{"gene":"UFSP2","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2006,"finding":"UFSP2 (and UFSP1) are novel thiol proteases that specifically process the C-terminal extension of UFM1 but not ubiquitin, SUMO-1, or ISG15. The active-site Cys residue is essential: replacement of conserved Cys with Ser abolishes activity, and UFSP2 can be covalently labeled with UFM1-vinylmethylester. Both enzymes are sensitive to sulfhydryl-blocking agents (e.g., N-ethylmaleimide) and can release UFM1 from UFM1-conjugated cellular proteins.","method":"In vitro protease activity assays with recombinant protein, active-site Cys→Ser mutagenesis, covalent labeling with UFM1-vinylmethylester, inhibitor (NEM) sensitivity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis and chemical probe labeling, foundational paper replicated across multiple subsequent studies","pmids":["17182609"],"is_preprint":false},{"year":2008,"finding":"The crystal structure of mouse UfSP1 at 1.7 Å reveals a novel cysteine protease with a papain-like fold; the catalytic triad is Cys53, Asp175, His177, with Tyr41 forming the oxyanion hole. The Asp-Pro-His configuration defines a new subfamily of cysteine proteases. Mutagenesis of active-site residues confirms their catalytic roles. ITC shows UFM1 binds UfSP1 with KD ≈ 1.6 μM; NMR shows the β3–α2 loop and C-terminal region of UFM1 are involved in binding.","method":"X-ray crystallography (1.7 Å), active-site mutagenesis, isothermal titration calorimetry, NMR","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, ITC binding measurement, NMR epitope mapping, mutagenesis validation, multiple orthogonal methods in one study","pmids":["18321862"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of mouse UfSP2 at 2.6 Å reveals two domains: a C-terminal catalytic domain (cysteine protease with Cys294, Asp418, His420, Tyr282, and a regulatory loop) and a novel N-terminal domain. The N-terminal domain is required for recognition of the cellular substrate C20orf116 (DDRGK1/UFBP1) and for recruitment of UfSP2 to the endoplasmic reticulum where C20orf116 localizes. The BHD-associated mutation Y290H (equivalent position) abolishes catalytic activity, providing structural basis for disease.","method":"X-ray crystallography (2.6 Å), active-site mutagenesis, subcellular localization experiments, domain deletion/mutation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and functional localization data, multiple orthogonal methods in one study","pmids":["21228277"],"is_preprint":false},{"year":2014,"finding":"In the absence of estradiol (E2), UFSP2 binds the nuclear receptor coactivator ASC1 and maintains it in a non-UFMylated state. E2 induces ERα binding to ASC1, which displaces UFSP2, allowing polyufmylation of ASC1. UFSP2 knockdown promotes ERα-mediated tumor formation in vivo, an effect abrogatable by tamoxifen.","method":"Co-immunoprecipitation, knockdown (siRNA/shRNA), in vivo tumor formation assays, promoter ChIP, reconstitution experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo tumor model, multiple orthogonal functional assays, widely replicated concept","pmids":["25219498"],"is_preprint":false},{"year":2014,"finding":"In C. elegans, ODR-8/UfSP2 (the ortholog of UFSP2) and ODR-4 physically interact at the ER membrane in chemosensory neurons and together promote GPCR (ODR-10) maturation/export from the ER. This function is independent of UfSP2 protease activity: catalytically dead ODR-8/UfSP2 mutants rescue all odr-8 phenotypes. Human ODR4 and UFSP2 also physically interact, suggesting evolutionary conservation. Deletion of C. elegans ufm-1 does not affect chemoreceptor trafficking, demonstrating a UFM1-independent role.","method":"Genetic epistasis (C. elegans mutants), co-immunoprecipitation (human proteins), rescue experiments with catalytic mutants, GPCR localization assays","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in C. elegans, catalytic-dead rescue, human Co-IP, UFM1-deletion negative control, multiple orthogonal methods","pmids":["24603482"],"is_preprint":false},{"year":2015,"finding":"A UFSP2 missense mutation p.Tyr290His (c.868T>C) segregates with Beukes hip dysplasia in all 17 affected family members. In vitro functional assays with purified recombinant wild-type and mutant UFSP2 demonstrate that the Y290H substitution abolishes UFSP2-mediated C-terminal cleavage of UFM1, establishing loss of proteolytic function as the disease mechanism.","method":"In vitro protease activity assay with recombinant wild-type and p.Y290H UFSP2, linkage analysis, Sanger sequencing","journal":"South African medical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro reconstitution of catalytic activity with disease mutant vs. wild-type protein, LOD score 10.4 in full family","pmids":["26428751"],"is_preprint":false},{"year":2016,"finding":"In the absence of UFSP2, ectopic co-expression of E3 components UFL1 and UFBP1 dramatically increases UFM1-conjugate formation at the ER, establishing UFSP2 as the primary deconjugating enzyme controlling UFM1 modification levels at the ER.","method":"UFSP2 knockout/knockdown combined with UFL1+UFBP1 overexpression, immunoblotting for UFM1 conjugates, subcellular fractionation","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined biochemical readout and overexpression, single lab, two orthogonal approaches","pmids":["27926783"],"is_preprint":false},{"year":2016,"finding":"The C. elegans UfSP crystal structure reveals an N-terminal MPN domain (136 residues extra vs. UfSP2) that lacks metalloprotease activity but is required for recognition and deufmylation of the substrate UfBP1, and also for ER localization of cUfSP.","method":"X-ray crystallography, domain deletion mutagenesis, substrate deufmylation assay, subcellular localization","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure, mutagenesis with functional deufmylation assay and localization, single lab but multiple orthogonal methods","pmids":["27240952"],"is_preprint":false},{"year":2018,"finding":"A novel UFSP2 heterozygous missense mutation p.D426A (c.1277A>C) causes spondyloepimetaphyseal dysplasia. Asp426 is a catalytic residue in the active site, and the mutation is predicted (and consistent with prior in vitro data for related mutations) to inactivate UFSP2 proteolytic activity, extending the genotype-phenotype relationship to different skeletal dysplasia presentations.","method":"Exome sequencing, structural mapping of mutant to catalytic site, comparison to prior in vitro functional data","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — structural/computational mapping with reference to prior reconstitution data; no new in vitro assay performed in this paper","pmids":["28892125"],"is_preprint":false},{"year":2021,"finding":"A homozygous UFSP2 variant p.V115E reduces UFSP2 protein abundance and increases UFMylated targets in patient-derived fibroblasts, indicating impaired de-UFMylation. Reconstitution of patient-derived fibroblasts with wild-type UFSP2 reduces UFMylation marks. Structural analysis shows V115 localizes to the N-terminal domain, distinct from catalytic-domain mutations causing skeletal dysplasia.","method":"Immunoblotting of patient fibroblasts, wild-type UFSP2 reconstitution rescue experiment, structural domain mapping","journal":"Genetics in medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rescue reconstitution experiment in patient cells with defined biochemical readout, single lab","pmids":["33473208"],"is_preprint":false},{"year":2022,"finding":"Human UFSP2, but not UFSP1, specifically removes UFM1 from the ribosomal subunit RPL26. UFSP2 is also the primary protease for UFM1 maturation, though UFSP1 (translated from a non-canonical start site) acts earlier in the pathway. Cells lacking both UFSPs show complete loss of UFMylation due to absence of mature UFM1.","method":"UFSP1/UFSP2 double-knockout cells, immunoblotting for UFM1 maturation and RPL26 UFMylation, reconstitution experiments, biochemical fractionation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR knockout of individual and combined UFSPs with specific substrate readouts and reconstitution, multiple orthogonal methods","pmids":["35926457"],"is_preprint":false},{"year":2022,"finding":"UFMylation of ERα at Lys171 and Lys180 promotes ERα stability by inhibiting its ubiquitination; UFSP2 knockdown (which elevates UFMylation) dramatically increases ERα stability, while the ufmylation-deficient ERα 2KR mutant is destabilized and shows abrogated E2-induced transactivity and downstream gene expression.","method":"UFSP2 knockdown, ERα K171R/K180R mutagenesis, ubiquitination assay, luciferase transactivation assay, colony formation","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis of UFMylation sites with multiple functional readouts, single lab","pmids":["35680375"],"is_preprint":false},{"year":2022,"finding":"A UFM1 α-chloroacetyl activity-based probe selectively and covalently modifies the active-site cysteine of UFSP2 in cellulo, demonstrating that UFSP2 is the dominant active de-UFMylase accessible in living cells.","method":"Chemical biology/activity-based protein profiling with UFM1 electrophilic probes in cell lysates and living cells","journal":"ACS central science","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — covalent active-site labeling in cellulo with high selectivity demonstrated, single lab","pmids":["35756382"],"is_preprint":false},{"year":2022,"finding":"UfSP2 binds to the MRN complex in the absence of DNA double-strand breaks (DSBs). After irradiation, ATM phosphorylates UfSP2, causing its dissociation from MRN. WIP1 phosphatase removes this phosphorylation, allowing UfSP2 recruitment to DSBs where it deufmylates histone H4 and suppresses ATM activation in a negative-feedback manner.","method":"Co-immunoprecipitation, irradiation-induced foci formation assays, phosphorylation assays, WIP1 inhibition/knockdown, ATM kinase assay","journal":"Genome instability & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, kinase/phosphatase functional dissection, single lab","pmids":["36042814"],"is_preprint":false},{"year":2023,"finding":"PD-L1 is a UFMylation substrate; UFMylation destabilizes PD-L1 by synergizing with its ubiquitination. UFSP2 inhibition (via a covalent inhibitor) promotes PD-L1 UFMylation and enhances anti-tumor immunity in combination with PD-1 blockade.","method":"UFMylation assay, PD-L1 stability assays, UFSP2 covalent inhibitor, in vitro and in vivo immune killing assays, immunoprecipitation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — UFMylation-defective mutants, UFSP2 inhibitor, in vivo tumor immune assays, single lab","pmids":["36893266"],"is_preprint":false},{"year":2024,"finding":"Overexpression of UFSP2 (de-UFMylase) protects nascent DNA strands from degradation and confers resistance to PARP inhibitors in BRCA1/2-deficient cells, by reversing PTIP UFMylation at K148 that would otherwise promote MRE11 recruitment and fork degradation.","method":"UFSP2 overexpression, fork degradation assay (DNA fiber), PARP inhibitor resistance assay, genetic epistasis with UFL1 knockdown","journal":"Nature chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — specific UFSP2 overexpression phenotype in defined genetic background with mechanistic epistasis, single study","pmids":["38649452"],"is_preprint":false},{"year":2024,"finding":"In UFSP2 knockout neurons, the DNA damage response and unfolded protein response are perturbed, linking UFSP2-mediated de-UFMylation to these cellular stress pathways.","method":"CRISPR-Cas9 UFSP2 knockout in gene-edited neurons, functional assays for DNA damage response and UPR","journal":"Molecular neurodegeneration","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO in relevant cell type with defined stress pathway readouts, single lab","pmids":["39696466"],"is_preprint":false},{"year":2026,"finding":"UFSP2 interacts with the ER-anchored protein C1orf27, which recruits UFSP2 to the ER. UFSP2 protease activity is dispensable for ER recruitment but required for ER export of GPCRs. UFSP2, C1orf27, and cargo GPCRs form a multi-protein complex at the ER, and GPCR interaction with C1orf27 is required for ER export.","method":"Co-immunoprecipitation, catalytic mutant rescue experiments, GPCR ER-export assays, structural analysis, subcellular localization","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, catalytic dead mutant analysis, functional ER export assays, single lab","pmids":["42088365"],"is_preprint":false},{"year":2026,"finding":"UFSP2 deficiency leads to accumulation of UFMylated mitochondrial ribosome, ETC, and pyruvate dehydrogenase (PDH) complex components. DLAT (E2 subunit of PDH) is a direct UFMylation substrate at K118; K118R mutation abolishes DLAT UFMylation and reduces pyruvate oxidation, establishing that UFMylation activates PDH and that UFSP2 negatively regulates this modification.","method":"Quantitative proteomics in UFSP2-deficient cells, site-directed mutagenesis (K118R), PDH enzymatic activity assay, glucose oxidation/TCA flux assays, mitochondrial respiration (Seahorse)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro/cell-based reconstitution with site mutagenesis and multiple functional metabolic assays; preprint, single lab","pmids":["41890053"],"is_preprint":true},{"year":2026,"finding":"Cryo-EM analysis and biochemical assays with synthetic ufmylated histones (H4K31UFM1) show that neither UFSP1 nor UFSP2 can efficiently cleave H4 UFMylation at K31 in the nucleosome context, likely due to steric hindrance imposed by the nucleosome around the isopeptide bond.","method":"Cryo-EM structure of H4K31UFM1-nucleosome, in vitro deufmylation assay with recombinant UFSP1 and UFSP2, chemoenzymatic synthesis of ufmylated histones","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-EM structural data with in vitro enzyme activity assay, single lab; finding is a negative result (UFSP2 cannot cleave H4K31UFM1 in nucleosome)","pmids":["41964563"],"is_preprint":false},{"year":2025,"finding":"UFM1 signaling promotes NHEJ by UFMylating Ku70; XRCC4 engages UFMylated Ku70 via a non-canonical UFM1-binding region (identified by NMR and photo-crosslinkable UFM1 probe) to promote chromatin assembly of NHEJ factors. UFSP2 depletion (which elevates UFMylation) was used to demonstrate that perturbation of UFM1 signaling impairs these DSB-repair processes.","method":"Proximity-dependent proteomics, photo-crosslinkable UFM1 probe, NMR, UFSP2 depletion, NHEJ reporter assay, patient-derived fibroblasts","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — structural NMR + chemical probe + proteomics + functional NHEJ assay; preprint, single study","pmids":["bio_10.1101_2025.06.16.659844"],"is_preprint":true}],"current_model":"UFSP2 is a cysteine protease (catalytic triad Cys294/Asp418/His420 in the C-terminal domain, ER-recruitment N-terminal domain) that specifically processes pro-UFM1 to its mature form and removes UFM1 from conjugated substrates including RPL26, ASC1, ERα, PD-L1, histone H4, and DLAT; its N-terminal domain recruits it to the ER via interactions with C20orf116/UFBP1 and C1orf27, where it also participates (protease-independently) in GPCR biogenesis, and at DNA double-strand breaks it is recruited in an ATM-phosphorylation/WIP1-regulated manner to deufmylate H4 and suppress ATM activation, with loss-of-function mutations causing skeletal dysplasias (Beukes hip dysplasia, SEMD) and neurodevelopmental disorders depending on which domain is disrupted."},"narrative":{"mechanistic_narrative":"UFSP2 is a papain-like cysteine protease that serves as the principal de-UFMylating enzyme of the cell, both maturing pro-UFM1 and removing UFM1 from conjugated substrates [PMID:17182609, PMID:35926457]. It is a thiol protease that specifically processes the UFM1 C-terminal extension—and not ubiquitin, SUMO-1, or ISG15—through an active-site cysteine that can be covalently trapped with UFM1-based probes [PMID:17182609, PMID:35756382]. Structurally, the enzyme comprises a C-terminal catalytic domain bearing a Cys294/Asp418/His420 triad and oxyanion-hole tyrosine, plus a novel N-terminal domain that recognizes the ER-resident substrate DDRGK1/UFBP1 (C20orf116) and recruits the enzyme to the endoplasmic reticulum, where it counteracts the UFL1/UFBP1 E3 machinery to set steady-state UFM1-conjugate levels [PMID:21228277, PMID:27926783]. Through this deconjugating activity UFSP2 regulates the stability and activity of diverse substrates, including the ribosomal protein RPL26 [PMID:35926457], the coactivator ASC1 and estrogen receptor ERα [PMID:25219498, PMID:35680375], PD-L1 [PMID:36893266], and the pyruvate dehydrogenase E2 subunit DLAT at K118 [PMID:41890053]. UFSP2 also functions at sites of DNA damage: it is recruited to double-strand breaks under ATM-phosphorylation and WIP1-phosphatase control to deufmylate histone H4 and suppress ATM activation, and its de-UFMylase activity modulates replication-fork protection and PARP-inhibitor sensitivity in BRCA1/2-deficient cells [PMID:36042814, PMID:38649452]. Separately from proteolysis, UFSP2 acts catalytically-independently in GPCR biogenesis, forming an ER complex with ODR4 and C1orf27 that promotes receptor export from the ER [PMID:24603482, PMID:42088365]. Loss-of-function mutations cause distinct human disorders depending on the affected domain: catalytic-domain mutations (p.Y290H, p.D426A) cause skeletal dysplasias including Beukes hip dysplasia and spondyloepimetaphyseal dysplasia, whereas an N-terminal-domain variant (p.V115E) underlies a neurodevelopmental presentation [PMID:26428751, PMID:28892125, PMID:33473208].","teleology":[{"year":2006,"claim":"Established that UFSP2 is a dedicated protease for the UFM1 modifier system, distinguishing it from ubiquitin/SUMO/ISG15 proteases and defining its catalytic chemistry.","evidence":"In vitro protease assays with recombinant protein, active-site Cys→Ser mutagenesis, UFM1-vinylmethylester covalent labeling, and NEM inhibitor sensitivity","pmids":["17182609"],"confidence":"High","gaps":["Cellular substrates beyond bulk UFM1 conjugates not yet identified","Domain organization and localization not resolved"]},{"year":2008,"claim":"Solved the catalytic fold and UFM1-binding mode for the UfSP family, defining a novel papain-like cysteine protease subfamily with an Asp-Pro-His configuration.","evidence":"X-ray crystallography of mouse UfSP1 at 1.7 Å, active-site mutagenesis, ITC and NMR epitope mapping of UFM1 binding","pmids":["18321862"],"confidence":"High","gaps":["Structure is of UfSP1, not UFSP2","Does not address the second domain and substrate recognition specific to UFSP2"]},{"year":2011,"claim":"Showed that UFSP2's two-domain architecture couples catalysis to localization—the N-terminal domain recognizes DDRGK1/UFBP1 and targets the enzyme to the ER—and provided a structural basis for disease mutation Y290H.","evidence":"X-ray crystallography of mouse UfSP2 at 2.6 Å with domain deletion/mutation and subcellular localization analysis","pmids":["21228277"],"confidence":"High","gaps":["How the N-terminal domain engages the broader ER E3 machinery not detailed","Full substrate range unknown"]},{"year":2014,"claim":"Identified physiological de-UFMylation substrates and a catalysis-independent function, showing UFSP2 controls ERα/ASC1 coactivation and that the ortholog mediates GPCR ER export without protease activity.","evidence":"Co-IP, knockdown, in vivo tumor models, and ChIP (ASC1/ERα); C. elegans genetic epistasis with catalytic-dead rescue and human Co-IP (ODR-4/GPCR maturation)","pmids":["25219498","24603482"],"confidence":"High","gaps":["Mechanism by which catalytic-dead UFSP2 supports GPCR export unresolved","Generality of UFM1-independent role across receptor families unknown"]},{"year":2016,"claim":"Positioned UFSP2 as the dominant ER de-conjugase opposing the UFL1/UFBP1 E3, and clarified that the N-terminal MPN-like domain is the substrate/ER-targeting module.","evidence":"UFSP2 KO/knockdown with UFL1+UFBP1 overexpression and immunoblotting; C. elegans UfSP crystal structure with domain deletion and deufmylation/localization assays","pmids":["27926783","27240952"],"confidence":"Medium","gaps":["Quantitative balance between conjugation and deconjugation in vivo not measured","MPN domain lacks metalloprotease activity; its precise recognition determinants unclear"]},{"year":2018,"claim":"Extended the genotype-phenotype map by linking another catalytic-residue mutation (D426A) to a distinct skeletal dysplasia.","evidence":"Exome sequencing with structural mapping of the variant to the active site and comparison to prior in vitro data","pmids":["28892125"],"confidence":"Medium","gaps":["No new in vitro activity assay performed for D426A","Tissue-specific basis of skeletal phenotype unexplained"]},{"year":2021,"claim":"Showed that disrupting the N-terminal domain (V115E) impairs de-UFMylation through reduced protein abundance and produces a phenotype distinct from catalytic-domain skeletal dysplasias.","evidence":"Patient-fibroblast immunoblotting, wild-type UFSP2 reconstitution rescue, and structural domain mapping","pmids":["33473208"],"confidence":"Medium","gaps":["Whether V115E destabilizes folding or only impairs localization not distinguished","Single lab/single family"]},{"year":2022,"claim":"Defined UFSP2's substrate selectivity and central role: it specifically deufmylates RPL26, is the dominant active de-UFMylase in living cells, and is required (with UFSP1) for UFM1 maturation.","evidence":"UFSP1/UFSP2 double-knockout cells with substrate-specific immunoblotting and reconstitution; UFM1 α-chloroacetyl activity-based probe labeling in cellulo","pmids":["35926457","35756382"],"confidence":"High","gaps":["Division of labor between UFSP1 and UFSP2 in maturation incompletely mapped","Substrate-specificity determinants for RPL26 vs other targets unknown"]},{"year":2022,"claim":"Demonstrated that UFSP2 tunes substrate stability by removing UFM1 marks that antagonize ubiquitination, exemplified by ERα stabilization and transactivity.","evidence":"UFSP2 knockdown, ERα K171R/K180R mutagenesis, ubiquitination, luciferase transactivation, and colony-formation assays","pmids":["35680375"],"confidence":"Medium","gaps":["Crosstalk geometry between UFMylation and ubiquitination sites not structurally resolved","Single lab"]},{"year":2022,"claim":"Established a regulated DNA-damage function: UFSP2 is recruited to double-strand breaks under ATM/WIP1 phosphorylation control to deufmylate histone H4 and dampen ATM activation.","evidence":"Reciprocal Co-IP with MRN, irradiation-induced foci, phosphorylation assays, WIP1 perturbation, and ATM kinase assays","pmids":["36042814"],"confidence":"Medium","gaps":["Direct H4 deufmylation in chromatin context not structurally validated","Single lab"]},{"year":2023,"claim":"Connected UFSP2 to immune evasion by showing its inhibition raises PD-L1 UFMylation, destabilizing PD-L1 and enhancing anti-tumor immunity.","evidence":"UFMylation and stability assays, UFSP2 covalent inhibitor, and in vitro/in vivo immune-killing assays with PD-1 blockade","pmids":["36893266"],"confidence":"Medium","gaps":["Whether UFSP2 directly deufmylates PD-L1 vs acts on the pathway not pinned down","Single lab"]},{"year":2024,"claim":"Linked UFSP2 de-UFMylase activity to replication-fork protection and PARP-inhibitor resistance via reversal of PTIP UFMylation, and to stress-pathway integrity in neurons.","evidence":"UFSP2 overexpression with DNA fiber fork-degradation and PARPi resistance assays plus UFL1-knockdown epistasis; CRISPR UFSP2 KO neurons with DDR and UPR readouts","pmids":["38649452","39696466"],"confidence":"Medium","gaps":["Direct PTIP deufmylation by UFSP2 not biochemically reconstituted","Neuronal stress-pathway link is phenotypic, not mechanistic"]},{"year":2026,"claim":"Resolved the catalysis-independent ER role mechanistically and revealed a mitochondrial-metabolic axis, identifying C1orf27 as an ER recruiter for GPCR export and DLAT-K118 as a UFMylation substrate controlling PDH activity.","evidence":"Co-IP and catalytic-mutant rescue with GPCR ER-export assays (C1orf27); quantitative proteomics, K118R mutagenesis, PDH activity and Seahorse assays in UFSP2-deficient cells (DLAT, preprint)","pmids":["42088365","41890053"],"confidence":"Medium","gaps":["DLAT findings are from a preprint, single lab","How UFSP2 partitions between ER, nucleus, and mitochondrial-substrate compartments is unclear"]},{"year":2026,"claim":"Defined a structural limit of UFSP2 activity: it cannot efficiently cleave H4K31-UFM1 within the nucleosome due to steric occlusion of the isopeptide bond.","evidence":"Cryo-EM of H4K31UFM1-nucleosome with in vitro deufmylation assays using chemoenzymatically synthesized ufmylated histones","pmids":["41964563"],"confidence":"Medium","gaps":["Reconciliation with prior H4 deufmylation at DSBs unresolved","Whether accessory factors relieve steric hindrance in cells unknown"]},{"year":null,"claim":"How UFSP2 is spatially and temporally partitioned among its ER, DNA-damage, and metabolic functions—and what determines substrate choice and the switch between catalytic and non-catalytic roles—remains unresolved.","evidence":"No single study integrates the localization, recruitment, and substrate-selection logic across compartments","pmids":[],"confidence":"Medium","gaps":["No unified model of compartment-specific recruitment","Substrate-specificity code for the protease undefined","Mechanism of catalysis-independent GPCR function not structurally explained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,10]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,10]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2,6,17]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[13]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[18]}],"pathway":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0]}],"complexes":["MRN complex (transient interaction)","UFSP2–C1orf27–GPCR ER export complex"],"partners":["DDRGK1","UFL1","ODR4","C1ORF27","ASC1","RPL26"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NUQ7","full_name":"Ufm1-specific protease 2","aliases":[],"length_aa":469,"mass_kda":53.3,"function":"Thiol-dependent isopeptidase that specifically cleaves UFM1, a ubiquitin-like modifier protein, from conjugated proteins, such as CD274/PD-L1, CYB5R3, DDRGK1, MRE11, RPL26/uL24, TRIP4 and RPL26/uL24 (PubMed:25219498, PubMed:27351204, PubMed:27926783, PubMed:30783677, PubMed:31595041, PubMed:32160526, PubMed:33473208, PubMed:35394863, PubMed:35926457, PubMed:36543799, PubMed:36893266, PubMed:37795761, PubMed:38383785). While it is also able to mediate the processing of UFM1 precursors, a prerequisite for conjugation reactions, UFSP2 mainly acts as a protein deUFMylase that mediates deconjugation of UFM1 from target proteins (PubMed:27926783). Mediates deUFMylation of RPL26/uL24, a critical step to release the UFM1 ribosome E3 ligase (UREL) complex during the recycling of 60S ribosome subunits from the endoplasmic reticulum (PubMed:38383785). Catalyzes deUFMylation of TRIP4, regulating intracellular nuclear receptors transactivation and thereby regulate cell proliferation and differentiation (PubMed:25219498)","subcellular_location":"Endoplasmic reticulum; Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NUQ7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UFSP2","classification":"Not Classified","n_dependent_lines":132,"n_total_lines":1208,"dependency_fraction":0.10927152317880795},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SEC61B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/UFSP2","total_profiled":1310},"omim":[{"mim_id":"620266","title":"RAB7A-INTERACTING MON1-CCZ1 COMPLEX SUBUNIT 1; RIMOC1","url":"https://www.omim.org/entry/620266"},{"mim_id":"620028","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 106; DEE106","url":"https://www.omim.org/entry/620028"},{"mim_id":"617974","title":"SPONDYLOEPIMETAPHYSEAL DYSPLASIA, DI ROCCO TYPE; SEMDDR","url":"https://www.omim.org/entry/617974"},{"mim_id":"611482","title":"UFM1-SPECIFIC PEPTIDASE 2; UFSP2","url":"https://www.omim.org/entry/611482"},{"mim_id":"611481","title":"UFM1-SPECIFIC PEPTIDASE 1; UFSP1","url":"https://www.omim.org/entry/611481"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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The active-site Cys residue is essential: replacement of conserved Cys with Ser abolishes activity, and UFSP2 can be covalently labeled with UFM1-vinylmethylester. Both enzymes are sensitive to sulfhydryl-blocking agents (e.g., N-ethylmaleimide) and can release UFM1 from UFM1-conjugated cellular proteins.\",\n      \"method\": \"In vitro protease activity assays with recombinant protein, active-site Cys→Ser mutagenesis, covalent labeling with UFM1-vinylmethylester, inhibitor (NEM) sensitivity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis and chemical probe labeling, foundational paper replicated across multiple subsequent studies\",\n      \"pmids\": [\"17182609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The crystal structure of mouse UfSP1 at 1.7 Å reveals a novel cysteine protease with a papain-like fold; the catalytic triad is Cys53, Asp175, His177, with Tyr41 forming the oxyanion hole. The Asp-Pro-His configuration defines a new subfamily of cysteine proteases. Mutagenesis of active-site residues confirms their catalytic roles. ITC shows UFM1 binds UfSP1 with KD ≈ 1.6 μM; NMR shows the β3–α2 loop and C-terminal region of UFM1 are involved in binding.\",\n      \"method\": \"X-ray crystallography (1.7 Å), active-site mutagenesis, isothermal titration calorimetry, NMR\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, ITC binding measurement, NMR epitope mapping, mutagenesis validation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"18321862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of mouse UfSP2 at 2.6 Å reveals two domains: a C-terminal catalytic domain (cysteine protease with Cys294, Asp418, His420, Tyr282, and a regulatory loop) and a novel N-terminal domain. The N-terminal domain is required for recognition of the cellular substrate C20orf116 (DDRGK1/UFBP1) and for recruitment of UfSP2 to the endoplasmic reticulum where C20orf116 localizes. The BHD-associated mutation Y290H (equivalent position) abolishes catalytic activity, providing structural basis for disease.\",\n      \"method\": \"X-ray crystallography (2.6 Å), active-site mutagenesis, subcellular localization experiments, domain deletion/mutation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and functional localization data, multiple orthogonal methods in one study\",\n      \"pmids\": [\"21228277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In the absence of estradiol (E2), UFSP2 binds the nuclear receptor coactivator ASC1 and maintains it in a non-UFMylated state. E2 induces ERα binding to ASC1, which displaces UFSP2, allowing polyufmylation of ASC1. UFSP2 knockdown promotes ERα-mediated tumor formation in vivo, an effect abrogatable by tamoxifen.\",\n      \"method\": \"Co-immunoprecipitation, knockdown (siRNA/shRNA), in vivo tumor formation assays, promoter ChIP, reconstitution experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo tumor model, multiple orthogonal functional assays, widely replicated concept\",\n      \"pmids\": [\"25219498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In C. elegans, ODR-8/UfSP2 (the ortholog of UFSP2) and ODR-4 physically interact at the ER membrane in chemosensory neurons and together promote GPCR (ODR-10) maturation/export from the ER. This function is independent of UfSP2 protease activity: catalytically dead ODR-8/UfSP2 mutants rescue all odr-8 phenotypes. Human ODR4 and UFSP2 also physically interact, suggesting evolutionary conservation. Deletion of C. elegans ufm-1 does not affect chemoreceptor trafficking, demonstrating a UFM1-independent role.\",\n      \"method\": \"Genetic epistasis (C. elegans mutants), co-immunoprecipitation (human proteins), rescue experiments with catalytic mutants, GPCR localization assays\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in C. elegans, catalytic-dead rescue, human Co-IP, UFM1-deletion negative control, multiple orthogonal methods\",\n      \"pmids\": [\"24603482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A UFSP2 missense mutation p.Tyr290His (c.868T>C) segregates with Beukes hip dysplasia in all 17 affected family members. In vitro functional assays with purified recombinant wild-type and mutant UFSP2 demonstrate that the Y290H substitution abolishes UFSP2-mediated C-terminal cleavage of UFM1, establishing loss of proteolytic function as the disease mechanism.\",\n      \"method\": \"In vitro protease activity assay with recombinant wild-type and p.Y290H UFSP2, linkage analysis, Sanger sequencing\",\n      \"journal\": \"South African medical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro reconstitution of catalytic activity with disease mutant vs. wild-type protein, LOD score 10.4 in full family\",\n      \"pmids\": [\"26428751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In the absence of UFSP2, ectopic co-expression of E3 components UFL1 and UFBP1 dramatically increases UFM1-conjugate formation at the ER, establishing UFSP2 as the primary deconjugating enzyme controlling UFM1 modification levels at the ER.\",\n      \"method\": \"UFSP2 knockout/knockdown combined with UFL1+UFBP1 overexpression, immunoblotting for UFM1 conjugates, subcellular fractionation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined biochemical readout and overexpression, single lab, two orthogonal approaches\",\n      \"pmids\": [\"27926783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The C. elegans UfSP crystal structure reveals an N-terminal MPN domain (136 residues extra vs. UfSP2) that lacks metalloprotease activity but is required for recognition and deufmylation of the substrate UfBP1, and also for ER localization of cUfSP.\",\n      \"method\": \"X-ray crystallography, domain deletion mutagenesis, substrate deufmylation assay, subcellular localization\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure, mutagenesis with functional deufmylation assay and localization, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"27240952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A novel UFSP2 heterozygous missense mutation p.D426A (c.1277A>C) causes spondyloepimetaphyseal dysplasia. Asp426 is a catalytic residue in the active site, and the mutation is predicted (and consistent with prior in vitro data for related mutations) to inactivate UFSP2 proteolytic activity, extending the genotype-phenotype relationship to different skeletal dysplasia presentations.\",\n      \"method\": \"Exome sequencing, structural mapping of mutant to catalytic site, comparison to prior in vitro functional data\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — structural/computational mapping with reference to prior reconstitution data; no new in vitro assay performed in this paper\",\n      \"pmids\": [\"28892125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A homozygous UFSP2 variant p.V115E reduces UFSP2 protein abundance and increases UFMylated targets in patient-derived fibroblasts, indicating impaired de-UFMylation. Reconstitution of patient-derived fibroblasts with wild-type UFSP2 reduces UFMylation marks. Structural analysis shows V115 localizes to the N-terminal domain, distinct from catalytic-domain mutations causing skeletal dysplasia.\",\n      \"method\": \"Immunoblotting of patient fibroblasts, wild-type UFSP2 reconstitution rescue experiment, structural domain mapping\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rescue reconstitution experiment in patient cells with defined biochemical readout, single lab\",\n      \"pmids\": [\"33473208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Human UFSP2, but not UFSP1, specifically removes UFM1 from the ribosomal subunit RPL26. UFSP2 is also the primary protease for UFM1 maturation, though UFSP1 (translated from a non-canonical start site) acts earlier in the pathway. Cells lacking both UFSPs show complete loss of UFMylation due to absence of mature UFM1.\",\n      \"method\": \"UFSP1/UFSP2 double-knockout cells, immunoblotting for UFM1 maturation and RPL26 UFMylation, reconstitution experiments, biochemical fractionation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR knockout of individual and combined UFSPs with specific substrate readouts and reconstitution, multiple orthogonal methods\",\n      \"pmids\": [\"35926457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UFMylation of ERα at Lys171 and Lys180 promotes ERα stability by inhibiting its ubiquitination; UFSP2 knockdown (which elevates UFMylation) dramatically increases ERα stability, while the ufmylation-deficient ERα 2KR mutant is destabilized and shows abrogated E2-induced transactivity and downstream gene expression.\",\n      \"method\": \"UFSP2 knockdown, ERα K171R/K180R mutagenesis, ubiquitination assay, luciferase transactivation assay, colony formation\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis of UFMylation sites with multiple functional readouts, single lab\",\n      \"pmids\": [\"35680375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A UFM1 α-chloroacetyl activity-based probe selectively and covalently modifies the active-site cysteine of UFSP2 in cellulo, demonstrating that UFSP2 is the dominant active de-UFMylase accessible in living cells.\",\n      \"method\": \"Chemical biology/activity-based protein profiling with UFM1 electrophilic probes in cell lysates and living cells\",\n      \"journal\": \"ACS central science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — covalent active-site labeling in cellulo with high selectivity demonstrated, single lab\",\n      \"pmids\": [\"35756382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UfSP2 binds to the MRN complex in the absence of DNA double-strand breaks (DSBs). After irradiation, ATM phosphorylates UfSP2, causing its dissociation from MRN. WIP1 phosphatase removes this phosphorylation, allowing UfSP2 recruitment to DSBs where it deufmylates histone H4 and suppresses ATM activation in a negative-feedback manner.\",\n      \"method\": \"Co-immunoprecipitation, irradiation-induced foci formation assays, phosphorylation assays, WIP1 inhibition/knockdown, ATM kinase assay\",\n      \"journal\": \"Genome instability & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, kinase/phosphatase functional dissection, single lab\",\n      \"pmids\": [\"36042814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PD-L1 is a UFMylation substrate; UFMylation destabilizes PD-L1 by synergizing with its ubiquitination. UFSP2 inhibition (via a covalent inhibitor) promotes PD-L1 UFMylation and enhances anti-tumor immunity in combination with PD-1 blockade.\",\n      \"method\": \"UFMylation assay, PD-L1 stability assays, UFSP2 covalent inhibitor, in vitro and in vivo immune killing assays, immunoprecipitation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — UFMylation-defective mutants, UFSP2 inhibitor, in vivo tumor immune assays, single lab\",\n      \"pmids\": [\"36893266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Overexpression of UFSP2 (de-UFMylase) protects nascent DNA strands from degradation and confers resistance to PARP inhibitors in BRCA1/2-deficient cells, by reversing PTIP UFMylation at K148 that would otherwise promote MRE11 recruitment and fork degradation.\",\n      \"method\": \"UFSP2 overexpression, fork degradation assay (DNA fiber), PARP inhibitor resistance assay, genetic epistasis with UFL1 knockdown\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific UFSP2 overexpression phenotype in defined genetic background with mechanistic epistasis, single study\",\n      \"pmids\": [\"38649452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In UFSP2 knockout neurons, the DNA damage response and unfolded protein response are perturbed, linking UFSP2-mediated de-UFMylation to these cellular stress pathways.\",\n      \"method\": \"CRISPR-Cas9 UFSP2 knockout in gene-edited neurons, functional assays for DNA damage response and UPR\",\n      \"journal\": \"Molecular neurodegeneration\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO in relevant cell type with defined stress pathway readouts, single lab\",\n      \"pmids\": [\"39696466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"UFSP2 interacts with the ER-anchored protein C1orf27, which recruits UFSP2 to the ER. UFSP2 protease activity is dispensable for ER recruitment but required for ER export of GPCRs. UFSP2, C1orf27, and cargo GPCRs form a multi-protein complex at the ER, and GPCR interaction with C1orf27 is required for ER export.\",\n      \"method\": \"Co-immunoprecipitation, catalytic mutant rescue experiments, GPCR ER-export assays, structural analysis, subcellular localization\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, catalytic dead mutant analysis, functional ER export assays, single lab\",\n      \"pmids\": [\"42088365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"UFSP2 deficiency leads to accumulation of UFMylated mitochondrial ribosome, ETC, and pyruvate dehydrogenase (PDH) complex components. DLAT (E2 subunit of PDH) is a direct UFMylation substrate at K118; K118R mutation abolishes DLAT UFMylation and reduces pyruvate oxidation, establishing that UFMylation activates PDH and that UFSP2 negatively regulates this modification.\",\n      \"method\": \"Quantitative proteomics in UFSP2-deficient cells, site-directed mutagenesis (K118R), PDH enzymatic activity assay, glucose oxidation/TCA flux assays, mitochondrial respiration (Seahorse)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro/cell-based reconstitution with site mutagenesis and multiple functional metabolic assays; preprint, single lab\",\n      \"pmids\": [\"41890053\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Cryo-EM analysis and biochemical assays with synthetic ufmylated histones (H4K31UFM1) show that neither UFSP1 nor UFSP2 can efficiently cleave H4 UFMylation at K31 in the nucleosome context, likely due to steric hindrance imposed by the nucleosome around the isopeptide bond.\",\n      \"method\": \"Cryo-EM structure of H4K31UFM1-nucleosome, in vitro deufmylation assay with recombinant UFSP1 and UFSP2, chemoenzymatic synthesis of ufmylated histones\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structural data with in vitro enzyme activity assay, single lab; finding is a negative result (UFSP2 cannot cleave H4K31UFM1 in nucleosome)\",\n      \"pmids\": [\"41964563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UFM1 signaling promotes NHEJ by UFMylating Ku70; XRCC4 engages UFMylated Ku70 via a non-canonical UFM1-binding region (identified by NMR and photo-crosslinkable UFM1 probe) to promote chromatin assembly of NHEJ factors. UFSP2 depletion (which elevates UFMylation) was used to demonstrate that perturbation of UFM1 signaling impairs these DSB-repair processes.\",\n      \"method\": \"Proximity-dependent proteomics, photo-crosslinkable UFM1 probe, NMR, UFSP2 depletion, NHEJ reporter assay, patient-derived fibroblasts\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural NMR + chemical probe + proteomics + functional NHEJ assay; preprint, single study\",\n      \"pmids\": [\"bio_10.1101_2025.06.16.659844\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"UFSP2 is a cysteine protease (catalytic triad Cys294/Asp418/His420 in the C-terminal domain, ER-recruitment N-terminal domain) that specifically processes pro-UFM1 to its mature form and removes UFM1 from conjugated substrates including RPL26, ASC1, ERα, PD-L1, histone H4, and DLAT; its N-terminal domain recruits it to the ER via interactions with C20orf116/UFBP1 and C1orf27, where it also participates (protease-independently) in GPCR biogenesis, and at DNA double-strand breaks it is recruited in an ATM-phosphorylation/WIP1-regulated manner to deufmylate H4 and suppress ATM activation, with loss-of-function mutations causing skeletal dysplasias (Beukes hip dysplasia, SEMD) and neurodevelopmental disorders depending on which domain is disrupted.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UFSP2 is a papain-like cysteine protease that serves as the principal de-UFMylating enzyme of the cell, both maturing pro-UFM1 and removing UFM1 from conjugated substrates [#0, #10]. It is a thiol protease that specifically processes the UFM1 C-terminal extension—and not ubiquitin, SUMO-1, or ISG15—through an active-site cysteine that can be covalently trapped with UFM1-based probes [#0, #12]. Structurally, the enzyme comprises a C-terminal catalytic domain bearing a Cys294/Asp418/His420 triad and oxyanion-hole tyrosine, plus a novel N-terminal domain that recognizes the ER-resident substrate DDRGK1/UFBP1 (C20orf116) and recruits the enzyme to the endoplasmic reticulum, where it counteracts the UFL1/UFBP1 E3 machinery to set steady-state UFM1-conjugate levels [#2, #6]. Through this deconjugating activity UFSP2 regulates the stability and activity of diverse substrates, including the ribosomal protein RPL26 [#10], the coactivator ASC1 and estrogen receptor ERα [#3, #11], PD-L1 [#14], and the pyruvate dehydrogenase E2 subunit DLAT at K118 [#18]. UFSP2 also functions at sites of DNA damage: it is recruited to double-strand breaks under ATM-phosphorylation and WIP1-phosphatase control to deufmylate histone H4 and suppress ATM activation, and its de-UFMylase activity modulates replication-fork protection and PARP-inhibitor sensitivity in BRCA1/2-deficient cells [#13, #15]. Separately from proteolysis, UFSP2 acts catalytically-independently in GPCR biogenesis, forming an ER complex with ODR4 and C1orf27 that promotes receptor export from the ER [#4, #17]. Loss-of-function mutations cause distinct human disorders depending on the affected domain: catalytic-domain mutations (p.Y290H, p.D426A) cause skeletal dysplasias including Beukes hip dysplasia and spondyloepimetaphyseal dysplasia, whereas an N-terminal-domain variant (p.V115E) underlies a neurodevelopmental presentation [#5, #8, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that UFSP2 is a dedicated protease for the UFM1 modifier system, distinguishing it from ubiquitin/SUMO/ISG15 proteases and defining its catalytic chemistry.\",\n      \"evidence\": \"In vitro protease assays with recombinant protein, active-site Cys\\u2192Ser mutagenesis, UFM1-vinylmethylester covalent labeling, and NEM inhibitor sensitivity\",\n      \"pmids\": [\"17182609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular substrates beyond bulk UFM1 conjugates not yet identified\", \"Domain organization and localization not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Solved the catalytic fold and UFM1-binding mode for the UfSP family, defining a novel papain-like cysteine protease subfamily with an Asp-Pro-His configuration.\",\n      \"evidence\": \"X-ray crystallography of mouse UfSP1 at 1.7 \\u00c5, active-site mutagenesis, ITC and NMR epitope mapping of UFM1 binding\",\n      \"pmids\": [\"18321862\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure is of UfSP1, not UFSP2\", \"Does not address the second domain and substrate recognition specific to UFSP2\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed that UFSP2's two-domain architecture couples catalysis to localization—the N-terminal domain recognizes DDRGK1/UFBP1 and targets the enzyme to the ER—and provided a structural basis for disease mutation Y290H.\",\n      \"evidence\": \"X-ray crystallography of mouse UfSP2 at 2.6 \\u00c5 with domain deletion/mutation and subcellular localization analysis\",\n      \"pmids\": [\"21228277\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the N-terminal domain engages the broader ER E3 machinery not detailed\", \"Full substrate range unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified physiological de-UFMylation substrates and a catalysis-independent function, showing UFSP2 controls ERα/ASC1 coactivation and that the ortholog mediates GPCR ER export without protease activity.\",\n      \"evidence\": \"Co-IP, knockdown, in vivo tumor models, and ChIP (ASC1/ER\\u03b1); C. elegans genetic epistasis with catalytic-dead rescue and human Co-IP (ODR-4/GPCR maturation)\",\n      \"pmids\": [\"25219498\", \"24603482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which catalytic-dead UFSP2 supports GPCR export unresolved\", \"Generality of UFM1-independent role across receptor families unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Positioned UFSP2 as the dominant ER de-conjugase opposing the UFL1/UFBP1 E3, and clarified that the N-terminal MPN-like domain is the substrate/ER-targeting module.\",\n      \"evidence\": \"UFSP2 KO/knockdown with UFL1+UFBP1 overexpression and immunoblotting; C. elegans UfSP crystal structure with domain deletion and deufmylation/localization assays\",\n      \"pmids\": [\"27926783\", \"27240952\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative balance between conjugation and deconjugation in vivo not measured\", \"MPN domain lacks metalloprotease activity; its precise recognition determinants unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended the genotype-phenotype map by linking another catalytic-residue mutation (D426A) to a distinct skeletal dysplasia.\",\n      \"evidence\": \"Exome sequencing with structural mapping of the variant to the active site and comparison to prior in vitro data\",\n      \"pmids\": [\"28892125\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No new in vitro activity assay performed for D426A\", \"Tissue-specific basis of skeletal phenotype unexplained\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed that disrupting the N-terminal domain (V115E) impairs de-UFMylation through reduced protein abundance and produces a phenotype distinct from catalytic-domain skeletal dysplasias.\",\n      \"evidence\": \"Patient-fibroblast immunoblotting, wild-type UFSP2 reconstitution rescue, and structural domain mapping\",\n      \"pmids\": [\"33473208\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether V115E destabilizes folding or only impairs localization not distinguished\", \"Single lab/single family\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined UFSP2's substrate selectivity and central role: it specifically deufmylates RPL26, is the dominant active de-UFMylase in living cells, and is required (with UFSP1) for UFM1 maturation.\",\n      \"evidence\": \"UFSP1/UFSP2 double-knockout cells with substrate-specific immunoblotting and reconstitution; UFM1 \\u03b1-chloroacetyl activity-based probe labeling in cellulo\",\n      \"pmids\": [\"35926457\", \"35756382\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Division of labor between UFSP1 and UFSP2 in maturation incompletely mapped\", \"Substrate-specificity determinants for RPL26 vs other targets unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that UFSP2 tunes substrate stability by removing UFM1 marks that antagonize ubiquitination, exemplified by ERα stabilization and transactivity.\",\n      \"evidence\": \"UFSP2 knockdown, ER\\u03b1 K171R/K180R mutagenesis, ubiquitination, luciferase transactivation, and colony-formation assays\",\n      \"pmids\": [\"35680375\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Crosstalk geometry between UFMylation and ubiquitination sites not structurally resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established a regulated DNA-damage function: UFSP2 is recruited to double-strand breaks under ATM/WIP1 phosphorylation control to deufmylate histone H4 and dampen ATM activation.\",\n      \"evidence\": \"Reciprocal Co-IP with MRN, irradiation-induced foci, phosphorylation assays, WIP1 perturbation, and ATM kinase assays\",\n      \"pmids\": [\"36042814\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct H4 deufmylation in chromatin context not structurally validated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected UFSP2 to immune evasion by showing its inhibition raises PD-L1 UFMylation, destabilizing PD-L1 and enhancing anti-tumor immunity.\",\n      \"evidence\": \"UFMylation and stability assays, UFSP2 covalent inhibitor, and in vitro/in vivo immune-killing assays with PD-1 blockade\",\n      \"pmids\": [\"36893266\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether UFSP2 directly deufmylates PD-L1 vs acts on the pathway not pinned down\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked UFSP2 de-UFMylase activity to replication-fork protection and PARP-inhibitor resistance via reversal of PTIP UFMylation, and to stress-pathway integrity in neurons.\",\n      \"evidence\": \"UFSP2 overexpression with DNA fiber fork-degradation and PARPi resistance assays plus UFL1-knockdown epistasis; CRISPR UFSP2 KO neurons with DDR and UPR readouts\",\n      \"pmids\": [\"38649452\", \"39696466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PTIP deufmylation by UFSP2 not biochemically reconstituted\", \"Neuronal stress-pathway link is phenotypic, not mechanistic\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Resolved the catalysis-independent ER role mechanistically and revealed a mitochondrial-metabolic axis, identifying C1orf27 as an ER recruiter for GPCR export and DLAT-K118 as a UFMylation substrate controlling PDH activity.\",\n      \"evidence\": \"Co-IP and catalytic-mutant rescue with GPCR ER-export assays (C1orf27); quantitative proteomics, K118R mutagenesis, PDH activity and Seahorse assays in UFSP2-deficient cells (DLAT, preprint)\",\n      \"pmids\": [\"42088365\", \"41890053\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DLAT findings are from a preprint, single lab\", \"How UFSP2 partitions between ER, nucleus, and mitochondrial-substrate compartments is unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined a structural limit of UFSP2 activity: it cannot efficiently cleave H4K31-UFM1 within the nucleosome due to steric occlusion of the isopeptide bond.\",\n      \"evidence\": \"Cryo-EM of H4K31UFM1-nucleosome with in vitro deufmylation assays using chemoenzymatically synthesized ufmylated histones\",\n      \"pmids\": [\"41964563\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with prior H4 deufmylation at DSBs unresolved\", \"Whether accessory factors relieve steric hindrance in cells unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UFSP2 is spatially and temporally partitioned among its ER, DNA-damage, and metabolic functions—and what determines substrate choice and the switch between catalytic and non-catalytic roles—remains unresolved.\",\n      \"evidence\": \"No single study integrates the localization, recruitment, and substrate-selection logic across compartments\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of compartment-specific recruitment\", \"Substrate-specificity code for the protease undefined\", \"Mechanism of catalysis-independent GPCR function not structurally explained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 10]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2, 6, 17]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"MRN complex (transient interaction)\", \"UFSP2\\u2013C1orf27\\u2013GPCR ER export complex\"],\n    \"partners\": [\"DDRGK1\", \"UFL1\", \"ODR4\", \"C1orf27\", \"ASC1\", \"RPL26\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}