{"gene":"UFSP1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2006,"finding":"UfSP1 (mouse) is a novel thiol protease that specifically cleaves the C-terminal extension of Ufm1 to generate mature Ufm1, and also releases Ufm1 from Ufm1-conjugated cellular proteins; it does not act on ubiquitin, SUMO-1, or ISG15. The active-site Cys residue is essential for catalysis, as its replacement by Ser abolishes activity, and the enzyme is sensitive to sulfhydryl-blocking agents (N-ethylmaleimide). The active-site Cys can be covalently labeled with Ufm1-vinylmethylester.","method":"In vitro cleavage assays with Ufm1 and other Ubl substrates; active-site Cys→Ser mutagenesis; inhibition by N-ethylmaleimide; covalent labeling with Ufm1-vinylmethylester (activity-based probe)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biochemical methods (in vitro assay, active-site mutagenesis, covalent probe labeling, inhibitor sensitivity), replicated for both proteases in same study","pmids":["17182609"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of mouse UfSP1 (1.7 Å) reveals a novel cysteine protease with a papain-like fold. The catalytic triad consists of Cys53, Asp175, and His177, arranged in an 'Asp-Pro-His' box configuration, with Tyr41 forming the oxyanion hole. Mutagenesis of active-site residues confirms their catalytic role. Isothermal titration calorimetry shows UfSP1 binds Ufm1 with KD ~1.6 µM. NMR data indicate that the loop between β3 and α2, plus the C-terminal region of Ufm1, are involved in binding to UfSP1.","method":"X-ray crystallography (1.7 Å resolution); active-site mutagenesis; isothermal titration calorimetry; NMR binding analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis confirmation and orthogonal binding measurements (ITC, NMR) in a single rigorous study","pmids":["18321862"],"is_preprint":false},{"year":2022,"finding":"Human UFSP1 is translated from a non-canonical start site to yield a catalytically active protease. In cells lacking both UFSP1 and UFSP2, UFM1 maturation is completely abolished. UFSP1, but not UFSP2, is responsible for maturing UFM1 and for cleaving a potential autoinhibitory UFMylation on UFC1, thereby controlling activation of the UFMylation pathway. UFSP2, but not UFSP1, removes UFM1 from the ribosomal subunit RPL26. The two proteases thus have distinct substrate specificities and localization-dependent functions.","method":"Double UFSP1/UFSP2 knockout cells; non-canonical translation start site identification; biochemical assays for UFM1 maturation, UFC1 deufmylation, and RPL26 deufmylation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic double KO with defined biochemical readouts for distinct substrates, multiple orthogonal experiments","pmids":["35926457"],"is_preprint":false},{"year":2023,"finding":"UfSP1 interacts with the autophagy receptor p62/SQSTM1 via p62's ubiquitin-associated (UBA) domain, colocalizes with p62, and promotes p62-mediated protein aggregate (p62 body) formation by enhancing the interaction between p62 and ubiquitinated proteins. This function is independent of UfSP1's protease activity, as both catalytically active and inactive UfSP1 promote p62 body formation equivalently.","method":"Proximity labeling (BioID)/quantitative proteomics; co-immunoprecipitation; immunofluorescence colocalization; catalytic mutant analysis","journal":"Journal of proteome research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal Co-IP and functional colocalization with catalytic mutant comparison, single lab, multiple orthogonal methods","pmids":["37285312"],"is_preprint":false},{"year":2025,"finding":"Ufsp1 knockout and catalytic-dead point-mutant mice are viable and born in normal Mendelian ratios with no gross phenotypic abnormalities, indicating Ufsp1 is dispensable for normal mouse development and homeostasis. Despite enrichment of Ufsp1 at the neuromuscular junction (NMJ), only mild NMJ structural alterations were detected with no impact on overall muscle function.","method":"Knockout mouse generation; catalytic-dead point-mutant mouse; Mendelian ratio analysis; NMJ structural and functional assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two genetic mouse models with defined phenotypic readouts, single lab, negative/mild phenotype result","pmids":["40748989"],"is_preprint":false},{"year":2026,"finding":"Neither UfSP1 nor UfSP2 can efficiently cleave H4 ufmylation at Lys31 (H4K31UFM1) in the nucleosome context. Cryo-EM analysis of the H4K31UFM1-nucleosome suggests that steric hindrance around the isopeptide bond within the nucleosome may underlie the weak deufmylation activity of both UfSPs on nucleosomal substrates.","method":"Semisynthetic ufmylated histone preparation; in vitro deufmylation assays with synthetic substrates; cryo-EM structural analysis of H4K31UFM1-nucleosome","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — reconstituted in vitro assay with cryo-EM structural analysis, single lab, no replication","pmids":["41964563"],"is_preprint":false},{"year":2015,"finding":"UFSP1/UFSP2 double-knockout HEK293T cells accumulate elevated UFMylated proteins and were validated as a cellular model for identifying UFMylated substrates, confirming that both proteases are required for efficient deufmylation in human cells.","method":"UFSP1KO/UFSP2KO cell line generation; western blot for UFMylation levels; large-scale UFMylated substrate enrichment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic double KO with defined biochemical readout, single lab, single method for this specific finding","pmids":["41759738"],"is_preprint":false}],"current_model":"UFSP1 is a cysteine protease (catalytic triad Cys53/Asp175/His177 in the mouse ortholog) that is translated from a non-canonical start site in humans to yield an active enzyme; it specifically processes pro-UFM1 to expose its C-terminal glycine required for conjugation, cleaves a potential autoinhibitory UFMylation on the E2 enzyme UFC1 to activate the UFMylation pathway, and has a distinct substrate specificity from UFSP2 (which handles RPL26 deufmylation); additionally, UFSP1 has a protease-independent role in promoting p62-mediated protein aggregate formation by binding the UBA domain of p62 and enhancing its interaction with ubiquitinated proteins, while being dispensable for normal mouse development and neuromuscular junction homeostasis."},"narrative":{"mechanistic_narrative":"UFSP1 is a papain-like cysteine protease that regulates the UFM1 conjugation (UFMylation) system by controlling both maturation of the UFM1 modifier and recycling of UFMylated targets [PMID:17182609, PMID:18321862, PMID:35926457]. It specifically cleaves the C-terminal extension of pro-UFM1 to generate the mature form competent for conjugation and releases UFM1 from conjugated proteins, acting on UFM1 but not on ubiquitin, SUMO-1, or ISG15; catalysis depends on an active-site cysteine within an Asp175-Pro-His177 triad and a Tyr41 oxyanion hole, and the enzyme binds UFM1 with sub-micromolar affinity [PMID:17182609, PMID:18321862]. In human cells UFSP1 is produced from a non-canonical translation start site and is the principal protease for UFM1 maturation and for cleaving an autoinhibitory UFMylation on the E2 enzyme UFC1, thereby gating activation of the pathway; this is a division of labor distinct from UFSP2, which deUFMylates the ribosomal subunit RPL26, while both proteases together are required for efficient deUFMylation and neither efficiently cleaves UFM1 from histone H4K31 within the nucleosome owing to steric occlusion [PMID:35926457, PMID:41964563, PMID:41759738]. Independently of its catalytic activity, UFSP1 binds the UBA domain of the autophagy receptor p62/SQSTM1 and promotes p62-body formation by enhancing p62 engagement with ubiquitinated proteins [PMID:37285312]. Loss of Ufsp1 in mice is compatible with normal development and homeostasis, with only mild structural alterations at the neuromuscular junction where the protein is enriched [PMID:40748989].","teleology":[{"year":2006,"claim":"Established that UFSP1 is the dedicated protease for the UFM1 modifier, answering whether a specific deconjugase/maturase exists for this Ubl.","evidence":"In vitro cleavage assays against multiple Ubl substrates, active-site Cys→Ser mutagenesis, NEM inhibition, and Ufm1-vinylmethylester activity-based labeling (mouse)","pmids":["17182609"],"confidence":"High","gaps":["Did not define structural basis of UFM1 recognition","Cellular substrates and physiological role not addressed"]},{"year":2008,"claim":"Defined the catalytic architecture and UFM1-binding determinants, explaining how UFSP1 achieves its specificity as a novel papain-like protease.","evidence":"1.7 Å crystal structure of mouse UfSP1 with active-site mutagenesis, ITC binding measurement, and NMR mapping of the UFM1 interface","pmids":["18321862"],"confidence":"High","gaps":["Structure is of the apo/mouse enzyme without bound substrate complex","Human enzyme behavior not addressed"]},{"year":2022,"claim":"Resolved how active human UFSP1 is generated and partitioned the pathway, showing it matures UFM1 and de-represses UFC1 while UFSP2 handles RPL26.","evidence":"Non-canonical start-site identification plus UFSP1/UFSP2 double-knockout cells with biochemical readouts for UFM1 maturation, UFC1 deUFMylation, and RPL26 deUFMylation","pmids":["35926457"],"confidence":"High","gaps":["Mechanism of non-canonical translation regulation unresolved","Physiological consequences of UFC1 autoinhibition control not defined in vivo"]},{"year":2023,"claim":"Uncovered a protease-independent function in selective autophagy, addressing whether UFSP1 acts only as a deconjugase.","evidence":"BioID proximity proteomics, reciprocal Co-IP, immunofluorescence colocalization, and catalytic-mutant comparison for p62-body formation","pmids":["37285312"],"confidence":"Medium","gaps":["Single-lab finding","Structural basis of UBA-domain binding not defined","In vivo relevance of p62-body promotion unknown"]},{"year":2025,"claim":"Tested the organismal requirement for UFSP1, showing it is dispensable for mouse development despite NMJ enrichment.","evidence":"Ufsp1 knockout and catalytic-dead point-mutant mice with Mendelian ratio analysis and NMJ structural/functional assays","pmids":["40748989"],"confidence":"Medium","gaps":["Mild NMJ phenotype mechanism unexplained","Possible redundancy with UFSP2 not tested in vivo","Single lab, negative/mild result"]},{"year":2026,"claim":"Defined a substrate context limit, showing nucleosomal H4K31-UFM1 resists both UFSPs due to steric hindrance.","evidence":"Semisynthetic ufmylated histone deUFMylation assays and cryo-EM of the H4K31UFM1-nucleosome","pmids":["41964563"],"confidence":"Medium","gaps":["Whether another factor removes nucleosomal UFM1 is unknown","Single lab, no replication","In vivo relevance of persistent histone UFMylation not addressed"]},{"year":null,"claim":"How UFSP1's enzymatic and protease-independent (p62) activities are coordinated, and what its physiological substrate repertoire and regulation are in vivo, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No in vivo substrate catalog","Coordination of dual functions unresolved","Regulation of non-canonical translation not mechanistically defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[0]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[3]}],"complexes":[],"partners":["UFM1","UFC1","SQSTM1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6NVU6","full_name":"Ufm1-specific protease 1","aliases":[],"length_aa":218,"mass_kda":23.3,"function":"Thiol-dependent isopeptidase that specifically mediate the processing of UFM1 precursors as well as the deconjugation of UFM1 from target proteins (PubMed:35525273, PubMed:35926457). Mainly responsible for the maturation of the UFM1 precursor, a prerequisite for conjugation reactions (PubMed:35525273, PubMed:35926457)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q6NVU6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UFSP1","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/UFSP1","total_profiled":1310},"omim":[{"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":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UFSP1"},"hgnc":{"alias_symbol":["UFSP"],"prev_symbol":[]},"alphafold":{"accession":"Q6NVU6","domains":[{"cath_id":"3.90.70.130","chopping":"18-142","consensus_level":"high","plddt":91.6028,"start":18,"end":142}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6NVU6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6NVU6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6NVU6-F1-predicted_aligned_error_v6.png","plddt_mean":92.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UFSP1","jax_strain_url":"https://www.jax.org/strain/search?query=UFSP1"},"sequence":{"accession":"Q6NVU6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6NVU6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6NVU6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6NVU6"}},"corpus_meta":[{"pmid":"17182609","id":"PMC_17182609","title":"Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17182609","citation_count":172,"is_preprint":false},{"pmid":"21228277","id":"PMC_21228277","title":"Structure of ubiquitin-fold modifier 1-specific protease UfSP2.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21228277","citation_count":60,"is_preprint":false},{"pmid":"35926457","id":"PMC_35926457","title":"Human UFSP1 is an active protease that regulates UFM1 maturation and UFMylation.","date":"2022","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/35926457","citation_count":59,"is_preprint":false},{"pmid":"18321862","id":"PMC_18321862","title":"Structural basis for Ufm1 processing by UfSP1.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18321862","citation_count":50,"is_preprint":false},{"pmid":"27193597","id":"PMC_27193597","title":"Mapping adipose and muscle tissue expression quantitative trait loci in African Americans to identify genes for type 2 diabetes and obesity.","date":"2016","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/27193597","citation_count":38,"is_preprint":false},{"pmid":"25290169","id":"PMC_25290169","title":"Mallory-Denk Body (MDB) formation modulates Ufmylation expression epigenetically in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH).","date":"2014","source":"Experimental and molecular pathology","url":"https://pubmed.ncbi.nlm.nih.gov/25290169","citation_count":35,"is_preprint":false},{"pmid":"24587462","id":"PMC_24587462","title":"Deletion of ubiquitin fold modifier protein Ufm1 processing peptidase Ufsp in L. donovani abolishes Ufm1 processing and alters pathogenesis.","date":"2014","source":"PLoS neglected tropical diseases","url":"https://pubmed.ncbi.nlm.nih.gov/24587462","citation_count":17,"is_preprint":false},{"pmid":"29251776","id":"PMC_29251776","title":"Structural basis for Ufm1 recognition by UfSP.","date":"2018","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/29251776","citation_count":11,"is_preprint":false},{"pmid":"27240952","id":"PMC_27240952","title":"The MPN domain of Caenorhabditis elegans UfSP modulates both substrate recognition and deufmylation activity.","date":"2016","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/27240952","citation_count":10,"is_preprint":false},{"pmid":"37285312","id":"PMC_37285312","title":"Proximity Proteomics and Biochemical Analysis Reveal a Noncanonical Function for UFM1-Specific Protease 1 in the p62 Body Formation.","date":"2023","source":"Journal of proteome research","url":"https://pubmed.ncbi.nlm.nih.gov/37285312","citation_count":10,"is_preprint":false},{"pmid":"39858578","id":"PMC_39858578","title":"Systematic Analysis of UFMylation Family Genes in Tissues of Mice with Metabolic Dysfunction-Associated Steatotic Liver Disease.","date":"2024","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/39858578","citation_count":4,"is_preprint":false},{"pmid":"36037969","id":"PMC_36037969","title":"Nontraditional translation is the key to UFMylation and beyond.","date":"2022","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36037969","citation_count":4,"is_preprint":false},{"pmid":"37543930","id":"PMC_37543930","title":"Potential of Autologous Adipose-Derived Mesenchymal Stem Cells in Peritoneal Fibrosis: A Pilot Study.","date":"2023","source":"Archives of Iranian medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37543930","citation_count":4,"is_preprint":false},{"pmid":"40748989","id":"PMC_40748989","title":"Loss of Ufsp1 does not cause major changes at the neuromuscular junction.","date":"2025","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/40748989","citation_count":1,"is_preprint":false},{"pmid":"41759738","id":"PMC_41759738","title":"Optimization of protein UFMylation modification method and its application in substrate identification in human cells.","date":"2026","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41759738","citation_count":1,"is_preprint":false},{"pmid":"32744966","id":"PMC_32744966","title":"Leveraging on Active Site Similarities; Identification of Potential Inhibitors of Zinc-Finger and UFSP domain Protein (ZUFSP).","date":"2021","source":"Current pharmaceutical biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/32744966","citation_count":0,"is_preprint":false},{"pmid":"41823810","id":"PMC_41823810","title":"The UFM1 Conjugation System: A Master Regulator of Cellular Stress Surveillance in Human Disease.","date":"2026","source":"Biology","url":"https://pubmed.ncbi.nlm.nih.gov/41823810","citation_count":0,"is_preprint":false},{"pmid":"41964563","id":"PMC_41964563","title":"Expedient Chemical Synthesis of Ufmylated Histones Using Chemoenzymatic C-Terminal Hydrazinolysis of Ubiquitin-Fold Modifier 1.","date":"2026","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/41964563","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10911,"output_tokens":2202,"usd":0.032882,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9149,"output_tokens":2395,"usd":0.05281,"stage2_stop_reason":"end_turn"},"total_usd":0.085692,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"UfSP1 (mouse) is a novel thiol protease that specifically cleaves the C-terminal extension of Ufm1 to generate mature Ufm1, and also releases Ufm1 from Ufm1-conjugated cellular proteins; it does not act on ubiquitin, SUMO-1, or ISG15. The active-site Cys residue is essential for catalysis, as its replacement by Ser abolishes activity, and the enzyme is sensitive to sulfhydryl-blocking agents (N-ethylmaleimide). The active-site Cys can be covalently labeled with Ufm1-vinylmethylester.\",\n      \"method\": \"In vitro cleavage assays with Ufm1 and other Ubl substrates; active-site Cys→Ser mutagenesis; inhibition by N-ethylmaleimide; covalent labeling with Ufm1-vinylmethylester (activity-based probe)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biochemical methods (in vitro assay, active-site mutagenesis, covalent probe labeling, inhibitor sensitivity), replicated for both proteases in same study\",\n      \"pmids\": [\"17182609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of mouse UfSP1 (1.7 Å) reveals a novel cysteine protease with a papain-like fold. The catalytic triad consists of Cys53, Asp175, and His177, arranged in an 'Asp-Pro-His' box configuration, with Tyr41 forming the oxyanion hole. Mutagenesis of active-site residues confirms their catalytic role. Isothermal titration calorimetry shows UfSP1 binds Ufm1 with KD ~1.6 µM. NMR data indicate that the loop between β3 and α2, plus the C-terminal region of Ufm1, are involved in binding to UfSP1.\",\n      \"method\": \"X-ray crystallography (1.7 Å resolution); active-site mutagenesis; isothermal titration calorimetry; NMR binding analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis confirmation and orthogonal binding measurements (ITC, NMR) in a single rigorous study\",\n      \"pmids\": [\"18321862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Human UFSP1 is translated from a non-canonical start site to yield a catalytically active protease. In cells lacking both UFSP1 and UFSP2, UFM1 maturation is completely abolished. UFSP1, but not UFSP2, is responsible for maturing UFM1 and for cleaving a potential autoinhibitory UFMylation on UFC1, thereby controlling activation of the UFMylation pathway. UFSP2, but not UFSP1, removes UFM1 from the ribosomal subunit RPL26. The two proteases thus have distinct substrate specificities and localization-dependent functions.\",\n      \"method\": \"Double UFSP1/UFSP2 knockout cells; non-canonical translation start site identification; biochemical assays for UFM1 maturation, UFC1 deufmylation, and RPL26 deufmylation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic double KO with defined biochemical readouts for distinct substrates, multiple orthogonal experiments\",\n      \"pmids\": [\"35926457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UfSP1 interacts with the autophagy receptor p62/SQSTM1 via p62's ubiquitin-associated (UBA) domain, colocalizes with p62, and promotes p62-mediated protein aggregate (p62 body) formation by enhancing the interaction between p62 and ubiquitinated proteins. This function is independent of UfSP1's protease activity, as both catalytically active and inactive UfSP1 promote p62 body formation equivalently.\",\n      \"method\": \"Proximity labeling (BioID)/quantitative proteomics; co-immunoprecipitation; immunofluorescence colocalization; catalytic mutant analysis\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal Co-IP and functional colocalization with catalytic mutant comparison, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"37285312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ufsp1 knockout and catalytic-dead point-mutant mice are viable and born in normal Mendelian ratios with no gross phenotypic abnormalities, indicating Ufsp1 is dispensable for normal mouse development and homeostasis. Despite enrichment of Ufsp1 at the neuromuscular junction (NMJ), only mild NMJ structural alterations were detected with no impact on overall muscle function.\",\n      \"method\": \"Knockout mouse generation; catalytic-dead point-mutant mouse; Mendelian ratio analysis; NMJ structural and functional assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two genetic mouse models with defined phenotypic readouts, single lab, negative/mild phenotype result\",\n      \"pmids\": [\"40748989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Neither UfSP1 nor UfSP2 can efficiently cleave H4 ufmylation at Lys31 (H4K31UFM1) in the nucleosome context. Cryo-EM analysis of the H4K31UFM1-nucleosome suggests that steric hindrance around the isopeptide bond within the nucleosome may underlie the weak deufmylation activity of both UfSPs on nucleosomal substrates.\",\n      \"method\": \"Semisynthetic ufmylated histone preparation; in vitro deufmylation assays with synthetic substrates; cryo-EM structural analysis of H4K31UFM1-nucleosome\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — reconstituted in vitro assay with cryo-EM structural analysis, single lab, no replication\",\n      \"pmids\": [\"41964563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"UFSP1/UFSP2 double-knockout HEK293T cells accumulate elevated UFMylated proteins and were validated as a cellular model for identifying UFMylated substrates, confirming that both proteases are required for efficient deufmylation in human cells.\",\n      \"method\": \"UFSP1KO/UFSP2KO cell line generation; western blot for UFMylation levels; large-scale UFMylated substrate enrichment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic double KO with defined biochemical readout, single lab, single method for this specific finding\",\n      \"pmids\": [\"41759738\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UFSP1 is a cysteine protease (catalytic triad Cys53/Asp175/His177 in the mouse ortholog) that is translated from a non-canonical start site in humans to yield an active enzyme; it specifically processes pro-UFM1 to expose its C-terminal glycine required for conjugation, cleaves a potential autoinhibitory UFMylation on the E2 enzyme UFC1 to activate the UFMylation pathway, and has a distinct substrate specificity from UFSP2 (which handles RPL26 deufmylation); additionally, UFSP1 has a protease-independent role in promoting p62-mediated protein aggregate formation by binding the UBA domain of p62 and enhancing its interaction with ubiquitinated proteins, while being dispensable for normal mouse development and neuromuscular junction homeostasis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UFSP1 is a papain-like cysteine protease that regulates the UFM1 conjugation (UFMylation) system by controlling both maturation of the UFM1 modifier and recycling of UFMylated targets [#0, #1, #2]. It specifically cleaves the C-terminal extension of pro-UFM1 to generate the mature form competent for conjugation and releases UFM1 from conjugated proteins, acting on UFM1 but not on ubiquitin, SUMO-1, or ISG15; catalysis depends on an active-site cysteine within an Asp175-Pro-His177 triad and a Tyr41 oxyanion hole, and the enzyme binds UFM1 with sub-micromolar affinity [#0, #1]. In human cells UFSP1 is produced from a non-canonical translation start site and is the principal protease for UFM1 maturation and for cleaving an autoinhibitory UFMylation on the E2 enzyme UFC1, thereby gating activation of the pathway; this is a division of labor distinct from UFSP2, which deUFMylates the ribosomal subunit RPL26, while both proteases together are required for efficient deUFMylation and neither efficiently cleaves UFM1 from histone H4K31 within the nucleosome owing to steric occlusion [#2, #5, #6]. Independently of its catalytic activity, UFSP1 binds the UBA domain of the autophagy receptor p62/SQSTM1 and promotes p62-body formation by enhancing p62 engagement with ubiquitinated proteins [#3]. Loss of Ufsp1 in mice is compatible with normal development and homeostasis, with only mild structural alterations at the neuromuscular junction where the protein is enriched [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that UFSP1 is the dedicated protease for the UFM1 modifier, answering whether a specific deconjugase/maturase exists for this Ubl.\",\n      \"evidence\": \"In vitro cleavage assays against multiple Ubl substrates, active-site Cys\\u2192Ser mutagenesis, NEM inhibition, and Ufm1-vinylmethylester activity-based labeling (mouse)\",\n      \"pmids\": [\"17182609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define structural basis of UFM1 recognition\", \"Cellular substrates and physiological role not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the catalytic architecture and UFM1-binding determinants, explaining how UFSP1 achieves its specificity as a novel papain-like protease.\",\n      \"evidence\": \"1.7 \\u00c5 crystal structure of mouse UfSP1 with active-site mutagenesis, ITC binding measurement, and NMR mapping of the UFM1 interface\",\n      \"pmids\": [\"18321862\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure is of the apo/mouse enzyme without bound substrate complex\", \"Human enzyme behavior not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved how active human UFSP1 is generated and partitioned the pathway, showing it matures UFM1 and de-represses UFC1 while UFSP2 handles RPL26.\",\n      \"evidence\": \"Non-canonical start-site identification plus UFSP1/UFSP2 double-knockout cells with biochemical readouts for UFM1 maturation, UFC1 deUFMylation, and RPL26 deUFMylation\",\n      \"pmids\": [\"35926457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of non-canonical translation regulation unresolved\", \"Physiological consequences of UFC1 autoinhibition control not defined in vivo\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Uncovered a protease-independent function in selective autophagy, addressing whether UFSP1 acts only as a deconjugase.\",\n      \"evidence\": \"BioID proximity proteomics, reciprocal Co-IP, immunofluorescence colocalization, and catalytic-mutant comparison for p62-body formation\",\n      \"pmids\": [\"37285312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding\", \"Structural basis of UBA-domain binding not defined\", \"In vivo relevance of p62-body promotion unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Tested the organismal requirement for UFSP1, showing it is dispensable for mouse development despite NMJ enrichment.\",\n      \"evidence\": \"Ufsp1 knockout and catalytic-dead point-mutant mice with Mendelian ratio analysis and NMJ structural/functional assays\",\n      \"pmids\": [\"40748989\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mild NMJ phenotype mechanism unexplained\", \"Possible redundancy with UFSP2 not tested in vivo\", \"Single lab, negative/mild result\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined a substrate context limit, showing nucleosomal H4K31-UFM1 resists both UFSPs due to steric hindrance.\",\n      \"evidence\": \"Semisynthetic ufmylated histone deUFMylation assays and cryo-EM of the H4K31UFM1-nucleosome\",\n      \"pmids\": [\"41964563\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether another factor removes nucleosomal UFM1 is unknown\", \"Single lab, no replication\", \"In vivo relevance of persistent histone UFMylation not addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UFSP1's enzymatic and protease-independent (p62) activities are coordinated, and what its physiological substrate repertoire and regulation are in vivo, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo substrate catalog\", \"Coordination of dual functions unresolved\", \"Regulation of non-canonical translation not mechanistically defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UFM1\", \"UFC1\", \"SQSTM1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}