{"gene":"UFM1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2004,"finding":"UFM1 is a novel ubiquitin-fold modifier that undergoes C-terminal cleavage to expose a conserved Gly residue, which is essential for conjugation. The exposed Gly is then activated by E1-like enzyme UBA5 via a high-energy thioester bond, and transferred to E2-like enzyme UFC1 in a similar thioester linkage, forming a three-enzyme conjugation cascade analogous to ubiquitylation.","method":"Biochemical reconstitution of thioester intermediates, C-terminal processing assays, in vitro conjugation assays, mutagenesis of the conserved Gly residue","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis confirming Gly requirement; foundational paper establishing the cascade","pmids":["15071506"],"is_preprint":false},{"year":2006,"finding":"Two UFM1-specific proteases, UfSP1 and UfSP2, cleave the C-terminal extension of UFM1 to expose the Gly residue required for conjugation, and also release UFM1 from UFM1-conjugated cellular proteins. Both are thiol proteases sensitive to sulfhydryl-blocking agents; mutation of the active-site Cys to Ser abolishes activity, and Ufm1-vinylmethylester labels the active-site Cys.","method":"Biochemical cleavage assays with recombinant proteins, active-site mutagenesis (Cys→Ser), activity-based probe labeling with Ufm1-vinylmethylester, inhibitor studies","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — active-site mutagenesis combined with substrate cleavage assays and covalent probe labeling in a single rigorous study","pmids":["17182609"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of human UFC1 (the UFM1 E2-conjugating enzyme) was determined at 1.6 Å resolution. The structure 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 mutational analysis; the N-terminal domain contributes to thermal stability.","method":"X-ray crystallography (1.6 Å), structural comparison, mutational analysis","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with mutational validation of binding site, single lab","pmids":["17825256"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of mouse UfSP1 at 1.7 Å resolution reveals it is a novel cysteine protease with a papain-like fold. The catalytic triad is formed by Cys53, Asp175, and His177 in a Asp-Pro-His configuration; Tyr41 participates in the oxyanion hole. NMR data indicate that the loop between β3 and α2, plus the C-terminal region of UFM1, are important for UFM1-UfSP1 binding (KD ~1.6 μM by ITC).","method":"X-ray crystallography (1.7 Å), active-site mutagenesis, isothermal titration calorimetry, NMR binding mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and ITC binding data, multiple orthogonal methods","pmids":["18321862"],"is_preprint":false},{"year":2009,"finding":"UFL1 (also called RCAD) was identified as the first E3 ligase for UFM1. UFL1 covalently conjugates UFM1 to the substrate C20orf116 (UFBP1/DDRGK1); this conjugation is greatly accelerated by UFL1, reversed by UFM1-specific proteases, and abolished in Uba5 knockout cells. UFL1 and UFBP1 localize mainly to the endoplasmic reticulum.","method":"Co-immunoprecipitation, in vitro conjugation assays, Uba5 knockout cells, subcellular fractionation, immunofluorescence localization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — enzymatic E3 activity demonstrated in vitro and in cells with genetic controls (Uba5 KO), replicated in subsequent studies","pmids":["20018847"],"is_preprint":false},{"year":2011,"finding":"UFBP1 (C20orf116) and CDK5RAP3 interact with UFM1. UFM1 co-localizes with UFBP1 at the endoplasmic reticulum, and this ER co-localization depends on UFBP1. siRNA knockdown of UFM1, UFBP1, or UFL1 enhances apoptosis upon ER stress in INS-1E beta cells, indicating that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis.","method":"Co-immunoprecipitation, immunofluorescence co-localization, siRNA knockdown, apoptosis assays under ER stress","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interactions, functional knockdown with specific apoptosis readout, localization tied to function, replicated by multiple groups","pmids":["21494687"],"is_preprint":false},{"year":2011,"finding":"Uba5 (UFM1 E1 activating enzyme) is indispensable for erythroid differentiation in mice. Uba5 knockout mice exhibit severe anaemia and die in utero. Genetic loss impairs development of megakaryocyte and erythroid progenitors from common myeloid progenitors in a cell-autonomous manner, as transgenic Uba5 expression restricted to erythroid lineage rescues the anaemia.","method":"Uba5 knockout mouse model, erythroid-specific transgenic rescue, hematopoietic progenitor analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with specific phenotype and rescue by lineage-specific transgene establishing cell-autonomous function","pmids":["21304510"],"is_preprint":false},{"year":2012,"finding":"The UFM1 system is transcriptionally upregulated in response to ER stress and inhibition of vesicle trafficking. Using luciferase reporter and ChIP assays, UFM1 was identified as a transcriptional target of Xbp-1. Conversely, knockdown of the UFM1 system in U2OS cells triggered the unfolded protein response and amplification of the ER network. RCAD/Ufl1 and its binding partner C53/LZAP are involved in ufmylation of endogenous targets.","method":"Luciferase reporter assays, ChIP assays, Xbp-1 knockout MEFs, siRNA knockdown with UPR readouts","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assays establish Xbp-1 regulation; single lab, multiple methods","pmids":["23152784"],"is_preprint":false},{"year":2014,"finding":"Ufmylation of the nuclear receptor coactivator ASC1 is required for ERα transactivation. In the absence of E2-estradiol, UfSP2 binds ASC1 and keeps it deufmylated. E2 binding causes ERα to associate with ASC1 and displace UfSP2, leading to polyufmylation of ASC1, which enhances association of p300, SRC1, and ASC1 at promoters of ERα target genes.","method":"Co-immunoprecipitation, ufmylation assays, ChIP, knockdown of UBA5 and UFSP2, ufmylation-deficient ASC1 mutant expression, in vivo tumor formation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, site-specific mutants, ChIP, and in vivo rescue experiments; multiple orthogonal methods","pmids":["25219498"],"is_preprint":false},{"year":2014,"finding":"Uba5 activates UFM1 via a two-step mechanism forming a binary Uba5~UFM1 thioester (unlike the three-step ternary complex of Uba1/ubiquitin). Uba5 shows random ordered binding with UFM1 and ATP; its transthiolation of UFM1 to UFC1 requires ATP binding to the Uba5~UFM1 thioester complex. The pan-E1 inhibitor adenosine 5'-sulfamate forms a covalent Ufm1-ADS adduct in Uba5's active site.","method":"In vitro kinetic assays, thioester formation assays, ATP-PPi exchange assays, mechanism-based inhibitor studies in cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — detailed mechanistic in vitro assays with multiple orthogonal methods and cellular validation","pmids":["24966333"],"is_preprint":false},{"year":2015,"finding":"RCAD/Ufl1 (UFM1 E3 ligase) is essential for hematopoietic stem cell survival and erythroid differentiation. Both germ-line and somatic deletion impaired hematopoietic development resulting in severe anaemia. Loss of RCAD/Ufl1 elevated ER stress, evoked UPR, blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response and p53 activation in HSCs.","method":"Germ-line and conditional knockout mouse models, bone marrow cell analysis, ER stress markers, autophagy assays, ROS measurement","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent genetic KO models with multiple mechanistic readouts, consistent with UFM1 E1 KO phenotype","pmids":["25952549"],"is_preprint":false},{"year":2015,"finding":"UFBP1 is indispensable for embryonic development and hematopoiesis. UFBP1 deficiency causes elevated ER stress and UPR activation leading to HSC/progenitor cell death, and suppresses expression of erythroid transcription factors GATA-1 and KLF1. ASC1 was found to associate with GATA-1 and Klf1 promoters in a UFBP1-dependent manner.","method":"Germ-line and conditional UFBP1 knockout mice, ChIP, K562 cell knockdowns, ER stress marker analysis","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent KO models, ChIP establishing UFBP1-dependence of ASC1 promoter association, multiple orthogonal readouts","pmids":["26544067"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of the non-canonical homodimeric E1 UBA5 in complex with UFM1 reveals that UFM1 interacts with distinct sites in both subunits of the UBA5 dimer via a trans-binding mechanism. UFM1 transfer from UBA5 to UFC1 also occurs via a trans mechanism requiring UBA5 homodimerization. A C-terminal region outside the adenylation domain recruits UFM1 to the active site of the adjacent subunit.","method":"X-ray crystallography, biochemical assays of trans-binding mechanism, mutagenesis","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with biochemical validation of trans-binding mechanism","pmids":["27653677"],"is_preprint":false},{"year":2016,"finding":"A combined LIR/UFM1-interacting motif (LIR/UFIM) at the C-terminus of UBA5 mediates binding to both UFM1 and LC3/GABARAP. This motif is required for full biological activity of UBA5 and for effective transfer of UFM1 to UFC1 and downstream protein substrates both in vitro and in cells.","method":"NMR structure, mutational analysis, in vitro UFM1 transfer assays, cellular ufmylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural determination with functional mutagenesis, in vitro and cellular validation","pmids":["26929408"],"is_preprint":false},{"year":2016,"finding":"Biallelic mutations in UBA5 impair ufmylation, resulting in abnormal endoplasmic reticulum structure. The UBA5 p.Ala371Thr variant is hypomorphic with attenuated ability to transfer activated UFM1 to UFC1 (shown by in vitro thioester formation assay). CNS-specific knockout of Ufm1 in mice causes neonatal death with microcephaly and neuronal apoptosis. In C. elegans, knockout of uba-5 alters cholinergic but not glutamatergic neurotransmission.","method":"In vitro thioester formation assay with mutant UBA5, patient fibroblast studies, Ufm1 CNS-specific knockout mice, C. elegans knockout, zebrafish uba5 silencing","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro biochemical assay of mutant protein, multiple model organism KO studies, independently replicated across two concurrent papers","pmids":["27545674","27545681"],"is_preprint":false},{"year":2018,"finding":"Biallelic mutations in UFM1 and UFC1 impair formation of UFM1-UBA5 and UFM1-UFC1 thioester intermediates, causing widespread reduction of cellular ufmylation. This establishes that mutations in the UFM1 modifier itself and its E2 enzyme cause severe early-onset encephalopathy with progressive microcephaly, demonstrating the essential role of ufmylation in human brain development.","method":"Thioester intermediate formation assays with mutant proteins, cellular ufmylation assays, clinical genetics","journal":"Brain : a journal of neurology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct biochemical assay of thioester formation with disease mutations, replicated across multiple families","pmids":["29868776"],"is_preprint":false},{"year":2018,"finding":"Ufl1 (UFM1 E3 ligase) deficiency in cardiac-specific knockout mice leads to age-dependent cardiomyopathy and heart failure, with excessive ER stress preceding and worsening with disease progression. Mechanistically, Ufl1 depletion impairs PERK (PKR-like ER-resident kinase) signaling and aggravates cardiomyocyte death after ER stress. Chemical ER chaperone tauroursodeoxycholic acid treatment alleviates the cardiac dysfunction.","method":"Cardiac-specific knockout mice, transcriptome analysis, ER stress markers, PERK signaling assays, pharmacological rescue","journal":"Circulation. Heart failure","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with transcriptome and biochemical analysis, pharmacological rescue confirming ER stress mechanism","pmids":["30354401"],"is_preprint":false},{"year":2018,"finding":"Trans-binding of UFM1 to UBA5 stabilizes the UBA5 homodimer, which in turn enhances ATP binding to UBA5. Dimerization of UBA5 is required for ATP binding; UFM1 binding promotes dimerization and thereby stimulates ATP binding necessary for UFM1 activation.","method":"Biochemical binding assays, dimerization analysis, ATP binding assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical assays establishing functional connection between UFM1 binding, dimerization, and ATP binding; single lab","pmids":["29295865"],"is_preprint":false},{"year":2018,"finding":"The N-terminal extension present in the long isoform of UBA5 is directly involved in ATP binding and affects how the adenylation domain interacts with ATP, changing the stoichiometry from 1:2 to 1:1 ATP:UBA5. The N-terminus also significantly increases ATP affinity and stimulates UFM1 transfer from UBA5 to UFC1, though it is not directly involved in E2 binding.","method":"Crystal structures of UBA5 long isoform with ATP and UFM1, ATP binding assays, UFC1 transfer assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with functional biochemical assays; single lab, multiple orthogonal methods","pmids":["30412706"],"is_preprint":false},{"year":2019,"finding":"The UFM1 cascade controls cell cycle entry at the G2/M transition in Drosophila neuroblasts. Disruption of ufmylation increases the mitotic index and extends G2/M phase. Impaired E3 ligase Ufl1 function causes premature mitotic entry and failed cellularization. The UFM1 cascade alters the phosphorylation level of Tyr15 on CDK1 (pY15-CDK1), a negative regulator of the G2-to-M transition.","method":"Drosophila neuroblast genetic knockdown/knockout, live imaging, mitotic index analysis, phosphorylation assays for CDK1-pY15","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic disruption in Drosophila with live imaging and CDK1 phosphorylation as mechanistic readout; single lab","pmids":["31914610"],"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 observed in other E1-E2 complexes. A region of UBA5 outside the adenylation domain, dispensable for UFC1 binding, is critical for UFM1 transfer by compensating for a missing loop in UFC1's active site.","method":"X-ray crystallography of UFC1-UBA5 C-terminal peptide complex, mutational analysis of transfer","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis establishing mechanistic basis for UFM1 transfer","pmids":["34588452"],"is_preprint":false},{"year":2022,"finding":"NADH-cytochrome b5 reductase 3 (CYB5R3) on the ER membrane is a UFM1 substrate. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1, converts CYB5R3 into its inactive form, and is recognized by UFBP1 through a UFM1-interacting motif on UFBP1. Ufmylated CYB5R3 is degraded in lysosomes in an Atg7- and CDK5RAP3-dependent manner, promoting ER-phagy. Ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly.","method":"Identification of ufmylation substrate, UFL1/UFBP1 dependency assays, in vitro ufmylation, lysosomal degradation assays, Atg7 dependency, Cyb5r3 ufmylation-defective knock-in mice","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including knock-in mouse model, substrate identification with mechanistic follow-up","pmids":["36543799"],"is_preprint":false},{"year":2022,"finding":"ERα (estrogen receptor α) is directly ufmylated at Lys171 and Lys180. UFMylation of ERα increases its stability by inhibiting ubiquitination and proteasomal degradation. UfSP2 knockdown increases ERα stability, while UBA5 silencing decreases it. Ufmylation-deficient ERα (2KR mutant) shows reduced stability, abolished E2-induced transactivity, and cannot form anchorage-independent colonies.","method":"Site-specific mutagenesis (Lys to Arg), ubiquitination assays, stability assays with UBA5/UFSP2 manipulation, ERα target gene expression analysis, colony formation assays","journal":"Molecules and cells","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — site-specific mutants, multiple functional assays; builds on prior ASC1 ufmylation work from same group","pmids":["35680375"],"is_preprint":false},{"year":2022,"finding":"UFSP1 is translated from a non-canonical start site and acts as an active protease that matures UFM1 (cleaves pro-UFM1) and cleaves a potential autoinhibitory modification on UFC1 to control activation of UFMylation. Cells lacking both UFSP1 and UFSP2 show complete loss of UFMylation due to absence of mature UFM1. UFSP2, but not UFSP1, removes UFM1 from ribosomal subunit RPL26, revealing distinct substrate specificities.","method":"UFSP1/UFSP2 double-knockout cells, identification of non-canonical start site, UFM1 maturation assays, UFC1 deufmylation assays, RPL26 deufmylation assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — double KO cells with specific biochemical readouts, discovery of non-canonical start site, distinct substrate specificity determination","pmids":["35926457"],"is_preprint":false},{"year":2022,"finding":"PLAC8 is post-translationally modified by UFM1 (ufmylated), and this modification maintains PLAC8 protein stability in triple-negative breast cancer cells. PLAC8 in turn regulates PD-L1 levels by affecting PD-L1 ubiquitination.","method":"Co-immunoprecipitation, ufmylation assays, protein stability assays","journal":"Journal for immunotherapy of cancer","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP and stability assays showing ufmylation of PLAC8; single lab, moderate mechanistic follow-up","pmids":["36543379"],"is_preprint":false},{"year":2022,"finding":"UFL1 and CDK5RAP3/C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or CDK5RAP3 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation with γ-tubulin accumulation. CDK5RAP3, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in UFL1-lacking cells.","method":"Knockout of UFL1 and CDK5RAP3, immunofluorescence, γ-tubulin complex co-immunoprecipitation, microtubule nucleation assays","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO phenotype with biochemical interaction data; single lab, indirect link to UFM1 itself via UFL1","pmids":["35159364"],"is_preprint":false},{"year":2022,"finding":"Neuron-specific deletion of either UFL1 or UFBP1 (UFM1 E3 ligase components) leads to significant neuronal loss in adult mice, elevated inflammatory response, and seizure-like events upon loss of one UFBP1 allele, demonstrating the indispensable role of the UFM1 E3 ligase in mature neuron survival.","method":"Neuron-specific conditional knockout mice for UFL1 and UFBP1, histological analysis, inflammatory marker measurement","journal":"Molecular neurobiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent conditional KO models with consistent neurodegeneration phenotype","pmids":["35931931"],"is_preprint":false},{"year":2023,"finding":"The UFM1 E3 complex (UFL1-UFBP1-CDK5RAP3) interacts with UFC1 (E2), then CDK5RAP3 acts as an adaptor for ufmylation of ribosomal subunit RPL26. Upon disome formation (colliding ribosomes), the E3 complex associates with ufmylated RPL26 on the 60S subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of UFBP1-ufmylated RPL26 interaction impairs ER-ribosome quality control.","method":"Co-immunoprecipitation, ribosome pulldown, disome analysis, mutational disruption of UFBP1-UFM1 interaction, ER-RQC reporter assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple Co-IP and functional assays, mutational analysis of key interactions with ER-RQC readout","pmids":["37595036"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structures of the UFM1 E3 ligase complex (UREL, comprising UFL1-UFBP1-CDK5RAP3) bound to 60S ribosomes reveal a C-shaped clamp architecture. UFL1 loops insert into and remodel the peptidyl transferase center. UREL functions as both a 'writer' (catalyzing UFMylation of RPL26/uL24) and subsequently a 'reader' of ufmylated 60S. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating UFMylation is necessary for releasing SEC61 from 60S subunits.","method":"Cryo-EM structures, biochemical reconstitution, genetic loss-of-function accumulation of 60S-SEC61 complexes","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structures with functional validation; independently replicated in two concurrent Nature papers","pmids":["38383789","38383785"],"is_preprint":false},{"year":2024,"finding":"Sequential cryo-EM snapshots show the UFM1 E3 ligase (E3(UFM1)) engages its substrate uL24 (RPL26) on free 60S ribosomes. E3(UFM1) binds the L1 stalk, empty tRNA-binding sites, and the peptidyl transferase center through C-terminal domains of UFL1, catalyzing UFM1 transfer to uL24 more than 150 Å away. After catalysis, E3(UFM1) remains stably bound to ufmylated 60S, forming the C-shaped clamp, suggesting a role in post-termination recycling of the large ribosomal subunit from the ER membrane.","method":"Cryo-EM (sequential snapshots), biochemical analysis of E3 binding and catalysis, substrate engagement mapping","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple high-resolution cryo-EM structures with biochemical validation; independently replicated","pmids":["38383785"],"is_preprint":false},{"year":2018,"finding":"Structural basis for UFM1 recognition by C. elegans UfSP: the structure reveals that the conserved Pro88-Val92 residues (P6-P2 positions from the cleavage site) plus extended β-structure at the UFM1 C-terminus are important for specific recognition of UFM1 by UfSP.","method":"X-ray crystallography of cUFM1-cUfSP complex","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure of complex; single lab, C. elegans ortholog","pmids":["29251776"],"is_preprint":false},{"year":2014,"finding":"In C. elegans, UfSP2 (odr-8) forms a physical complex with ODR-4 at the ER membrane in chemosensory neurons. This complex promotes GPCR (ODR-10) maturation and ER export independently of UfSP2's catalytic activity and independently of UFM1, since catalytically dead UfSP2 rescues all odr-8 mutant phenotypes and deletion of ufm-1 does not alter chemoreceptor trafficking.","method":"Genetic epistasis in C. elegans, co-immunoprecipitation of ODR-4/ODR-8, catalytic-dead mutant rescue, ufm-1 null mutant analysis","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with Co-IP and catalytic mutant rescue; C. elegans ortholog, establishes protease-independent UfSP2 function","pmids":["24603482"],"is_preprint":false},{"year":2021,"finding":"The NMR structure of UFC1 bound to a peptide spanning the last 20 residues of UBA5 reveals the molecular basis of UBA5-UFC1 interaction. These last 20 residues are pivotal for UFC1 binding and accelerate UFM1 transfer to UFC1; a proposed regulatory role involves the C-terminal unstructured region controlling cellular localization and interaction of ufmylation cascade elements.","method":"NMR structure of UFC1-UBA5 peptide complex, ITC binding assays, UFM1 transfer assays","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with ITC and functional transfer assays; single lab","pmids":["34299007"],"is_preprint":false},{"year":2021,"finding":"The UFM1 pathway impacts ER-associated protein degradation (ERAD). A genome-wide CRISPR/Cas9 screen using US2-mediated HLA-I degradation as ERAD model identified the UFM1 pathway. Interference with the UFM1 pathway inhibits ER-to-cytosol dislocation of HLA-I specifically; no UFMylation of HLA-I was detected, suggesting UFM1 impacts ERAD indirectly. Ribosomal proteins are a major class of UFMylated proteins in US2-expressing cells.","method":"Genome-wide CRISPR/Cas9 screen, mass spectrometry, HLA-I dislocation assays","journal":"Molecules (Basel, Switzerland)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen plus biochemical follow-up; mechanistic link is indirect","pmids":["33430125"],"is_preprint":false},{"year":2023,"finding":"UfSP1 binds to the ubiquitin-associated (UBA) domain of p62/SQSTM1 and promotes the interaction between p62 and ubiquitinated proteins, thereby increasing p62 body formation. This function is independent of UfSP1's protease activity, as both catalytically active and inactive UfSP1 promote p62 body formation through the same mechanism.","method":"Proximity labeling (TurboID), co-immunoprecipitation, immunofluorescence co-localization, catalytic-dead mutant analysis","journal":"Journal of proteome research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity proteomics with Co-IP and catalytic dead mutant validation; single lab","pmids":["37285312"],"is_preprint":false},{"year":2022,"finding":"UFM1 suppresses gastric cancer cell invasion by increasing ubiquitination of PDK1 (decreasing PDK1 protein level), thereby inhibiting AKT phosphorylation at Ser473 and the PI3K/AKT signaling pathway. The effect of UFM1 on cancer cell function depends on PDK1 expression.","method":"Co-immunoprecipitation, protein kinase array, stable UFM1 overexpression/knockdown, PDK1 ubiquitination assays, AKT phosphorylation analysis","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP and ubiquitination assays with genetic dependency analysis; single lab, mechanistic depth is partial","pmids":["31533855"],"is_preprint":false},{"year":2025,"finding":"UFM1 loss in neurons causes ER stress, activation of the unfolded protein response (UPR), and reduced protein translation, leading to impaired neuron development and synapse function. The pathogenic UFM1-R81C variant partially rescues UFM1 loss phenotypes but displays distinct ER stress responses, suggesting it is not merely a loss-of-function variant. The UPR inhibitor Trazodone restores protein translation only in UFM1-R81C-expressing neurons, and increases synapse numbers in both UFM1-KO and R81C neurons.","method":"UFM1-deficient mouse neurons, UFM1-R81C knock-in neurons, UPR pathway analysis, protein translation assays, synapse counting, pharmacological rescue with Trazodone","journal":"EMBO molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models with mechanistic biochemical readouts (ER stress, translation, UPR), pharmacological rescue","pmids":["41731076"],"is_preprint":false},{"year":2025,"finding":"UFMylation promotes non-homologous end-joining (NHEJ) of DNA double-strand breaks. Ku70 is a UFMylation substrate; XRCC4 engages UFMylated Ku70 via non-canonical UFM1-binding regions to promote chromatin assembly of NHEJ factors. Perturbation of UFM1 signaling (via UFSP2 depletion or hypomorphic UBA5 allele in patient fibroblasts) impairs NHEJ.","method":"Photo-crosslinkable UFM1 probe, NMR, proximity-dependent proteomics, NHEJ assays, patient fibroblast analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel probe combined with NMR and proximity proteomics; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.06.16.659844"],"is_preprint":true},{"year":2025,"finding":"UFMylation of 14-3-3ε at Lys50 and Lys215 promotes RIG-I/MAVS antiviral signaling. K50R/K215R mutations abolish UFMylation and reduce type I and III interferon induction following RIG-I activation. These mutations do not disrupt the 14-3-3ε-RIG-I interaction but paradoxically enhance RIG-I-MAVS interaction while reducing 14-3-3ε-MAVS interaction, indicating UFMylation controls MAVS signaling complex architecture.","method":"In vitro and cellular UFMylation assays, site-specific mutagenesis, co-immunoprecipitation, interferon induction assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutants with biochemical and functional assays; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.03.19.644084"],"is_preprint":true},{"year":2025,"finding":"Loss of UFMylation reroutes glucose metabolism and promotes prostate cancer cell invasion. Phosphofructokinase (PFKAP) was identified as a UFMylation substrate. Loss of UFMylation reduces glycolytic flux and increases hexosamine biosynthesis, elevating protein glycosylation relevant to invasion.","method":"Biotin-based UFMylation proteomics, transcriptomics, metabolic flux analysis, pharmacological pathway inhibition","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — substrate identified by proteomics with metabolic readout; preprint, mechanistic depth of UFMylation on PFKAP function not fully established","pmids":["bio_10.1101_2025.06.02.657324"],"is_preprint":true},{"year":2025,"finding":"Canonical RQC factors associate with ribosomes stalled at the ER. Ribosome splitting is a prerequisite for UFMylation of RPL26. The UFM1 E3 ligase complex binds and UFMylates the 60S-peptidyl-tRNA complex; UFMylation of RPL26 persists without late RQC components (NEMF, LTN1). UFMylation and the canonical RQC pathway act in concert to facilitate clearance of arrested polypeptides at the ER.","method":"ER-targeted stalling reporters, ribosome fractionation, UFMylation and RQC factor dependency assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cellular reporters with biochemical validation; preprint, consistent with peer-reviewed cryo-EM structural work","pmids":["bio_10.1101_2025.01.17.633636"],"is_preprint":true}],"current_model":"UFM1 is a ubiquitin-like modifier that undergoes C-terminal processing by UFSPs (UFSP1/2) to expose a conserved Gly, then is activated by E1 enzyme UBA5 via a trans-binding, homodimer-dependent thioester mechanism, transferred to E2 enzyme UFC1, and conjugated to substrates by the trimeric E3 ligase complex (UFL1-UFBP1-CDK5RAP3); the best-characterized substrate is ribosomal protein RPL26 (uL24) on ER-associated 60S ribosomes, where the E3 complex wraps around 60S as a C-shaped clamp to remodel the peptidyl transferase center, catalyze UFMylation, and then remain bound to UFMylated 60S to release stalled 60S subunits from the SEC61 translocon—thereby coupling ribosome-associated quality control and ER-phagy to ER proteostasis—while additional substrates including ASC1, ERα, CYB5R3, PLAC8, PDK1, and 14-3-3ε link ufmylation to transcription, ER-phagy, antiviral immunity, and cancer cell biology."},"narrative":{"mechanistic_narrative":"UFM1 is a ubiquitin-fold modifier that is covalently conjugated to target proteins through a dedicated three-enzyme cascade analogous to ubiquitylation, coupling protein quality control at the endoplasmic reticulum to cellular proteostasis [PMID:15071506]. Newly synthesized UFM1 is matured by the cysteine proteases UFSP1 and UFSP2, which cleave its C-terminal extension to expose the conjugation-essential Gly; these same proteases also reverse the modification by releasing UFM1 from substrates [PMID:17182609, PMID:35926457]. The exposed Gly is activated by the non-canonical homodimeric E1 enzyme UBA5 through an ATP-dependent thioester mechanism in which UFM1 binds in trans across the dimer interface and stabilizes the dimer to promote ATP loading, then is transthiolated to the E2 enzyme UFC1 [PMID:15071506, PMID:27653677, PMID:29295865]. Conjugation to substrates is catalyzed by a trimeric E3 ligase complex of UFL1, UFBP1 (DDRGK1) and CDK5RAP3, which assembles at the ER membrane [PMID:20018847, PMID:21494687]. The best-characterized substrate is the 60S ribosomal protein RPL26 (uL24): the E3 complex engages stalled and split 60S subunits as a C-shaped clamp, inserting loops of UFL1 to remodel the peptidyl transferase center, catalyzing UFMylation of uL24 across a large distance, and then remaining bound to ufmylated 60S to release the SEC61 translocon and recycle the subunit—thereby integrating ER-associated ribosome quality control with ER-phagy [PMID:37595036, PMID:38383789, PMID:38383785]. Beyond ribosome quality control, UFMylation modifies additional ER and signaling substrates including CYB5R3, where modification drives ER-phagy [PMID:36543799], and the transcriptional regulators ASC1 and ERα, linking ufmylation to estrogen-receptor target-gene activation and hematopoietic transcription [PMID:25219498, PMID:35680375, PMID:26544067]. The pathway is transcriptionally induced as part of the ER stress response downstream of Xbp-1, and its disruption activates the unfolded protein response, impairs erythroid and hematopoietic development, and causes neuronal loss [PMID:23152784, PMID:21304510, PMID:25952549, PMID:35931931]. Biallelic mutations in UFM1, UBA5, and UFC1 that impair thioester intermediate formation cause severe early-onset encephalopathy with progressive microcephaly, establishing ufmylation as essential for human brain development [PMID:27545674, PMID:27545681, PMID:29868776].","teleology":[{"year":2004,"claim":"Established UFM1 as a bona fide ubiquitin-like modifier by demonstrating it operates through a three-enzyme conjugation cascade, answering whether this ubiquitin-fold protein is a functional modifier.","evidence":"In vitro reconstitution of thioester intermediates with UBA5/UFC1 and Gly mutagenesis","pmids":["15071506"],"confidence":"High","gaps":["No physiological substrates or E3 ligase identified","Cellular processes regulated by UFMylation unknown"]},{"year":2006,"claim":"Identified the deconjugating/maturing machinery, showing UFSP1 and UFSP2 both expose the conjugation Gly on pro-UFM1 and reverse the modification, establishing ufmylation as a reversible cycle.","evidence":"Cleavage assays with recombinant proteins, Cys→Ser active-site mutagenesis, and activity-based probe labeling","pmids":["17182609"],"confidence":"High","gaps":["Distinct roles of UFSP1 vs UFSP2 not resolved","Substrate specificity of deconjugation undefined"]},{"year":2008,"claim":"Structural and biochemical work defined the catalytic chemistry and UFM1-recognition basis of the proteases, explaining how UFSP enzymes engage the UFM1 C-terminus.","evidence":"X-ray crystallography of UfSP1, active-site mutagenesis, ITC and NMR binding mapping (mouse; C. elegans complex in #30)","pmids":["18321862","29251776"],"confidence":"High","gaps":["Recognition of substrate-conjugated UFM1 versus free UFM1 not distinguished","Regulation of protease activity in cells unaddressed"]},{"year":2009,"claim":"Identified UFL1 as the first UFM1 E3 ligase and its substrate UFBP1, locating the conjugation machinery at the ER and answering how UFM1 is transferred to substrates.","evidence":"Co-IP, in vitro conjugation acceleration, Uba5 knockout cells, and subcellular localization","pmids":["20018847"],"confidence":"High","gaps":["Full E3 complex composition not yet defined","Functional consequence of UFBP1 modification unknown"]},{"year":2012,"claim":"Placed the UFM1 system within the ER stress response by showing it is a transcriptional target of Xbp-1 and that its loss triggers the UPR, framing ufmylation as an ER proteostasis pathway.","evidence":"Luciferase reporters, ChIP, Xbp-1 knockout MEFs, and siRNA knockdown with UPR readouts","pmids":["23152784"],"confidence":"Medium","gaps":["Direct molecular targets mediating UPR effects not identified","Single-lab transcriptional regulation evidence"]},{"year":2015,"claim":"Demonstrated physiological essentiality of the pathway in hematopoiesis, linking E1 (UBA5), E3 (UFL1) and substrate-receptor (UFBP1) loss to ER stress, blocked differentiation, and HSC death.","evidence":"Germline and conditional knockout mice for Uba5, Ufl1, and UFBP1 with ER stress, autophagy, ROS, and ChIP analyses","pmids":["21304510","25952549","26544067"],"confidence":"High","gaps":["Molecular substrates driving erythroid phenotypes not pinned to specific UFMylation events","Whether phenotypes are conjugation-dependent in every case unresolved"]},{"year":2016,"claim":"Resolved the unusual E1 mechanism, showing UBA5 is a homodimer that binds and transfers UFM1 in trans, with a dual LIR/UFIM motif linking activation to autophagy machinery.","evidence":"Crystal and NMR structures of UBA5–UFM1, mutagenesis, and in vitro/cellular transfer assays","pmids":["27653677","26929408"],"confidence":"High","gaps":["Functional significance of LC3/GABARAP binding to ufmylation outputs incomplete","Regulation of dimerization in cells unaddressed"]},{"year":2016,"claim":"Established UFM1 ufmylation as essential for human development by linking biallelic UBA5 hypomorphic mutations to abnormal ER and CNS phenotypes across model organisms.","evidence":"In vitro thioester assay of mutant UBA5, patient fibroblasts, Ufm1 CNS-specific knockout mice, and C. elegans/zebrafish models","pmids":["27545674","27545681"],"confidence":"High","gaps":["Substrate(s) whose mis-ufmylation drives neuropathology not identified","Tissue-selective vulnerability mechanism unknown"]},{"year":2018,"claim":"Confirmed UFM1 and UFC1 mutations cause severe encephalopathy by impairing thioester intermediate formation, cementing ufmylation as essential for brain development.","evidence":"Thioester formation assays with mutant proteins, cellular ufmylation assays, and clinical genetics","pmids":["29868776"],"confidence":"High","gaps":["Genotype–phenotype relationship not mechanistically explained at substrate level"]},{"year":2018,"claim":"Refined the catalytic logic of UFM1 activation, showing UFM1 binding stabilizes the UBA5 dimer to enhance ATP binding and that an N-terminal extension tunes ATP stoichiometry and transfer.","evidence":"Biochemical binding/dimerization assays and crystal structures of the UBA5 long isoform with ATP","pmids":["29295865","30412706"],"confidence":"Medium","gaps":["In-cell relevance of isoform-specific ATP handling untested","Some claims rest on single-lab biochemistry"]},{"year":2018,"claim":"Extended physiological roles to the heart and identified substrate-specificity logic in the protease layer, linking UFL1 loss to ER-stress cardiomyopathy and UFSP1/2 to differential UFM1 maturation and RPL26 deufmylation.","evidence":"Cardiac-specific Ufl1 knockout mice with PERK signaling analysis and pharmacological rescue; UFSP1/UFSP2 double-knockout cells","pmids":["30354401","35926457"],"confidence":"High","gaps":["Cardiac substrates mediating ER stress not identified","Regulation of UFSP1 non-canonical translation in vivo unaddressed"]},{"year":2021,"claim":"Defined the molecular basis of UFM1 transfer from E1 to E2, showing a short linear UBA5 C-terminal sequence binds UFC1 and a separate UBA5 region compensates for a missing UFC1 active-site loop.","evidence":"X-ray and NMR structures of UFC1–UBA5 peptide complexes with mutational transfer assays","pmids":["34588452","34299007"],"confidence":"High","gaps":["How transfer is coupled to E3 handoff not structurally resolved"]},{"year":2014,"claim":"Identified transcriptional substrates of ufmylation, showing UFMylation of ASC1 (and later ERα) regulates estrogen-receptor target gene activation and stability, expanding ufmylation beyond the ER membrane.","evidence":"Co-IP, ufmylation assays, ChIP, site-specific Lys-to-Arg mutants, stability assays, and tumor formation assays","pmids":["25219498","35680375"],"confidence":"High","gaps":["How nuclear UFMylation is spatially coordinated with ER-localized E3 unclear","Generality across nuclear receptors untested"]},{"year":2022,"claim":"Identified ER-membrane substrates linking ufmylation to organelle turnover, showing CYB5R3 UFMylation inactivates it and targets it for lysosomal ER-phagy via UFBP1 recognition and CDK5RAP3.","evidence":"Substrate identification, UFL1/UFBP1 dependency, in vitro ufmylation, Atg7-dependent degradation, and ufmylation-defective Cyb5r3 knock-in mice","pmids":["36543799"],"confidence":"High","gaps":["Selectivity of CYB5R3 among ER substrates not explained","Link between CYB5R3 ER-phagy and microcephaly phenotype mechanistic gap"]},{"year":2023,"claim":"Defined the central ribosomal substrate pathway, showing the UFL1-UFBP1-CDK5RAP3 E3 complex UFMylates RPL26 on colliding/disome 60S and that UFBP1 reads ufmylated RPL26 to drive ER-ribosome quality control.","evidence":"Co-IP, ribosome pulldown, disome analysis, and mutational disruption of UFBP1–UFM1 interaction with ER-RQC reporters","pmids":["37595036"],"confidence":"High","gaps":["Trigger coupling collision sensing to E3 recruitment incompletely defined"]},{"year":2024,"claim":"Provided the structural mechanism of ribosomal UFMylation, showing the E3 complex clamps split 60S as a C-shaped writer/reader that remodels the PTC, UFMylates uL24 at long range, and releases SEC61 for subunit recycling.","evidence":"Cryo-EM structures of UREL–60S complexes and sequential snapshots with biochemical validation and 60S-SEC61 accumulation upon loss of function","pmids":["38383789","38383785"],"confidence":"High","gaps":["How the reader function feeds into downstream ER-phagy/recycling steps not fully traced","Regulation of writer-to-reader transition unclear"]},{"year":2025,"claim":"Connected ufmylation to additional substrates and processes—DNA double-strand break repair (Ku70/NHEJ), antiviral RIG-I/MAVS signaling (14-3-3ε), and cancer metabolism—and dissected neuronal UPR consequences of UFM1 loss with pharmacological rescue.","evidence":"UFM1-deficient and UFM1-R81C knock-in neurons with UPR/translation/synapse readouts and Trazodone rescue; preprint probe/proteomics studies of Ku70, 14-3-3ε, and metabolic substrates","pmids":["41731076","bio_10.1101_2025.06.16.659844","bio_10.1101_2025.03.19.644084","bio_10.1101_2025.06.02.657324"],"confidence":"Medium","gaps":["Several substrates rest on single-lab preprint evidence awaiting peer review","Whether non-ribosomal substrates are direct E3 targets in vivo not fully established"]},{"year":null,"claim":"How substrate selection is partitioned between the dominant ribosomal UFMylation pathway and the growing list of non-ribosomal substrates, and which substrates underlie the neurodevelopmental and hematopoietic disease phenotypes, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model for E3 substrate targeting outside the ribosome","Causal substrate(s) for human encephalopathy not identified","Cell-type-specific vulnerability mechanisms undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[0,4,8,21,22]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,27,28]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[4,5,21,27,28]},{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[23,27,28,29]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,11,22]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,27,28]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[27,28,29]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[5,7,16,36]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[21]}],"complexes":["UFM1 E3 ligase complex (UFL1-UFBP1-CDK5RAP3)"],"partners":["UBA5","UFC1","UFL1","UFBP1","CDK5RAP3","UFSP2","UFSP1","RPL26"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P61960","full_name":"Ubiquitin-fold modifier 1","aliases":[],"length_aa":85,"mass_kda":9.1,"function":"Ubiquitin-like modifier which can be covalently attached via an isopeptide bond to lysine residues of substrate proteins as a monomer or a lysine-linked polymer (PubMed:15071506, PubMed:20018847, PubMed:27653677, PubMed:29868776, PubMed:30626644, PubMed:38377992, PubMed:38383785). The so-called ufmylation, requires the UFM1-activating E1 enzyme UBA5, the UFM1-conjugating E2 enzyme UFC1, and the UFM1-ligase E3 enzyme UFL1 (PubMed:15071506, PubMed:20018847, PubMed:27653677, PubMed:29868776). Ufmylation is involved in various processes, such as ribosome recycling, response to DNA damage, transcription or reticulophagy (also called ER-phagy) induced in response to endoplasmic reticulum stress (PubMed:25219498, PubMed:32160526, PubMed:38383785)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P61960/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UFM1","classification":"Not Classified","n_dependent_lines":730,"n_total_lines":1208,"dependency_fraction":0.6043046357615894},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PSPC1","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2},{"gene":"SAR1B","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2},{"gene":"TPT1","stoichiometry":0.2},{"gene":"UBA5","stoichiometry":0.2},{"gene":"UFC1","stoichiometry":0.2},{"gene":"UPF1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/UFM1","total_profiled":1310},"omim":[{"mim_id":"620543","title":"ZINC FINGER CONTAINING UBIQUITIN PEPTIDASE 1; ZUP1","url":"https://www.omim.org/entry/620543"},{"mim_id":"620028","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 106; DEE106","url":"https://www.omim.org/entry/620028"},{"mim_id":"618076","title":"NEURODEVELOPMENTAL DISORDER WITH SPASTICITY AND POOR GROWTH; NEDSG","url":"https://www.omim.org/entry/618076"},{"mim_id":"617974","title":"SPONDYLOEPIMETAPHYSEAL DYSPLASIA, DI ROCCO TYPE; SEMDDR","url":"https://www.omim.org/entry/617974"},{"mim_id":"617899","title":"LEUKODYSTROPHY, HYPOMYELINATING, 14; HLD14","url":"https://www.omim.org/entry/617899"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UFM1"},"hgnc":{"alias_symbol":["bA131P10.1"],"prev_symbol":["C13orf20"]},"alphafold":{"accession":"P61960","domains":[{"cath_id":"3.10.20.90","chopping":"2-77","consensus_level":"high","plddt":94.9337,"start":2,"end":77}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P61960","model_url":"https://alphafold.ebi.ac.uk/files/AF-P61960-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P61960-F1-predicted_aligned_error_v6.png","plddt_mean":91.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UFM1","jax_strain_url":"https://www.jax.org/strain/search?query=UFM1"},"sequence":{"accession":"P61960","fasta_url":"https://rest.uniprot.org/uniprotkb/P61960.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P61960/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P61960"}},"corpus_meta":[{"pmid":"15071506","id":"PMC_15071506","title":"A novel protein-conjugating 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The exposed Gly is then activated by E1-like enzyme UBA5 via a high-energy thioester bond, and transferred to E2-like enzyme UFC1 in a similar thioester linkage, forming a three-enzyme conjugation cascade analogous to ubiquitylation.\",\n      \"method\": \"Biochemical reconstitution of thioester intermediates, C-terminal processing assays, in vitro conjugation assays, mutagenesis of the conserved Gly residue\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis confirming Gly requirement; foundational paper establishing the cascade\",\n      \"pmids\": [\"15071506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Two UFM1-specific proteases, UfSP1 and UfSP2, cleave the C-terminal extension of UFM1 to expose the Gly residue required for conjugation, and also release UFM1 from UFM1-conjugated cellular proteins. Both are thiol proteases sensitive to sulfhydryl-blocking agents; mutation of the active-site Cys to Ser abolishes activity, and Ufm1-vinylmethylester labels the active-site Cys.\",\n      \"method\": \"Biochemical cleavage assays with recombinant proteins, active-site mutagenesis (Cys→Ser), activity-based probe labeling with Ufm1-vinylmethylester, inhibitor studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — active-site mutagenesis combined with substrate cleavage assays and covalent probe labeling in a single rigorous study\",\n      \"pmids\": [\"17182609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of human UFC1 (the UFM1 E2-conjugating enzyme) was determined at 1.6 Å resolution. The structure 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 mutational analysis; the N-terminal domain contributes to thermal stability.\",\n      \"method\": \"X-ray crystallography (1.6 Å), structural comparison, mutational analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with mutational validation of binding site, single lab\",\n      \"pmids\": [\"17825256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of mouse UfSP1 at 1.7 Å resolution reveals it is a novel cysteine protease with a papain-like fold. The catalytic triad is formed by Cys53, Asp175, and His177 in a Asp-Pro-His configuration; Tyr41 participates in the oxyanion hole. NMR data indicate that the loop between β3 and α2, plus the C-terminal region of UFM1, are important for UFM1-UfSP1 binding (KD ~1.6 μM by ITC).\",\n      \"method\": \"X-ray crystallography (1.7 Å), active-site mutagenesis, isothermal titration calorimetry, NMR binding mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and ITC binding data, multiple orthogonal methods\",\n      \"pmids\": [\"18321862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"UFL1 (also called RCAD) was identified as the first E3 ligase for UFM1. UFL1 covalently conjugates UFM1 to the substrate C20orf116 (UFBP1/DDRGK1); this conjugation is greatly accelerated by UFL1, reversed by UFM1-specific proteases, and abolished in Uba5 knockout cells. UFL1 and UFBP1 localize mainly to the endoplasmic reticulum.\",\n      \"method\": \"Co-immunoprecipitation, in vitro conjugation assays, Uba5 knockout cells, subcellular fractionation, immunofluorescence localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — enzymatic E3 activity demonstrated in vitro and in cells with genetic controls (Uba5 KO), replicated in subsequent studies\",\n      \"pmids\": [\"20018847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"UFBP1 (C20orf116) and CDK5RAP3 interact with UFM1. UFM1 co-localizes with UFBP1 at the endoplasmic reticulum, and this ER co-localization depends on UFBP1. siRNA knockdown of UFM1, UFBP1, or UFL1 enhances apoptosis upon ER stress in INS-1E beta cells, indicating that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, siRNA knockdown, apoptosis assays under ER stress\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interactions, functional knockdown with specific apoptosis readout, localization tied to function, replicated by multiple groups\",\n      \"pmids\": [\"21494687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Uba5 (UFM1 E1 activating enzyme) is indispensable for erythroid differentiation in mice. Uba5 knockout mice exhibit severe anaemia and die in utero. Genetic loss impairs development of megakaryocyte and erythroid progenitors from common myeloid progenitors in a cell-autonomous manner, as transgenic Uba5 expression restricted to erythroid lineage rescues the anaemia.\",\n      \"method\": \"Uba5 knockout mouse model, erythroid-specific transgenic rescue, hematopoietic progenitor analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with specific phenotype and rescue by lineage-specific transgene establishing cell-autonomous function\",\n      \"pmids\": [\"21304510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The UFM1 system is transcriptionally upregulated in response to ER stress and inhibition of vesicle trafficking. Using luciferase reporter and ChIP assays, UFM1 was identified as a transcriptional target of Xbp-1. Conversely, knockdown of the UFM1 system in U2OS cells triggered the unfolded protein response and amplification of the ER network. RCAD/Ufl1 and its binding partner C53/LZAP are involved in ufmylation of endogenous targets.\",\n      \"method\": \"Luciferase reporter assays, ChIP assays, Xbp-1 knockout MEFs, siRNA knockdown with UPR readouts\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assays establish Xbp-1 regulation; single lab, multiple methods\",\n      \"pmids\": [\"23152784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Ufmylation of the nuclear receptor coactivator ASC1 is required for ERα transactivation. In the absence of E2-estradiol, UfSP2 binds ASC1 and keeps it deufmylated. E2 binding causes ERα to associate with ASC1 and displace UfSP2, leading to polyufmylation of ASC1, which enhances association of p300, SRC1, and ASC1 at promoters of ERα target genes.\",\n      \"method\": \"Co-immunoprecipitation, ufmylation assays, ChIP, knockdown of UBA5 and UFSP2, ufmylation-deficient ASC1 mutant expression, in vivo tumor formation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, site-specific mutants, ChIP, and in vivo rescue experiments; multiple orthogonal methods\",\n      \"pmids\": [\"25219498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Uba5 activates UFM1 via a two-step mechanism forming a binary Uba5~UFM1 thioester (unlike the three-step ternary complex of Uba1/ubiquitin). Uba5 shows random ordered binding with UFM1 and ATP; its transthiolation of UFM1 to UFC1 requires ATP binding to the Uba5~UFM1 thioester complex. The pan-E1 inhibitor adenosine 5'-sulfamate forms a covalent Ufm1-ADS adduct in Uba5's active site.\",\n      \"method\": \"In vitro kinetic assays, thioester formation assays, ATP-PPi exchange assays, mechanism-based inhibitor studies in cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — detailed mechanistic in vitro assays with multiple orthogonal methods and cellular validation\",\n      \"pmids\": [\"24966333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RCAD/Ufl1 (UFM1 E3 ligase) is essential for hematopoietic stem cell survival and erythroid differentiation. Both germ-line and somatic deletion impaired hematopoietic development resulting in severe anaemia. Loss of RCAD/Ufl1 elevated ER stress, evoked UPR, blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response and p53 activation in HSCs.\",\n      \"method\": \"Germ-line and conditional knockout mouse models, bone marrow cell analysis, ER stress markers, autophagy assays, ROS measurement\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent genetic KO models with multiple mechanistic readouts, consistent with UFM1 E1 KO phenotype\",\n      \"pmids\": [\"25952549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"UFBP1 is indispensable for embryonic development and hematopoiesis. UFBP1 deficiency causes elevated ER stress and UPR activation leading to HSC/progenitor cell death, and suppresses expression of erythroid transcription factors GATA-1 and KLF1. ASC1 was found to associate with GATA-1 and Klf1 promoters in a UFBP1-dependent manner.\",\n      \"method\": \"Germ-line and conditional UFBP1 knockout mice, ChIP, K562 cell knockdowns, ER stress marker analysis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent KO models, ChIP establishing UFBP1-dependence of ASC1 promoter association, multiple orthogonal readouts\",\n      \"pmids\": [\"26544067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of the non-canonical homodimeric E1 UBA5 in complex with UFM1 reveals that UFM1 interacts with distinct sites in both subunits of the UBA5 dimer via a trans-binding mechanism. UFM1 transfer from UBA5 to UFC1 also occurs via a trans mechanism requiring UBA5 homodimerization. A C-terminal region outside the adenylation domain recruits UFM1 to the active site of the adjacent subunit.\",\n      \"method\": \"X-ray crystallography, biochemical assays of trans-binding mechanism, mutagenesis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with biochemical validation of trans-binding mechanism\",\n      \"pmids\": [\"27653677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A combined LIR/UFM1-interacting motif (LIR/UFIM) at the C-terminus of UBA5 mediates binding to both UFM1 and LC3/GABARAP. This motif is required for full biological activity of UBA5 and for effective transfer of UFM1 to UFC1 and downstream protein substrates both in vitro and in cells.\",\n      \"method\": \"NMR structure, mutational analysis, in vitro UFM1 transfer assays, cellular ufmylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural determination with functional mutagenesis, in vitro and cellular validation\",\n      \"pmids\": [\"26929408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Biallelic mutations in UBA5 impair ufmylation, resulting in abnormal endoplasmic reticulum structure. The UBA5 p.Ala371Thr variant is hypomorphic with attenuated ability to transfer activated UFM1 to UFC1 (shown by in vitro thioester formation assay). CNS-specific knockout of Ufm1 in mice causes neonatal death with microcephaly and neuronal apoptosis. In C. elegans, knockout of uba-5 alters cholinergic but not glutamatergic neurotransmission.\",\n      \"method\": \"In vitro thioester formation assay with mutant UBA5, patient fibroblast studies, Ufm1 CNS-specific knockout mice, C. elegans knockout, zebrafish uba5 silencing\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro biochemical assay of mutant protein, multiple model organism KO studies, independently replicated across two concurrent papers\",\n      \"pmids\": [\"27545674\", \"27545681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Biallelic mutations in UFM1 and UFC1 impair formation of UFM1-UBA5 and UFM1-UFC1 thioester intermediates, causing widespread reduction of cellular ufmylation. This establishes that mutations in the UFM1 modifier itself and its E2 enzyme cause severe early-onset encephalopathy with progressive microcephaly, demonstrating the essential role of ufmylation in human brain development.\",\n      \"method\": \"Thioester intermediate formation assays with mutant proteins, cellular ufmylation assays, clinical genetics\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct biochemical assay of thioester formation with disease mutations, replicated across multiple families\",\n      \"pmids\": [\"29868776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Ufl1 (UFM1 E3 ligase) deficiency in cardiac-specific knockout mice leads to age-dependent cardiomyopathy and heart failure, with excessive ER stress preceding and worsening with disease progression. Mechanistically, Ufl1 depletion impairs PERK (PKR-like ER-resident kinase) signaling and aggravates cardiomyocyte death after ER stress. Chemical ER chaperone tauroursodeoxycholic acid treatment alleviates the cardiac dysfunction.\",\n      \"method\": \"Cardiac-specific knockout mice, transcriptome analysis, ER stress markers, PERK signaling assays, pharmacological rescue\",\n      \"journal\": \"Circulation. Heart failure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with transcriptome and biochemical analysis, pharmacological rescue confirming ER stress mechanism\",\n      \"pmids\": [\"30354401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Trans-binding of UFM1 to UBA5 stabilizes the UBA5 homodimer, which in turn enhances ATP binding to UBA5. Dimerization of UBA5 is required for ATP binding; UFM1 binding promotes dimerization and thereby stimulates ATP binding necessary for UFM1 activation.\",\n      \"method\": \"Biochemical binding assays, dimerization analysis, ATP binding assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical assays establishing functional connection between UFM1 binding, dimerization, and ATP binding; single lab\",\n      \"pmids\": [\"29295865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The N-terminal extension present in the long isoform of UBA5 is directly involved in ATP binding and affects how the adenylation domain interacts with ATP, changing the stoichiometry from 1:2 to 1:1 ATP:UBA5. The N-terminus also significantly increases ATP affinity and stimulates UFM1 transfer from UBA5 to UFC1, though it is not directly involved in E2 binding.\",\n      \"method\": \"Crystal structures of UBA5 long isoform with ATP and UFM1, ATP binding assays, UFC1 transfer assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with functional biochemical assays; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"30412706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The UFM1 cascade controls cell cycle entry at the G2/M transition in Drosophila neuroblasts. Disruption of ufmylation increases the mitotic index and extends G2/M phase. Impaired E3 ligase Ufl1 function causes premature mitotic entry and failed cellularization. The UFM1 cascade alters the phosphorylation level of Tyr15 on CDK1 (pY15-CDK1), a negative regulator of the G2-to-M transition.\",\n      \"method\": \"Drosophila neuroblast genetic knockdown/knockout, live imaging, mitotic index analysis, phosphorylation assays for CDK1-pY15\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic disruption in Drosophila with live imaging and CDK1 phosphorylation as mechanistic readout; single lab\",\n      \"pmids\": [\"31914610\"],\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 observed in other E1-E2 complexes. A region of UBA5 outside the adenylation domain, dispensable for UFC1 binding, is critical for UFM1 transfer by compensating for a missing loop in UFC1's active site.\",\n      \"method\": \"X-ray crystallography of UFC1-UBA5 C-terminal peptide complex, mutational analysis of transfer\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis establishing mechanistic basis for UFM1 transfer\",\n      \"pmids\": [\"34588452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NADH-cytochrome b5 reductase 3 (CYB5R3) on the ER membrane is a UFM1 substrate. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1, converts CYB5R3 into its inactive form, and is recognized by UFBP1 through a UFM1-interacting motif on UFBP1. Ufmylated CYB5R3 is degraded in lysosomes in an Atg7- and CDK5RAP3-dependent manner, promoting ER-phagy. Ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly.\",\n      \"method\": \"Identification of ufmylation substrate, UFL1/UFBP1 dependency assays, in vitro ufmylation, lysosomal degradation assays, Atg7 dependency, Cyb5r3 ufmylation-defective knock-in mice\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including knock-in mouse model, substrate identification with mechanistic follow-up\",\n      \"pmids\": [\"36543799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ERα (estrogen receptor α) is directly ufmylated at Lys171 and Lys180. UFMylation of ERα increases its stability by inhibiting ubiquitination and proteasomal degradation. UfSP2 knockdown increases ERα stability, while UBA5 silencing decreases it. Ufmylation-deficient ERα (2KR mutant) shows reduced stability, abolished E2-induced transactivity, and cannot form anchorage-independent colonies.\",\n      \"method\": \"Site-specific mutagenesis (Lys to Arg), ubiquitination assays, stability assays with UBA5/UFSP2 manipulation, ERα target gene expression analysis, colony formation assays\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — site-specific mutants, multiple functional assays; builds on prior ASC1 ufmylation work from same group\",\n      \"pmids\": [\"35680375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UFSP1 is translated from a non-canonical start site and acts as an active protease that matures UFM1 (cleaves pro-UFM1) and cleaves a potential autoinhibitory modification on UFC1 to control activation of UFMylation. Cells lacking both UFSP1 and UFSP2 show complete loss of UFMylation due to absence of mature UFM1. UFSP2, but not UFSP1, removes UFM1 from ribosomal subunit RPL26, revealing distinct substrate specificities.\",\n      \"method\": \"UFSP1/UFSP2 double-knockout cells, identification of non-canonical start site, UFM1 maturation assays, UFC1 deufmylation assays, RPL26 deufmylation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — double KO cells with specific biochemical readouts, discovery of non-canonical start site, distinct substrate specificity determination\",\n      \"pmids\": [\"35926457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PLAC8 is post-translationally modified by UFM1 (ufmylated), and this modification maintains PLAC8 protein stability in triple-negative breast cancer cells. PLAC8 in turn regulates PD-L1 levels by affecting PD-L1 ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, ufmylation assays, protein stability assays\",\n      \"journal\": \"Journal for immunotherapy of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP and stability assays showing ufmylation of PLAC8; single lab, moderate mechanistic follow-up\",\n      \"pmids\": [\"36543379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UFL1 and CDK5RAP3/C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or CDK5RAP3 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation with γ-tubulin accumulation. CDK5RAP3, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in UFL1-lacking cells.\",\n      \"method\": \"Knockout of UFL1 and CDK5RAP3, immunofluorescence, γ-tubulin complex co-immunoprecipitation, microtubule nucleation assays\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO phenotype with biochemical interaction data; single lab, indirect link to UFM1 itself via UFL1\",\n      \"pmids\": [\"35159364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Neuron-specific deletion of either UFL1 or UFBP1 (UFM1 E3 ligase components) leads to significant neuronal loss in adult mice, elevated inflammatory response, and seizure-like events upon loss of one UFBP1 allele, demonstrating the indispensable role of the UFM1 E3 ligase in mature neuron survival.\",\n      \"method\": \"Neuron-specific conditional knockout mice for UFL1 and UFBP1, histological analysis, inflammatory marker measurement\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent conditional KO models with consistent neurodegeneration phenotype\",\n      \"pmids\": [\"35931931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The UFM1 E3 complex (UFL1-UFBP1-CDK5RAP3) interacts with UFC1 (E2), then CDK5RAP3 acts as an adaptor for ufmylation of ribosomal subunit RPL26. Upon disome formation (colliding ribosomes), the E3 complex associates with ufmylated RPL26 on the 60S subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of UFBP1-ufmylated RPL26 interaction impairs ER-ribosome quality control.\",\n      \"method\": \"Co-immunoprecipitation, ribosome pulldown, disome analysis, mutational disruption of UFBP1-UFM1 interaction, ER-RQC reporter assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple Co-IP and functional assays, mutational analysis of key interactions with ER-RQC readout\",\n      \"pmids\": [\"37595036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structures of the UFM1 E3 ligase complex (UREL, comprising UFL1-UFBP1-CDK5RAP3) bound to 60S ribosomes reveal a C-shaped clamp architecture. UFL1 loops insert into and remodel the peptidyl transferase center. UREL functions as both a 'writer' (catalyzing UFMylation of RPL26/uL24) and subsequently a 'reader' of ufmylated 60S. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating UFMylation is necessary for releasing SEC61 from 60S subunits.\",\n      \"method\": \"Cryo-EM structures, biochemical reconstitution, genetic loss-of-function accumulation of 60S-SEC61 complexes\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structures with functional validation; independently replicated in two concurrent Nature papers\",\n      \"pmids\": [\"38383789\", \"38383785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Sequential cryo-EM snapshots show the UFM1 E3 ligase (E3(UFM1)) engages its substrate uL24 (RPL26) on free 60S ribosomes. E3(UFM1) binds the L1 stalk, empty tRNA-binding sites, and the peptidyl transferase center through C-terminal domains of UFL1, catalyzing UFM1 transfer to uL24 more than 150 Å away. After catalysis, E3(UFM1) remains stably bound to ufmylated 60S, forming the C-shaped clamp, suggesting a role in post-termination recycling of the large ribosomal subunit from the ER membrane.\",\n      \"method\": \"Cryo-EM (sequential snapshots), biochemical analysis of E3 binding and catalysis, substrate engagement mapping\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple high-resolution cryo-EM structures with biochemical validation; independently replicated\",\n      \"pmids\": [\"38383785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Structural basis for UFM1 recognition by C. elegans UfSP: the structure reveals that the conserved Pro88-Val92 residues (P6-P2 positions from the cleavage site) plus extended β-structure at the UFM1 C-terminus are important for specific recognition of UFM1 by UfSP.\",\n      \"method\": \"X-ray crystallography of cUFM1-cUfSP complex\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of complex; single lab, C. elegans ortholog\",\n      \"pmids\": [\"29251776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In C. elegans, UfSP2 (odr-8) forms a physical complex with ODR-4 at the ER membrane in chemosensory neurons. This complex promotes GPCR (ODR-10) maturation and ER export independently of UfSP2's catalytic activity and independently of UFM1, since catalytically dead UfSP2 rescues all odr-8 mutant phenotypes and deletion of ufm-1 does not alter chemoreceptor trafficking.\",\n      \"method\": \"Genetic epistasis in C. elegans, co-immunoprecipitation of ODR-4/ODR-8, catalytic-dead mutant rescue, ufm-1 null mutant analysis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with Co-IP and catalytic mutant rescue; C. elegans ortholog, establishes protease-independent UfSP2 function\",\n      \"pmids\": [\"24603482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The NMR structure of UFC1 bound to a peptide spanning the last 20 residues of UBA5 reveals the molecular basis of UBA5-UFC1 interaction. These last 20 residues are pivotal for UFC1 binding and accelerate UFM1 transfer to UFC1; a proposed regulatory role involves the C-terminal unstructured region controlling cellular localization and interaction of ufmylation cascade elements.\",\n      \"method\": \"NMR structure of UFC1-UBA5 peptide complex, ITC binding assays, UFM1 transfer assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with ITC and functional transfer assays; single lab\",\n      \"pmids\": [\"34299007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The UFM1 pathway impacts ER-associated protein degradation (ERAD). A genome-wide CRISPR/Cas9 screen using US2-mediated HLA-I degradation as ERAD model identified the UFM1 pathway. Interference with the UFM1 pathway inhibits ER-to-cytosol dislocation of HLA-I specifically; no UFMylation of HLA-I was detected, suggesting UFM1 impacts ERAD indirectly. Ribosomal proteins are a major class of UFMylated proteins in US2-expressing cells.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen, mass spectrometry, HLA-I dislocation assays\",\n      \"journal\": \"Molecules (Basel, Switzerland)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen plus biochemical follow-up; mechanistic link is indirect\",\n      \"pmids\": [\"33430125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UfSP1 binds to the ubiquitin-associated (UBA) domain of p62/SQSTM1 and promotes the interaction between p62 and ubiquitinated proteins, thereby increasing p62 body formation. This function is independent of UfSP1's protease activity, as both catalytically active and inactive UfSP1 promote p62 body formation through the same mechanism.\",\n      \"method\": \"Proximity labeling (TurboID), co-immunoprecipitation, immunofluorescence co-localization, catalytic-dead mutant analysis\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity proteomics with Co-IP and catalytic dead mutant validation; single lab\",\n      \"pmids\": [\"37285312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UFM1 suppresses gastric cancer cell invasion by increasing ubiquitination of PDK1 (decreasing PDK1 protein level), thereby inhibiting AKT phosphorylation at Ser473 and the PI3K/AKT signaling pathway. The effect of UFM1 on cancer cell function depends on PDK1 expression.\",\n      \"method\": \"Co-immunoprecipitation, protein kinase array, stable UFM1 overexpression/knockdown, PDK1 ubiquitination assays, AKT phosphorylation analysis\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP and ubiquitination assays with genetic dependency analysis; single lab, mechanistic depth is partial\",\n      \"pmids\": [\"31533855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UFM1 loss in neurons causes ER stress, activation of the unfolded protein response (UPR), and reduced protein translation, leading to impaired neuron development and synapse function. The pathogenic UFM1-R81C variant partially rescues UFM1 loss phenotypes but displays distinct ER stress responses, suggesting it is not merely a loss-of-function variant. The UPR inhibitor Trazodone restores protein translation only in UFM1-R81C-expressing neurons, and increases synapse numbers in both UFM1-KO and R81C neurons.\",\n      \"method\": \"UFM1-deficient mouse neurons, UFM1-R81C knock-in neurons, UPR pathway analysis, protein translation assays, synapse counting, pharmacological rescue with Trazodone\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models with mechanistic biochemical readouts (ER stress, translation, UPR), pharmacological rescue\",\n      \"pmids\": [\"41731076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UFMylation promotes non-homologous end-joining (NHEJ) of DNA double-strand breaks. Ku70 is a UFMylation substrate; XRCC4 engages UFMylated Ku70 via non-canonical UFM1-binding regions to promote chromatin assembly of NHEJ factors. Perturbation of UFM1 signaling (via UFSP2 depletion or hypomorphic UBA5 allele in patient fibroblasts) impairs NHEJ.\",\n      \"method\": \"Photo-crosslinkable UFM1 probe, NMR, proximity-dependent proteomics, NHEJ assays, patient fibroblast analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel probe combined with NMR and proximity proteomics; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.06.16.659844\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UFMylation of 14-3-3ε at Lys50 and Lys215 promotes RIG-I/MAVS antiviral signaling. K50R/K215R mutations abolish UFMylation and reduce type I and III interferon induction following RIG-I activation. These mutations do not disrupt the 14-3-3ε-RIG-I interaction but paradoxically enhance RIG-I-MAVS interaction while reducing 14-3-3ε-MAVS interaction, indicating UFMylation controls MAVS signaling complex architecture.\",\n      \"method\": \"In vitro and cellular UFMylation assays, site-specific mutagenesis, co-immunoprecipitation, interferon induction assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutants with biochemical and functional assays; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.03.19.644084\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Loss of UFMylation reroutes glucose metabolism and promotes prostate cancer cell invasion. Phosphofructokinase (PFKAP) was identified as a UFMylation substrate. Loss of UFMylation reduces glycolytic flux and increases hexosamine biosynthesis, elevating protein glycosylation relevant to invasion.\",\n      \"method\": \"Biotin-based UFMylation proteomics, transcriptomics, metabolic flux analysis, pharmacological pathway inhibition\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — substrate identified by proteomics with metabolic readout; preprint, mechanistic depth of UFMylation on PFKAP function not fully established\",\n      \"pmids\": [\"bio_10.1101_2025.06.02.657324\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Canonical RQC factors associate with ribosomes stalled at the ER. Ribosome splitting is a prerequisite for UFMylation of RPL26. The UFM1 E3 ligase complex binds and UFMylates the 60S-peptidyl-tRNA complex; UFMylation of RPL26 persists without late RQC components (NEMF, LTN1). UFMylation and the canonical RQC pathway act in concert to facilitate clearance of arrested polypeptides at the ER.\",\n      \"method\": \"ER-targeted stalling reporters, ribosome fractionation, UFMylation and RQC factor dependency assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cellular reporters with biochemical validation; preprint, consistent with peer-reviewed cryo-EM structural work\",\n      \"pmids\": [\"bio_10.1101_2025.01.17.633636\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"UFM1 is a ubiquitin-like modifier that undergoes C-terminal processing by UFSPs (UFSP1/2) to expose a conserved Gly, then is activated by E1 enzyme UBA5 via a trans-binding, homodimer-dependent thioester mechanism, transferred to E2 enzyme UFC1, and conjugated to substrates by the trimeric E3 ligase complex (UFL1-UFBP1-CDK5RAP3); the best-characterized substrate is ribosomal protein RPL26 (uL24) on ER-associated 60S ribosomes, where the E3 complex wraps around 60S as a C-shaped clamp to remodel the peptidyl transferase center, catalyze UFMylation, and then remain bound to UFMylated 60S to release stalled 60S subunits from the SEC61 translocon—thereby coupling ribosome-associated quality control and ER-phagy to ER proteostasis—while additional substrates including ASC1, ERα, CYB5R3, PLAC8, PDK1, and 14-3-3ε link ufmylation to transcription, ER-phagy, antiviral immunity, and cancer cell biology.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UFM1 is a ubiquitin-fold modifier that is covalently conjugated to target proteins through a dedicated three-enzyme cascade analogous to ubiquitylation, coupling protein quality control at the endoplasmic reticulum to cellular proteostasis [#0]. Newly synthesized UFM1 is matured by the cysteine proteases UFSP1 and UFSP2, which cleave its C-terminal extension to expose the conjugation-essential Gly; these same proteases also reverse the modification by releasing UFM1 from substrates [#1, #23]. The exposed Gly is activated by the non-canonical homodimeric E1 enzyme UBA5 through an ATP-dependent thioester mechanism in which UFM1 binds in trans across the dimer interface and stabilizes the dimer to promote ATP loading, then is transthiolated to the E2 enzyme UFC1 [#0, #12, #17]. Conjugation to substrates is catalyzed by a trimeric E3 ligase complex of UFL1, UFBP1 (DDRGK1) and CDK5RAP3, which assembles at the ER membrane [#4, #5]. The best-characterized substrate is the 60S ribosomal protein RPL26 (uL24): the E3 complex engages stalled and split 60S subunits as a C-shaped clamp, inserting loops of UFL1 to remodel the peptidyl transferase center, catalyzing UFMylation of uL24 across a large distance, and then remaining bound to ufmylated 60S to release the SEC61 translocon and recycle the subunit\\u2014thereby integrating ER-associated ribosome quality control with ER-phagy [#27, #28, #29]. Beyond ribosome quality control, UFMylation modifies additional ER and signaling substrates including CYB5R3, where modification drives ER-phagy [#21], and the transcriptional regulators ASC1 and ER\\u03b1, linking ufmylation to estrogen-receptor target-gene activation and hematopoietic transcription [#8, #22, #11]. The pathway is transcriptionally induced as part of the ER stress response downstream of Xbp-1, and its disruption activates the unfolded protein response, impairs erythroid and hematopoietic development, and causes neuronal loss [#7, #6, #10, #26]. Biallelic mutations in UFM1, UBA5, and UFC1 that impair thioester intermediate formation cause severe early-onset encephalopathy with progressive microcephaly, establishing ufmylation as essential for human brain development [#14, #15].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established UFM1 as a bona fide ubiquitin-like modifier by demonstrating it operates through a three-enzyme conjugation cascade, answering whether this ubiquitin-fold protein is a functional modifier.\",\n      \"evidence\": \"In vitro reconstitution of thioester intermediates with UBA5/UFC1 and Gly mutagenesis\",\n      \"pmids\": [\"15071506\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological substrates or E3 ligase identified\", \"Cellular processes regulated by UFMylation unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified the deconjugating/maturing machinery, showing UFSP1 and UFSP2 both expose the conjugation Gly on pro-UFM1 and reverse the modification, establishing ufmylation as a reversible cycle.\",\n      \"evidence\": \"Cleavage assays with recombinant proteins, Cys\\u2192Ser active-site mutagenesis, and activity-based probe labeling\",\n      \"pmids\": [\"17182609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Distinct roles of UFSP1 vs UFSP2 not resolved\", \"Substrate specificity of deconjugation undefined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Structural and biochemical work defined the catalytic chemistry and UFM1-recognition basis of the proteases, explaining how UFSP enzymes engage the UFM1 C-terminus.\",\n      \"evidence\": \"X-ray crystallography of UfSP1, active-site mutagenesis, ITC and NMR binding mapping (mouse; C. elegans complex in #30)\",\n      \"pmids\": [\"18321862\", \"29251776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recognition of substrate-conjugated UFM1 versus free UFM1 not distinguished\", \"Regulation of protease activity in cells unaddressed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified UFL1 as the first UFM1 E3 ligase and its substrate UFBP1, locating the conjugation machinery at the ER and answering how UFM1 is transferred to substrates.\",\n      \"evidence\": \"Co-IP, in vitro conjugation acceleration, Uba5 knockout cells, and subcellular localization\",\n      \"pmids\": [\"20018847\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full E3 complex composition not yet defined\", \"Functional consequence of UFBP1 modification unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed the UFM1 system within the ER stress response by showing it is a transcriptional target of Xbp-1 and that its loss triggers the UPR, framing ufmylation as an ER proteostasis pathway.\",\n      \"evidence\": \"Luciferase reporters, ChIP, Xbp-1 knockout MEFs, and siRNA knockdown with UPR readouts\",\n      \"pmids\": [\"23152784\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular targets mediating UPR effects not identified\", \"Single-lab transcriptional regulation evidence\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated physiological essentiality of the pathway in hematopoiesis, linking E1 (UBA5), E3 (UFL1) and substrate-receptor (UFBP1) loss to ER stress, blocked differentiation, and HSC death.\",\n      \"evidence\": \"Germline and conditional knockout mice for Uba5, Ufl1, and UFBP1 with ER stress, autophagy, ROS, and ChIP analyses\",\n      \"pmids\": [\"21304510\", \"25952549\", \"26544067\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular substrates driving erythroid phenotypes not pinned to specific UFMylation events\", \"Whether phenotypes are conjugation-dependent in every case unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the unusual E1 mechanism, showing UBA5 is a homodimer that binds and transfers UFM1 in trans, with a dual LIR/UFIM motif linking activation to autophagy machinery.\",\n      \"evidence\": \"Crystal and NMR structures of UBA5\\u2013UFM1, mutagenesis, and in vitro/cellular transfer assays\",\n      \"pmids\": [\"27653677\", \"26929408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional significance of LC3/GABARAP binding to ufmylation outputs incomplete\", \"Regulation of dimerization in cells unaddressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established UFM1 ufmylation as essential for human development by linking biallelic UBA5 hypomorphic mutations to abnormal ER and CNS phenotypes across model organisms.\",\n      \"evidence\": \"In vitro thioester assay of mutant UBA5, patient fibroblasts, Ufm1 CNS-specific knockout mice, and C. elegans/zebrafish models\",\n      \"pmids\": [\"27545674\", \"27545681\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate(s) whose mis-ufmylation drives neuropathology not identified\", \"Tissue-selective vulnerability mechanism unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Confirmed UFM1 and UFC1 mutations cause severe encephalopathy by impairing thioester intermediate formation, cementing ufmylation as essential for brain development.\",\n      \"evidence\": \"Thioester formation assays with mutant proteins, cellular ufmylation assays, and clinical genetics\",\n      \"pmids\": [\"29868776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genotype\\u2013phenotype relationship not mechanistically explained at substrate level\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Refined the catalytic logic of UFM1 activation, showing UFM1 binding stabilizes the UBA5 dimer to enhance ATP binding and that an N-terminal extension tunes ATP stoichiometry and transfer.\",\n      \"evidence\": \"Biochemical binding/dimerization assays and crystal structures of the UBA5 long isoform with ATP\",\n      \"pmids\": [\"29295865\", \"30412706\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In-cell relevance of isoform-specific ATP handling untested\", \"Some claims rest on single-lab biochemistry\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended physiological roles to the heart and identified substrate-specificity logic in the protease layer, linking UFL1 loss to ER-stress cardiomyopathy and UFSP1/2 to differential UFM1 maturation and RPL26 deufmylation.\",\n      \"evidence\": \"Cardiac-specific Ufl1 knockout mice with PERK signaling analysis and pharmacological rescue; UFSP1/UFSP2 double-knockout cells\",\n      \"pmids\": [\"30354401\", \"35926457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cardiac substrates mediating ER stress not identified\", \"Regulation of UFSP1 non-canonical translation in vivo unaddressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the molecular basis of UFM1 transfer from E1 to E2, showing a short linear UBA5 C-terminal sequence binds UFC1 and a separate UBA5 region compensates for a missing UFC1 active-site loop.\",\n      \"evidence\": \"X-ray and NMR structures of UFC1\\u2013UBA5 peptide complexes with mutational transfer assays\",\n      \"pmids\": [\"34588452\", \"34299007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How transfer is coupled to E3 handoff not structurally resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified transcriptional substrates of ufmylation, showing UFMylation of ASC1 (and later ER\\u03b1) regulates estrogen-receptor target gene activation and stability, expanding ufmylation beyond the ER membrane.\",\n      \"evidence\": \"Co-IP, ufmylation assays, ChIP, site-specific Lys-to-Arg mutants, stability assays, and tumor formation assays\",\n      \"pmids\": [\"25219498\", \"35680375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How nuclear UFMylation is spatially coordinated with ER-localized E3 unclear\", \"Generality across nuclear receptors untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified ER-membrane substrates linking ufmylation to organelle turnover, showing CYB5R3 UFMylation inactivates it and targets it for lysosomal ER-phagy via UFBP1 recognition and CDK5RAP3.\",\n      \"evidence\": \"Substrate identification, UFL1/UFBP1 dependency, in vitro ufmylation, Atg7-dependent degradation, and ufmylation-defective Cyb5r3 knock-in mice\",\n      \"pmids\": [\"36543799\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity of CYB5R3 among ER substrates not explained\", \"Link between CYB5R3 ER-phagy and microcephaly phenotype mechanistic gap\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined the central ribosomal substrate pathway, showing the UFL1-UFBP1-CDK5RAP3 E3 complex UFMylates RPL26 on colliding/disome 60S and that UFBP1 reads ufmylated RPL26 to drive ER-ribosome quality control.\",\n      \"evidence\": \"Co-IP, ribosome pulldown, disome analysis, and mutational disruption of UFBP1\\u2013UFM1 interaction with ER-RQC reporters\",\n      \"pmids\": [\"37595036\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger coupling collision sensing to E3 recruitment incompletely defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided the structural mechanism of ribosomal UFMylation, showing the E3 complex clamps split 60S as a C-shaped writer/reader that remodels the PTC, UFMylates uL24 at long range, and releases SEC61 for subunit recycling.\",\n      \"evidence\": \"Cryo-EM structures of UREL\\u201360S complexes and sequential snapshots with biochemical validation and 60S-SEC61 accumulation upon loss of function\",\n      \"pmids\": [\"38383789\", \"38383785\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the reader function feeds into downstream ER-phagy/recycling steps not fully traced\", \"Regulation of writer-to-reader transition unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected ufmylation to additional substrates and processes\\u2014DNA double-strand break repair (Ku70/NHEJ), antiviral RIG-I/MAVS signaling (14-3-3\\u03b5), and cancer metabolism\\u2014and dissected neuronal UPR consequences of UFM1 loss with pharmacological rescue.\",\n      \"evidence\": \"UFM1-deficient and UFM1-R81C knock-in neurons with UPR/translation/synapse readouts and Trazodone rescue; preprint probe/proteomics studies of Ku70, 14-3-3\\u03b5, and metabolic substrates\",\n      \"pmids\": [\"41731076\", \"bio_10.1101_2025.06.16.659844\", \"bio_10.1101_2025.03.19.644084\", \"bio_10.1101_2025.06.02.657324\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Several substrates rest on single-lab preprint evidence awaiting peer review\", \"Whether non-ribosomal substrates are direct E3 targets in vivo not fully established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How substrate selection is partitioned between the dominant ribosomal UFMylation pathway and the growing list of non-ribosomal substrates, and which substrates underlie the neurodevelopmental and hematopoietic disease phenotypes, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model for E3 substrate targeting outside the ribosome\", \"Causal substrate(s) for human encephalopathy not identified\", \"Cell-type-specific vulnerability mechanisms undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [0, 4, 8, 21, 22]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 27, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [4, 5, 21, 27, 28]},\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [23, 27, 28, 29]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 11, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 27, 28]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [27, 28, 29]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [5, 7, 16, 36]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"complexes\": [\n      \"UFM1 E3 ligase complex (UFL1-UFBP1-CDK5RAP3)\"\n    ],\n    \"partners\": [\n      \"UBA5\",\n      \"UFC1\",\n      \"UFL1\",\n      \"UFBP1\",\n      \"CDK5RAP3\",\n      \"UFSP2\",\n      \"UFSP1\",\n      \"RPL26\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}