"The long intergenic noncoding RNA UFC1, a target of MicroRNA 34a, interacts with the mRNA stabilizing protein HuR to increase levels of β-catenin in HCC cells.","date":"2014","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/25449213","citation_count":226,"is_preprint":false},{"pmid":"32242003","id":"PMC_32242003","title":"Exosome-transmitted lncRNA UFC1 promotes non-small-cell lung cancer progression by EZH2-mediated epigenetic silencing of PTEN expression.","date":"2020","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/32242003","citation_count":157,"is_preprint":false},{"pmid":"29868776","id":"PMC_29868776","title":"Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development.","date":"2018","source":"Brain : a journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/29868776","citation_count":98,"is_preprint":false},{"pmid":"27529373","id":"PMC_27529373","title":"Long Noncoding RNA UFC1 Promotes Proliferation of Chondrocyte in Osteoarthritis by Acting as a Sponge for miR-34a.","date":"2016","source":"DNA and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27529373","citation_count":64,"is_preprint":false},{"pmid":"17825256","id":"PMC_17825256","title":"Crystal structure of Ufc1, the Ufm1-conjugating enzyme.","date":"2007","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17825256","citation_count":44,"is_preprint":false},{"pmid":"29970131","id":"PMC_29970131","title":"Long non-coding RNA UFC1 promotes gastric cancer progression by regulating miR-498/Lin28b.","date":"2018","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/29970131","citation_count":41,"is_preprint":false},{"pmid":"34588452","id":"PMC_34588452","title":"Structural basis for UFM1 transfer from UBA5 to UFC1.","date":"2021","source":"Nature 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Section F, Structural biology communications","url":"https://pubmed.ncbi.nlm.nih.gov/25084390","citation_count":17,"is_preprint":false},{"pmid":"34299007","id":"PMC_34299007","title":"A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34299007","citation_count":15,"is_preprint":false},{"pmid":"31544897","id":"PMC_31544897","title":"Long Non-Coding RNA (LncRNA) UFC1/miR-34a Contributes to Proliferation and Migration in Breast Cancer.","date":"2019","source":"Medical science monitor : international medical journal of experimental and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/31544897","citation_count":14,"is_preprint":false},{"pmid":"39393561","id":"PMC_39393561","title":"Yiqi Yangxue formula inhibits cartilage degeneration in knee osteoarthritis by regulating LncRNA-UFC1/miR-34a/MMP-13 axis.","date":"2024","source":"Journal of ethnopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/39393561","citation_count":10,"is_preprint":false},{"pmid":"40280917","id":"PMC_40280917","title":"UFC1 reveals the multifactorial and plastic nature of oxyanion holes in E2 conjugating enzymes.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40280917","citation_count":5,"is_preprint":false},{"pmid":"37329532","id":"PMC_37329532","title":"Long noncoding RNA UFC1 acts as an oncogene via stimulating EZH2-induced inhibition of APC expression in renal cell carcinoma.","date":"2023","source":"Cellular and molecular biology (Noisy-le-Grand, France)","url":"https://pubmed.ncbi.nlm.nih.gov/37329532","citation_count":3,"is_preprint":false},{"pmid":"39078589","id":"PMC_39078589","title":"A novel compound heterozygous mutation of UFC1 in a patient with neurodevelopmental disorder.","date":"2024","source":"Genes & genomics","url":"https://pubmed.ncbi.nlm.nih.gov/39078589","citation_count":1,"is_preprint":false},{"pmid":"31270046","id":"PMC_31270046","title":"[Long noncoding RNA UFC1 promotes metastasis and invasion of hepatocellular carcinoma cells in vitro via GSK-3β/β-catenin axis].","date":"2019","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/31270046","citation_count":0,"is_preprint":false},{"pmid":"39352191","id":"PMC_39352191","title":"RETRACTION: Long Noncoding RNA UFC1 is Activated by E2F1 and Exerts Oncogenic Properties by Functioning as a CeRNA of FOXP3.","date":"2024","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39352191","citation_count":0,"is_preprint":false},{"pmid":"39846712","id":"PMC_39846712","title":"Hypomyelinating Leukodystrophy 14 (HLD14)-Related UFC1 p.Arg23Gln Decreases Cell Morphogenesis: A Phenotype Reversable with Hesperetin.","date":"2025","source":"Medicines (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/39846712","citation_count":0,"is_preprint":false},{"pmid":"25213431","id":"PMC_25213431","title":"Autoantibodies binding to ubiquitin-fold modifier-conjugating enzyme 1 (Ufc1) and pleckstrin homology domain containing, family G (with RhoGef domain) member 2 (Plekhg2) are associated with mycobacterial infections.","date":"2014","source":"HIV medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25213431","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.01.09.632082","title":"UFMylation promotes orthoflavivirus infectious particle production","date":"2025-01-09","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.09.632082","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14689,"output_tokens":2794,"usd":0.042988,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10146,"output_tokens":3440,"usd":0.068365,"stage2_stop_reason":"end_turn"},"total_usd":0.111353,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of human UFC1 determined at 1.6 Å resolution reveals a canonical E2 domain plus an additional N-terminal domain. The UBA5 binding site on UFC1 was mapped by structural comparison with Ubc12 and confirmed by mutational analyses. The N-terminal unique domain contributes to thermal stability.\",\n      \"method\": \"X-ray crystallography (1.6 Å resolution) + site-directed mutagenesis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure combined with mutagenesis to define binding site and functional domain\",\n      \"pmids\": [\"17825256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"NMR and X-ray structures of human UFC1 show the catalytic core domain with active-site Cys116 in a flexible, solvent-accessible loop forming a thioester bond with UFM1. The N-terminal helix adopts different conformations in crystal vs. solution, suggesting a role in mediating specificity. A model of the UFC1–UFM1 complex positions UFM1 C-terminal Gly83 near Cys116 for thioester formation.\",\n      \"method\": \"NMR spectroscopy + X-ray crystallography + structural modeling\",\n      \"journal\": \"Journal of structural and functional genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — two orthogonal structural methods (NMR + X-ray) identifying active site residue and thioester chemistry, replicated independently from PMID:17825256\",\n      \"pmids\": [\"19101823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Biallelic UFC1 mutations in humans impair UFM1–UFC1 thioester intermediate formation, resulting in widespread reduction of cellular ufmylation and severe early-onset encephalopathy with progressive microcephaly, establishing UFC1 as the E2-like enzyme essential for ufmylation in human brain development.\",\n      \"method\": \"Biochemical thioester intermediate formation assays + human genetics (patient-derived mutations)\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct biochemical assay of thioester intermediate with patient mutations, multiple families, replicated biochemical defect matching UFM1 mutation phenotype\",\n      \"pmids\": [\"29868776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structure of UFC1 bound to the C-terminus of UBA5 reveals that UBA5 interacts with UFC1 via a short linear sequence not found in other E1–E2 complexes. A region of UBA5 outside the adenylation domain is dispensable for UFC1 binding but critical for UFM1 transfer, and moves adjacent to UFC1's active-site Cys to compensate for a missing loop in UFC1 that exists in other E2s.\",\n      \"method\": \"X-ray crystallography of UFC1–UBA5 C-terminal peptide complex + biochemical transfer assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure plus functional biochemical assays defining mechanistic basis of UFM1 transfer\",\n      \"pmids\": [\"34588452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The last 20 residues of UBA5's unstructured C-terminal region are pivotal for binding to UFC1 and accelerate UFM1 transfer to UFC1. The NMR structure of UFC1 bound to this 20-residue UBA5 peptide combined with ITC confirmed the interaction mechanism.\",\n      \"method\": \"NMR spectroscopy + isothermal titration calorimetry + biochemical UFM1 transfer assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus ITC and functional transfer assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"34299007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UBA5 residues 381–404 constitute the minimal region required for UFC1 recognition, and residues 364–404 are necessary for transthiolation of UFM1 to UFC1; the UBA5 C-terminus–UFC1 fusion complex crystallized and diffracted to 3.0 Å.\",\n      \"method\": \"Deletion mutagenesis + biochemical transthiolation assay + X-ray crystallography (preliminary)\",\n      \"journal\": \"Acta crystallographica. Section F, Structural biology communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — functional deletion mapping with biochemical assay confirmed minimal binding region; crystal analysis preliminary (3.0 Å, no full structure reported)\",\n      \"pmids\": [\"25084390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UFC1 (Ufc1) interacts with the cytoplasmic domain of NCAM140, co-localizes with NCAM140 and UFM1, and Ufm1 increases endocytosis of NCAM140, suggesting possible ufmylation of NCAM140 as a cell-surface protein.\",\n      \"method\": \"Protein macroarray + co-localization imaging + endocytosis assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, macroarray pulldown with co-localization, ufmylation of NCAM140 not directly demonstrated\",\n      \"pmids\": [\"24726913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UFL1 (E3) binds UFC1 (E2) via the UFL1 N-terminal helix; crystal structure of UFL1–DDRGK1 fusion and UFC1 together with NMR and biochemical assays reveals a conserved UFL1–UFC1 interface and competition between UFL1 and UBA5 for binding to UFC1 that shifts in favor of UFL1 after UFM1 charging of UFC1.\",\n      \"method\": \"X-ray crystallography (UFL1–DDRGK1 structure) + NMR + biochemical competition assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus NMR and biochemical assays with multiple orthogonal methods establishing the E3–E2 interaction and regulatory mechanism\",\n      \"pmids\": [\"37988244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UFC1 contains a TAK motif rather than the canonical HPN motif found in other E2 enzymes. Crystal structures of UFC1 mutants including one mimicking the oxyanion intermediate, combined with in vitro ufmylation assays, demonstrate that UFC1 employs two distinct oxyanion holes: one stabilizing the oxyanion intermediate during E3-mediated trans-ufmylation and another for cis-driven auto-ufmylation, with C-alpha hydrogen bonding contributing to stabilization.\",\n      \"method\": \"X-ray crystallography of UFC1 mutants (including oxyanion-intermediate mimic) + in vitro ufmylation activity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures of multiple mutants plus functional activity assays dissecting catalytic mechanism, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"40280917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Depletion of UFC1 (along with other ufmylation machinery components UBA5, UFL1, UFBP1, UFM1) significantly reduces infectious virion production for orthoflaviviruses (dengue, Zika, West Nile, yellow fever) but does not affect viral RNA translation or replication, placing UFC1/ufmylation at a post-replication step in viral assembly.\",\n      \"method\": \"siRNA knockdown of UFC1 + viral infectivity assay + RNA replication assay (orthoflavivirus cell biology)\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with defined phenotypic readout and epistatic placement, but preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2025.01.09.632082\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The HLD14-associated UFC1 p.Arg23Gln missense mutation causes aggregation of UFC1 primarily in lysosomes in oligodendroglial FBD-102b cells, reduces Akt kinase phosphorylation, decreases expression of differentiation and myelination marker proteins, and impairs morphological differentiation (membrane extension).\",\n      \"method\": \"Mutant UFC1 expression in differentiating oligodendroglial cell line + immunofluorescence localization + Western blotting for Akt phosphorylation and myelination markers\",\n      \"journal\": \"Medicines (Basel, Switzerland)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — direct localization with functional consequences (signaling and morphogenesis phenotype) but single lab, single cell model\",\n      \"pmids\": [\"39846712\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UFC1 is the E2-like conjugating enzyme of the ufmylation pathway that accepts activated UFM1 from the E1 enzyme UBA5 via thioester bond formation at its active-site Cys116, facilitated by a short linear C-terminal sequence of UBA5 that docks onto UFC1 in a manner unique among E1–E2 pairs; UFC1 then transfers UFM1 to substrate proteins in concert with the E3 ligase UFL1, which competes with UBA5 for UFC1 binding via its N-terminal helix; the reaction proceeds through dual oxyanion holes (one for E3-driven trans-ufmylation, one for cis auto-ufmylation) defined by UFC1's atypical TAK motif, and loss-of-function mutations in UFC1 cause severe early-onset encephalopathy with progressive microcephaly in humans by globally impairing cellular ufmylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UFC1 is the E2-like conjugating enzyme of the ufmylation pathway, accepting activated UFM1 from the E1 enzyme UBA5 and relaying it to substrate proteins [#2, #3]. It comprises a canonical E2 catalytic core plus a stabilizing N-terminal domain, and carries out thioester chemistry through its active-site Cys116, which sits in a flexible, solvent-exposed loop positioned to capture the C-terminal Gly83 of UFM1 [#0, #1]. Charging of UFC1 depends on an unusual mode of E1 docking: a short linear sequence in the unstructured C-terminus of UBA5 (its last ~20 residues, within region 364–404) binds UFC1 and accelerates transthiolation, with part of UBA5 reaching toward the UFC1 active-site Cys to compensate for a loop absent in UFC1 but present in other E2s [#3, #4, #5]. Hand-off to substrates is directed by the E3 ligase UFL1, whose N-terminal helix engages a conserved interface on UFC1 and competes with UBA5 for binding; this competition shifts toward UFL1 once UFC1 is UFM1-charged, coupling E1 release to E3 engagement [#7]. Catalysis is mediated by an atypical TAK motif (in place of the canonical HPN motif) that builds two distinct oxyanion holes—one supporting E3-driven trans-ufmylation and one for cis auto-ufmylation [#8]. Biallelic loss-of-function UFC1 mutations that impair UFM1–UFC1 thioester formation cause severe early-onset encephalopathy with progressive microcephaly by globally reducing cellular ufmylation [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing the architecture of UFC1 was the first step in defining how this enzyme could function within the ufmylation cascade, by identifying both its E2 core and where it engages the upstream E1.\",\n      \"evidence\": \"X-ray crystallography at 1.6 Å with site-directed mutagenesis mapping the UBA5 binding site by comparison to Ubc12\",\n      \"pmids\": [\"17825256\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the catalytic active-site residue or thioester chemistry\", \"UBA5 binding mode inferred by comparison, not from a complex structure\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying Cys116 in a flexible loop poised to receive UFM1 established the catalytic basis for UFC1's E2 thioester chemistry.\",\n      \"evidence\": \"NMR and X-ray structures plus modeling of the UFC1–UFM1 complex positioning UFM1 Gly83 near Cys116\",\n      \"pmids\": [\"19101823\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The UFC1–UFM1 complex was modeled, not experimentally solved\", \"Role of the N-terminal helix conformational change in specificity not functionally tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapping the minimal UBA5 segment needed for UFC1 recognition versus transthiolation separated the binding determinant from the transfer-competent region, beginning to explain the unusual E1–E2 interaction.\",\n      \"evidence\": \"Deletion mutagenesis with biochemical transthiolation assays and preliminary crystallization of a UBA5 C-terminus–UFC1 fusion\",\n      \"pmids\": [\"25084390\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full crystal structure of the complex not reported (preliminary 3.0 Å)\", \"Atomic basis of why a region outside the binding determinant is needed for transfer unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"A candidate substrate link was explored by showing UFC1 interacts with and co-localizes with the NCAM140 cytoplasmic domain, raising the possibility of ufmylation of a cell-surface protein.\",\n      \"evidence\": \"Protein macroarray, co-localization imaging, and NCAM140 endocytosis assays\",\n      \"pmids\": [\"24726913\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Ufmylation of NCAM140 was not directly demonstrated\", \"Single lab, macroarray-based interaction without reciprocal validation\", \"Functional relevance to ufmylation pathway unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Patient mutations that block UFM1–UFC1 thioester formation tied UFC1 enzymatic activity directly to human brain development, establishing it as the essential E2 of ufmylation in vivo.\",\n      \"evidence\": \"Biochemical thioester intermediate assays with patient-derived mutations across multiple families\",\n      \"pmids\": [\"29868776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrates whose loss of ufmylation drives the neurological phenotype not identified\", \"Tissue-specific reasons for brain vulnerability unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Solving the UFC1–UBA5 C-terminus complex revealed a short linear docking motif unique among E1–E2 pairs and showed how UBA5 substitutes for a missing UFC1 loop to enable transfer.\",\n      \"evidence\": \"X-ray crystallography of a UFC1–UBA5 C-terminal peptide complex plus NMR, ITC, and biochemical transfer assays (two independent studies)\",\n      \"pmids\": [\"34588452\", \"34299007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of the charged intermediate during transfer not visualized\", \"How UBA5 release is triggered after charging not defined here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defining the UFL1 N-terminal helix interface on UFC1 and its competition with UBA5 explained how UFM1-charging switches the enzyme from E1 acceptor to E3-directed donor.\",\n      \"evidence\": \"Crystal structure of a UFL1–DDRGK1 fusion with UFC1, plus NMR and biochemical competition assays\",\n      \"pmids\": [\"37988244\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full E2–E3–substrate transfer complex not resolved\", \"How charging mechanistically increases UFL1 affinity not detailed at atomic level\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovery of UFC1's atypical TAK motif and dual oxyanion holes explained how a single E2 supports both E3-driven trans-ufmylation and cis auto-ufmylation.\",\n      \"evidence\": \"Crystal structures of UFC1 mutants including an oxyanion-intermediate mimic, with in vitro ufmylation assays\",\n      \"pmids\": [\"40280917\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological balance between trans- and cis-ufmylation not quantified in cells\", \"Substrate determinants selecting each pathway unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linking UFC1 to orthoflavivirus assembly placed ufmylation at a post-replication step required for infectious virion production.\",\n      \"evidence\": \"siRNA knockdown of UFC1 (and other ufmylation components) with viral infectivity and RNA replication assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.01.09.632082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"The ufmylated substrate mediating the assembly defect not identified\", \"Direct evidence of ufmylation at the assembly step lacking\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A disease-associated missense mutation was shown to mislocalize UFC1 and disrupt oligodendroglial differentiation signaling, connecting UFC1 dysfunction to myelination defects.\",\n      \"evidence\": \"Mutant UFC1 expression in a differentiating oligodendroglial cell line with immunofluorescence and Western blotting for Akt phosphorylation and myelination markers\",\n      \"pmids\": [\"39846712\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cell model, single lab\", \"Whether mislocalization reflects loss of ufmylation activity not established\", \"Link between Akt signaling and ufmylation not mechanistically defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The physiological substrates ufmylated by UFC1 and how their modification underlies neurodevelopmental phenotypes and viral assembly remain unresolved.\",\n      \"evidence\": \"No discovery in the timeline directly identifies a validated UFC1 ufmylation substrate driving disease or viral phenotypes\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No validated endogenous substrate identified\", \"Mechanism linking global ufmylation loss to tissue-specific defects unknown\", \"Cellular localization context of ufmylation activity uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 3, 8]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [2, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 3, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UBA5\", \"UFM1\", \"UFL1\", \"DDRGK1\", \"NCAM140\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}