{"gene":"DDI1","run_date":"2026-06-09T23:54:41","timeline":{"discoveries":[{"year":2006,"finding":"X-ray crystal structure of the central domain of Saccharomyces cerevisiae Ddi1 revealed it forms a homodimer with a fold similar to retroviral proteases, with the double Asp-Thr-Gly-Ala active site motif in identical geometry to HIV protease, establishing Ddi1 as a dimeric aspartyl protease-like domain flanked by UBL and UBA domains.","method":"X-ray crystallography","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with active-site detail, replicated and extended by subsequent structural studies","pmids":["17010377"],"is_preprint":false},{"year":2005,"finding":"Ddi1 is specifically required for degradation of ubiquitylated Ho endonuclease; Ho interacts only with Ddi1 (not Rad23 or Dsk2) and must be ubiquitylated for this interaction; Ddi1 binds the proteasome via its UbL domain and interacts with ubiquitylated Ho via its UbA domain; both domains are required for proteasomal association of Ho.","method":"Co-immunoprecipitation, genetic epistasis (ddi1Δ, mec1, ufo1Δ mutants), subcellular fractionation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and biochemical dissection with multiple domain-specific mutants; replicated in subsequent in vitro reconstitution studies","pmids":["15964793"],"is_preprint":false},{"year":2006,"finding":"Ddi1 is required for turnover of the F-box protein Ufo1; Ufo1 interacts with Ddi1 via its ubiquitin-interacting motifs (UIMs), and loss of Ddi1 stabilizes Ufo1 and causes cell cycle arrest at G1/S, implicating Ddi1 in SCF complex recycling.","method":"Co-immunoprecipitation, genetic loss-of-function (ddi1Δ), cell cycle analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus phenotypic rescue, single lab","pmids":["16478980"],"is_preprint":false},{"year":2008,"finding":"Domain dissection of Ddi1/Vsm1 showed: (1) the RVP domain mediates homodimerization; (2) UBL and UBA domains are required for nuclear enrichment and rescue of the pds1-128 checkpoint mutant; (3) an aspartate-220 mutation abolishing putative protease activity blocked checkpoint rescue but not dimerization; (4) a C-terminal region (residues 344–395) binds the Sso1 t-SNARE and its phosphorylation on T348 is required for exocytic function.","method":"GFP live imaging, domain-deletion mutagenesis, phosphorylation analysis, genetic rescue assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (imaging, mutagenesis, genetic rescue), single lab","pmids":["18562697"],"is_preprint":false},{"year":2011,"finding":"A specific docking site on the proteasome subunit Rpn1 (residue D517) is required for recruitment of Ddi1 (and Dsk2) to the proteasome; the D517A mutation in Rpn1 impairs delivery of ubiquitin conjugates, Ddi1 docking, and degradation of the Ddi1-dependent substrate Ufo1, while Rad23 recruitment is unaffected, indicating distinct docking mechanisms for different UBA-UBL proteins.","method":"Site-directed mutagenesis of Rpn1, genetic screening, binding assays, proteasome substrate degradation assays","journal":"BMC biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis + functional substrate degradation assay + binding assay, multiple orthogonal approaches","pmids":["21627799"],"is_preprint":false},{"year":2012,"finding":"In vitro reconstitution showed that Ddi1 can simultaneously bind Ho, Ufo1, and Rpn1, forming a ternary transfer complex; Ddi1-UbL binds Rpn1 while Ddi1-UbA binds ubiquitin chains on Ufo1; Ho substrate protects Ufo1 from displacement by Rpn1, establishing a substrate-shield mechanism.","method":"Complex reconstitution in vitro, pull-down assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution but single lab, single study","pmids":["22815701"],"is_preprint":false},{"year":2015,"finding":"NMR and binding studies of yeast Ddi1 UBL and UBA domains showed: (1) Ddi1-UBL does not interact with typical UBL receptors but instead binds ubiquitin via hydrophobic contacts and salt bridges, a unique interface; (2) Ddi1-UBA forms a canonical UBA:ubiquitin complex; suggesting a dual ubiquitin-binding mechanism for proteasomal shuttling.","method":"NMR structure determination, isothermal titration calorimetry, binding assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with ITC binding validation, two orthogonal methods, single lab","pmids":["25703377"],"is_preprint":false},{"year":2016,"finding":"In C. elegans, the aspartic protease DDI-1 is required to cleave and activate an ER-associated isoform of the transcription factor SKN-1A/Nrf1 in response to proteasome dysfunction; DDI-1 expression is itself induced by proteasome dysfunction; genetic analyses placed DDI-1 in a pathway with ER traffic regulators and a peptide N-glycanase.","method":"Genetic epistasis (C. elegans), loss-of-function, immunoblotting for SKN-1 cleavage products","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — comprehensive genetic analysis with direct proteolytic cleavage readout, replicated in mammalian context by subsequent studies","pmids":["27528192"],"is_preprint":false},{"year":2016,"finding":"Multi-domain structural analysis of yeast Ddi1 by X-ray crystallography (RVP domain), NMR (helical domain preceding RVP), and SAXS showed: (1) the RVP domain has a conserved loop forming a putative substrate recognition site; (2) both UBL and UBA domains bind ubiquitin by ITC, with enhanced affinity for K48-linked diubiquitin; (3) the helical domain (HDD) has structural similarity to DNA-binding domains of transcription regulators.","method":"X-ray crystallography, NMR, SAXS, isothermal titration calorimetry","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple complementary structural and biophysical methods, confirms and extends earlier structural work","pmids":["27646017"],"is_preprint":false},{"year":2019,"finding":"The helical domain (HDD) of Ddi1 preceding the RVP protease domain is required for the cellular response to DNA replication stress caused by hydroxyurea; catalytically competent Ddi1 protease is required to complement the hypersensitivity of ddi1Δ wss1Δ double-deleted yeast.","method":"Genetic complementation, domain deletion mutagenesis, yeast growth assays under hydroxyurea","journal":"DNA repair","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with domain-specific mutants, single lab","pmids":["31276951"],"is_preprint":false},{"year":2020,"finding":"Ddi1 is a ubiquitin-dependent protease: it cleaves substrate proteins only when tagged with long polyubiquitin chains (>~8 ubiquitins); the RVP domain alone is inactive and requires the HDD domain for proteolytic activity; the atypical UBL domain stimulates activity by mediating high-affinity binding to polyubiquitin chains; loss of Ddi1 activity in yeast causes accumulation of polyubiquitinated proteins.","method":"In vitro protease assay with ubiquitinated substrates, domain deletion and mutagenesis, yeast loss-of-function","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis and cellular validation, multiple orthogonal methods in single rigorous study","pmids":["32193351"],"is_preprint":false},{"year":2020,"finding":"Ddi1 is recruited to a persistent DNA-protein crosslink (DPC) lesion in S phase in yeast; loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC-trapping agents independently of Wss1 and the 26S proteasome; the core component of RNA Pol II is a Ddi1 target, as its genotoxin-induced degradation is impaired in ddi1Δ cells.","method":"Genetic screen, chromatin immunoprecipitation, genetic epistasis (ddi1Δ, wss1Δ, proteasome mutants), immunoblot for Pol II degradation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal approaches (genetic screen, ChIP, epistasis, substrate degradation assay), single lab but rigorous","pmids":["31902667"],"is_preprint":false},{"year":2018,"finding":"Human DDI1 is ubiquitinated by the E3 ligase UBE3A in neuroblastoma SH-SY5Y cells without being targeted for degradation; ubiquitination of DDI1 by UBE3A was confirmed by immunoblotting.","method":"Ubiquitin proteomics (bioUb strategy), immunoblotting, cell-based overexpression","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass-spectrometry proteomics plus immunoblot validation, single lab","pmids":["29788202"],"is_preprint":false},{"year":2019,"finding":"UBE3A-dependent ubiquitination sites and ubiquitin chain types on DDI1 were identified; a deubiquitinating enzyme capable of reversing UBE3A-mediated ubiquitination of DDI1 was identified.","method":"Ubiquitin proteomics (site mapping), deubiquitinase activity assay","journal":"Frontiers in physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry site mapping plus enzymatic reversal assay, single lab","pmids":["31130875"],"is_preprint":false},{"year":2010,"finding":"The retroviral proteinase active site (aspartate residue) and N-terminal region of yeast Ddi1 are both required for repression of protein secretion, demonstrating that Ddi1 functions in vivo as a catalytically active aspartic proteinase in the context of exocytosis regulation.","method":"Site-directed mutagenesis, yeast secretion assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — active-site mutagenesis with functional readout, single lab","pmids":["21094643"],"is_preprint":false},{"year":2025,"finding":"Ddi1 acts as a ubiquitin-activated protease that cleaves K48-ubiquitinated integral membrane proteins at post-ER compartments, generating cytosolic fragments; the HDD-RVP catalytic core is sufficient for ubiquitin-dependent proteolysis; Ddi1 binds ubiquitin directly; activity is amplified by the atypical UBL and UBA auxiliary ubiquitin-binding domains.","method":"In vitro protease assay, domain truncation mutagenesis, ubiquitin binding assays, cell-based substrate cleavage assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with domain mutagenesis, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.04.13.648637"],"is_preprint":true},{"year":2025,"finding":"In Saccharomyces cerevisiae and Candida albicans, Ddi1 (together with Wss1) is required for DNA-protein crosslink repair under oxidative stress and for survival in response to hydrogen peroxide, sodium hypochlorite, menadione, and plumbagin; CaDdi1 plays a partially redundant role with CaWss1 in resistance to macrophage killing.","method":"DPC measurement (SDS/KCl precipitation), genetic deletion (ddi1Δ, wss1Δ), complementation assays, macrophage killing assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct DPC measurement plus genetic epistasis, two orthogonal methods, single study","pmids":["40702097"],"is_preprint":false}],"current_model":"DDI1/Ddi1 is a multidomain ubiquitin receptor and ubiquitin-dependent aspartyl protease containing an N-terminal UBL domain (which unusually binds ubiquitin directly and also contacts the proteasome subunit Rpn1), a central helical domain (HDD) essential for protease activity, a retroviral-like protease (RVP) domain that is inactive in isolation but active together with HDD, and a C-terminal UBA domain that binds polyubiquitin chains; Ddi1 functions as a proteasome shuttle factor that delivers specific ubiquitylated substrates (e.g., Ho endonuclease, Ufo1/SCF complexes) to the 26S proteasome via Rpn1, and also acts as an autonomous ubiquitin-dependent endoprotease that cleaves substrates bearing long K48-linked polyubiquitin chains, contributes to DNA-protein crosslink repair (targeting stalled RNA Pol II and other DPC moieties in an S-phase-dependent manner), and in higher eukaryotes cleaves the ER-associated Nrf1/SKN-1 transcription factor to activate proteasome subunit gene transcription in response to proteasome stress."},"narrative":{"mechanistic_narrative":"DDI1/Ddi1 is a multidomain ubiquitin receptor and ubiquitin-dependent aspartyl protease that couples ubiquitin recognition to substrate proteolysis and proteasomal delivery [PMID:17010377, PMID:32193351]. Its central RVP domain adopts a retroviral protease-like fold, dimerizing with paired Asp-Thr-Gly-Ala active sites arranged like HIV protease, establishing the catalytic core flanked by UBL and UBA ubiquitin-binding modules [PMID:17010377]. The RVP domain is inactive in isolation and requires the preceding helical domain (HDD) for proteolytic activity, while the atypical UBL domain—which binds ubiquitin directly through a non-canonical interface rather than acting as a conventional proteasome-targeting tag—and the UBA domain together confer high-affinity binding to long K48-linked polyubiquitin chains, restricting cleavage to substrates bearing such chains [PMID:25703377, PMID:27646017, PMID:32193351]. As a proteasome shuttle factor, Ddi1 docks on the proteasome subunit Rpn1 (at residue D517) via its UBL domain and captures ubiquitylated substrates such as the Ho endonuclease and the F-box protein Ufo1 via its UBA domain, simultaneously bridging substrate and proteasome in a ternary transfer complex to drive SCF recycling and cell-cycle progression [PMID:15964793, PMID:16478980, PMID:21627799, PMID:22815701]. Independently of the proteasome, Ddi1 functions in DNA-protein crosslink repair, being recruited to persistent crosslinks in S phase and degrading trapped RNA Pol II in a manner that requires its protease activity [PMID:31276951, PMID:31902667, PMID:40702097]. In higher eukaryotes the protease cleaves and activates the ER-associated transcription factor SKN-1A/Nrf1 in response to proteasome dysfunction, linking Ddi1 to a feedback program that restores proteasome capacity [PMID:27528192].","teleology":[{"year":2005,"claim":"Established Ddi1 as a substrate-selective proteasomal shuttle, answering whether dedicated UBL-UBA proteins deliver specific ubiquitylated clients to the proteasome.","evidence":"Co-immunoprecipitation and genetic epistasis dissecting Ho endonuclease degradation in yeast","pmids":["15964793"],"confidence":"High","gaps":["Did not resolve the structural basis of UBL-proteasome docking","Did not test whether Ddi1 has intrinsic catalytic activity on substrates"]},{"year":2006,"claim":"Defined the catalytic core's architecture, showing Ddi1's central domain is a dimeric retroviral protease-like aspartyl protease fold.","evidence":"X-ray crystallography of the S. cerevisiae central domain","pmids":["17010377"],"confidence":"High","gaps":["Catalytic activity on substrates not demonstrated structurally","Role of flanking UBL/UBA domains in activity not addressed"]},{"year":2006,"claim":"Extended the shuttle role to SCF complex turnover, linking Ddi1 to cell-cycle control via Ufo1 degradation.","evidence":"Co-IP and cell cycle analysis in ddi1Δ yeast","pmids":["16478980"],"confidence":"Medium","gaps":["Single-lab Co-IP plus phenotype without reciprocal validation","Mechanism of Ufo1 capture not biochemically reconstituted"]},{"year":2008,"claim":"Mapped domain-specific functions, implicating the putative protease active site in checkpoint rescue and a separate C-terminal region in t-SNARE binding.","evidence":"GFP imaging, domain-deletion mutagenesis and genetic rescue in yeast","pmids":["18562697"],"confidence":"Medium","gaps":["Protease activity inferred from active-site mutant, not measured directly","Single lab"]},{"year":2011,"claim":"Identified the precise proteasome docking site, showing distinct receptors use distinct Rpn1 surfaces.","evidence":"Site-directed mutagenesis of Rpn1 (D517A) with binding and substrate degradation assays","pmids":["21627799"],"confidence":"High","gaps":["Structural details of the Ddi1-UBL:Rpn1 interface not resolved here","Did not address catalytic versus shuttle contributions"]},{"year":2012,"claim":"Demonstrated simultaneous substrate-and-proteasome bridging, defining a ternary transfer complex with a substrate-shield mechanism.","evidence":"In vitro reconstitution and pull-down assays with Ho, Ufo1 and Rpn1","pmids":["22815701"],"confidence":"Medium","gaps":["Single in vitro study","Kinetics of substrate handoff not quantified"]},{"year":2015,"claim":"Revealed the atypical UBL domain binds ubiquitin directly, establishing a dual ubiquitin-binding mode distinct from canonical UBL receptors.","evidence":"NMR structure determination and ITC of yeast UBL and UBA domains","pmids":["25703377"],"confidence":"High","gaps":["Functional consequence of UBL-ubiquitin binding for catalysis not tested","Single lab"]},{"year":2016,"claim":"Extended the protease function to higher eukaryotes, showing DDI-1 cleaves and activates SKN-1A/Nrf1 during proteasome stress.","evidence":"C. elegans genetic epistasis and immunoblotting for SKN-1 cleavage products","pmids":["27528192"],"confidence":"High","gaps":["Direct enzyme-substrate cleavage not reconstituted in vitro here","Cleavage site on SKN-1A not mapped"]},{"year":2016,"claim":"Provided full-length structural context showing K48-diubiquitin preference and a putative substrate-recognition loop in the RVP domain.","evidence":"X-ray crystallography, NMR, SAXS and ITC of yeast Ddi1 domains","pmids":["27646017"],"confidence":"High","gaps":["Catalytic mechanism on physiological substrates not shown","HDD similarity to DNA-binding folds not functionally tested"]},{"year":2020,"claim":"Defined Ddi1 as an autonomous ubiquitin-activated protease requiring long polyubiquitin chains and the HDD for activity, resolving how chain length gates catalysis.","evidence":"In vitro protease assays with ubiquitinated substrates, domain mutagenesis and yeast loss-of-function","pmids":["32193351"],"confidence":"High","gaps":["Endogenous physiological substrate repertoire not defined","Cleavage specificity rules within substrates not determined"]},{"year":2020,"claim":"Established a proteasome-independent role in DNA-protein crosslink repair, identifying RNA Pol II as an S-phase Ddi1 target.","evidence":"Genetic screen, ChIP, epistasis and Pol II degradation immunoblots in yeast","pmids":["31902667"],"confidence":"High","gaps":["Direct cleavage of crosslinked Pol II not reconstituted","Recruitment mechanism to crosslink lesions unclear"]},{"year":2025,"claim":"Extended the protease function to integral membrane proteins, showing cleavage of K48-ubiquitinated substrates at post-ER compartments by the HDD-RVP core.","evidence":"In vitro protease, domain truncation and cell-based cleavage assays (preprint)","pmids":["bio_10.1101_2025.04.13.648637"],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Membrane substrate range and physiological role not established"]},{"year":2025,"claim":"Connected Ddi1-mediated DPC repair to oxidative-stress survival and host defense across yeast species.","evidence":"DPC precipitation, ddi1Δ/wss1Δ genetics, complementation and macrophage killing assays","pmids":["40702097"],"confidence":"Medium","gaps":["Molecular targets in oxidative DPC repair not identified","Single study"]},{"year":null,"claim":"The full endogenous substrate spectrum of Ddi1's protease activity and the rules governing its cleavage-site selection within ubiquitin-tagged substrates remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No global substrate census across organisms","Cleavage-site determinants beyond chain-length requirement unknown","Relative contributions of shuttle versus protease functions in vivo unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[10,11,15]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,10,14]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5,15]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,4,10]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[11,16]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[7]}],"complexes":["26S proteasome (Rpn1-docked receptor)"],"partners":["RPN1","HO","UFO1","SKN-1A/NRF1","UBE3A","WSS1","SSO1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8WTU0","full_name":"Protein DDI1 homolog 1","aliases":[],"length_aa":396,"mass_kda":44.1,"function":"Probable aspartic protease (Probable). Seems to act as a proteasomal shuttle which links the proteasome and replication fork proteins like RTF2 (Probable). Required, with DDI2, for cellular survival following replication stress. Together or redudantly with DDI2, removes RTF2 from stalled forks to allow cell cycle progression after replication stress and maintains genome integrity (PubMed:29290612)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8WTU0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DDI1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DDI1","total_profiled":1310},"omim":[{"mim_id":"621498","title":"REPLICATION TERMINATION FACTOR 2; RTF2","url":"https://www.omim.org/entry/621498"},{"mim_id":"620871","title":"DNA DAMAGE-INDUCIBLE 1 HOMOLOG 2; DDI2","url":"https://www.omim.org/entry/620871"},{"mim_id":"620870","title":"DNA DAMAGE-INDUCIBLE 1 HOMOLOG 1; DDI1","url":"https://www.omim.org/entry/620870"},{"mim_id":"610661","title":"N-GLYCANASE 1; NGLY1","url":"https://www.omim.org/entry/610661"},{"mim_id":"607394","title":"POU DOMAIN, CLASS 2, TRANSCRIPTION FACTOR 3; POU2F3","url":"https://www.omim.org/entry/607394"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"testis","ntpm":24.1}],"url":"https://www.proteinatlas.org/search/DDI1"},"hgnc":{"alias_symbol":["FLJ36017"],"prev_symbol":[]},"alphafold":{"accession":"Q8WTU0","domains":[{"cath_id":"3.10.20.90","chopping":"3-77","consensus_level":"high","plddt":92.7365,"start":3,"end":77},{"cath_id":"-","chopping":"144-203","consensus_level":"high","plddt":83.1345,"start":144,"end":203},{"cath_id":"2.40.70.10","chopping":"246-368","consensus_level":"high","plddt":95.5201,"start":246,"end":368}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WTU0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WTU0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WTU0-F1-predicted_aligned_error_v6.png","plddt_mean":77.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DDI1","jax_strain_url":"https://www.jax.org/strain/search?query=DDI1"},"sequence":{"accession":"Q8WTU0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WTU0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WTU0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WTU0"}},"corpus_meta":[{"pmid":"27528192","id":"PMC_27528192","title":"Proteasome 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Interactions and Correlates with Evolutionary Relationships of Retroviral and Retroviral-Like Ddi1 and Ddi2 Proteases.","date":"2020","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32079302","citation_count":10,"is_preprint":false},{"pmid":"34832620","id":"PMC_34832620","title":"Artemisinin Binds and Inhibits the Activity of Plasmodium falciparum Ddi1, a Retroviral Aspartyl Protease.","date":"2021","source":"Pathogens (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/34832620","citation_count":9,"is_preprint":false},{"pmid":"36657610","id":"PMC_36657610","title":"Plasmodium DDI1 is a potential therapeutic target and important chromatin-associated protein.","date":"2023","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/36657610","citation_count":4,"is_preprint":false},{"pmid":"37511326","id":"PMC_37511326","title":"The Effects of DDI1 on Inducing Differentiation in Ovine Preadipocytes via Oar-miR-432.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37511326","citation_count":4,"is_preprint":false},{"pmid":"38612889","id":"PMC_38612889","title":"Domains in Action: Understanding Ddi1's Diverse Functions in the Ubiquitin-Proteasome System.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38612889","citation_count":3,"is_preprint":false},{"pmid":"35677776","id":"PMC_35677776","title":"Structural and functional insights into the DNA damage-inducible protein 1 (Ddi1) from protozoa.","date":"2022","source":"Current research in structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/35677776","citation_count":2,"is_preprint":false},{"pmid":"40702097","id":"PMC_40702097","title":"Wss1 and Ddi1 DNA-Protein crosslink repair proteases protect Saccharomyces cerevisiae and Candida albicans against oxidative stress.","date":"2025","source":"Scientific 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similar to retroviral proteases, with the double Asp-Thr-Gly-Ala active site motif in identical geometry to HIV protease, establishing Ddi1 as a dimeric aspartyl protease-like domain flanked by UBL and UBA domains.\",\n      \"method\": \"X-ray crystallography\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with active-site detail, replicated and extended by subsequent structural studies\",\n      \"pmids\": [\"17010377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Ddi1 is specifically required for degradation of ubiquitylated Ho endonuclease; Ho interacts only with Ddi1 (not Rad23 or Dsk2) and must be ubiquitylated for this interaction; Ddi1 binds the proteasome via its UbL domain and interacts with ubiquitylated Ho via its UbA domain; both domains are required for proteasomal association of Ho.\",\n      \"method\": \"Co-immunoprecipitation, genetic epistasis (ddi1Δ, mec1, ufo1Δ mutants), subcellular fractionation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and biochemical dissection with multiple domain-specific mutants; replicated in subsequent in vitro reconstitution studies\",\n      \"pmids\": [\"15964793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Ddi1 is required for turnover of the F-box protein Ufo1; Ufo1 interacts with Ddi1 via its ubiquitin-interacting motifs (UIMs), and loss of Ddi1 stabilizes Ufo1 and causes cell cycle arrest at G1/S, implicating Ddi1 in SCF complex recycling.\",\n      \"method\": \"Co-immunoprecipitation, genetic loss-of-function (ddi1Δ), cell cycle analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus phenotypic rescue, single lab\",\n      \"pmids\": [\"16478980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Domain dissection of Ddi1/Vsm1 showed: (1) the RVP domain mediates homodimerization; (2) UBL and UBA domains are required for nuclear enrichment and rescue of the pds1-128 checkpoint mutant; (3) an aspartate-220 mutation abolishing putative protease activity blocked checkpoint rescue but not dimerization; (4) a C-terminal region (residues 344–395) binds the Sso1 t-SNARE and its phosphorylation on T348 is required for exocytic function.\",\n      \"method\": \"GFP live imaging, domain-deletion mutagenesis, phosphorylation analysis, genetic rescue assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (imaging, mutagenesis, genetic rescue), single lab\",\n      \"pmids\": [\"18562697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A specific docking site on the proteasome subunit Rpn1 (residue D517) is required for recruitment of Ddi1 (and Dsk2) to the proteasome; the D517A mutation in Rpn1 impairs delivery of ubiquitin conjugates, Ddi1 docking, and degradation of the Ddi1-dependent substrate Ufo1, while Rad23 recruitment is unaffected, indicating distinct docking mechanisms for different UBA-UBL proteins.\",\n      \"method\": \"Site-directed mutagenesis of Rpn1, genetic screening, binding assays, proteasome substrate degradation assays\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis + functional substrate degradation assay + binding assay, multiple orthogonal approaches\",\n      \"pmids\": [\"21627799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In vitro reconstitution showed that Ddi1 can simultaneously bind Ho, Ufo1, and Rpn1, forming a ternary transfer complex; Ddi1-UbL binds Rpn1 while Ddi1-UbA binds ubiquitin chains on Ufo1; Ho substrate protects Ufo1 from displacement by Rpn1, establishing a substrate-shield mechanism.\",\n      \"method\": \"Complex reconstitution in vitro, pull-down assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution but single lab, single study\",\n      \"pmids\": [\"22815701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NMR and binding studies of yeast Ddi1 UBL and UBA domains showed: (1) Ddi1-UBL does not interact with typical UBL receptors but instead binds ubiquitin via hydrophobic contacts and salt bridges, a unique interface; (2) Ddi1-UBA forms a canonical UBA:ubiquitin complex; suggesting a dual ubiquitin-binding mechanism for proteasomal shuttling.\",\n      \"method\": \"NMR structure determination, isothermal titration calorimetry, binding assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with ITC binding validation, two orthogonal methods, single lab\",\n      \"pmids\": [\"25703377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In C. elegans, the aspartic protease DDI-1 is required to cleave and activate an ER-associated isoform of the transcription factor SKN-1A/Nrf1 in response to proteasome dysfunction; DDI-1 expression is itself induced by proteasome dysfunction; genetic analyses placed DDI-1 in a pathway with ER traffic regulators and a peptide N-glycanase.\",\n      \"method\": \"Genetic epistasis (C. elegans), loss-of-function, immunoblotting for SKN-1 cleavage products\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — comprehensive genetic analysis with direct proteolytic cleavage readout, replicated in mammalian context by subsequent studies\",\n      \"pmids\": [\"27528192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Multi-domain structural analysis of yeast Ddi1 by X-ray crystallography (RVP domain), NMR (helical domain preceding RVP), and SAXS showed: (1) the RVP domain has a conserved loop forming a putative substrate recognition site; (2) both UBL and UBA domains bind ubiquitin by ITC, with enhanced affinity for K48-linked diubiquitin; (3) the helical domain (HDD) has structural similarity to DNA-binding domains of transcription regulators.\",\n      \"method\": \"X-ray crystallography, NMR, SAXS, isothermal titration calorimetry\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple complementary structural and biophysical methods, confirms and extends earlier structural work\",\n      \"pmids\": [\"27646017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The helical domain (HDD) of Ddi1 preceding the RVP protease domain is required for the cellular response to DNA replication stress caused by hydroxyurea; catalytically competent Ddi1 protease is required to complement the hypersensitivity of ddi1Δ wss1Δ double-deleted yeast.\",\n      \"method\": \"Genetic complementation, domain deletion mutagenesis, yeast growth assays under hydroxyurea\",\n      \"journal\": \"DNA repair\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with domain-specific mutants, single lab\",\n      \"pmids\": [\"31276951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Ddi1 is a ubiquitin-dependent protease: it cleaves substrate proteins only when tagged with long polyubiquitin chains (>~8 ubiquitins); the RVP domain alone is inactive and requires the HDD domain for proteolytic activity; the atypical UBL domain stimulates activity by mediating high-affinity binding to polyubiquitin chains; loss of Ddi1 activity in yeast causes accumulation of polyubiquitinated proteins.\",\n      \"method\": \"In vitro protease assay with ubiquitinated substrates, domain deletion and mutagenesis, yeast loss-of-function\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis and cellular validation, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"32193351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Ddi1 is recruited to a persistent DNA-protein crosslink (DPC) lesion in S phase in yeast; loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC-trapping agents independently of Wss1 and the 26S proteasome; the core component of RNA Pol II is a Ddi1 target, as its genotoxin-induced degradation is impaired in ddi1Δ cells.\",\n      \"method\": \"Genetic screen, chromatin immunoprecipitation, genetic epistasis (ddi1Δ, wss1Δ, proteasome mutants), immunoblot for Pol II degradation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal approaches (genetic screen, ChIP, epistasis, substrate degradation assay), single lab but rigorous\",\n      \"pmids\": [\"31902667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Human DDI1 is ubiquitinated by the E3 ligase UBE3A in neuroblastoma SH-SY5Y cells without being targeted for degradation; ubiquitination of DDI1 by UBE3A was confirmed by immunoblotting.\",\n      \"method\": \"Ubiquitin proteomics (bioUb strategy), immunoblotting, cell-based overexpression\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass-spectrometry proteomics plus immunoblot validation, single lab\",\n      \"pmids\": [\"29788202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"UBE3A-dependent ubiquitination sites and ubiquitin chain types on DDI1 were identified; a deubiquitinating enzyme capable of reversing UBE3A-mediated ubiquitination of DDI1 was identified.\",\n      \"method\": \"Ubiquitin proteomics (site mapping), deubiquitinase activity assay\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry site mapping plus enzymatic reversal assay, single lab\",\n      \"pmids\": [\"31130875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The retroviral proteinase active site (aspartate residue) and N-terminal region of yeast Ddi1 are both required for repression of protein secretion, demonstrating that Ddi1 functions in vivo as a catalytically active aspartic proteinase in the context of exocytosis regulation.\",\n      \"method\": \"Site-directed mutagenesis, yeast secretion assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — active-site mutagenesis with functional readout, single lab\",\n      \"pmids\": [\"21094643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ddi1 acts as a ubiquitin-activated protease that cleaves K48-ubiquitinated integral membrane proteins at post-ER compartments, generating cytosolic fragments; the HDD-RVP catalytic core is sufficient for ubiquitin-dependent proteolysis; Ddi1 binds ubiquitin directly; activity is amplified by the atypical UBL and UBA auxiliary ubiquitin-binding domains.\",\n      \"method\": \"In vitro protease assay, domain truncation mutagenesis, ubiquitin binding assays, cell-based substrate cleavage assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with domain mutagenesis, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.04.13.648637\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Saccharomyces cerevisiae and Candida albicans, Ddi1 (together with Wss1) is required for DNA-protein crosslink repair under oxidative stress and for survival in response to hydrogen peroxide, sodium hypochlorite, menadione, and plumbagin; CaDdi1 plays a partially redundant role with CaWss1 in resistance to macrophage killing.\",\n      \"method\": \"DPC measurement (SDS/KCl precipitation), genetic deletion (ddi1Δ, wss1Δ), complementation assays, macrophage killing assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct DPC measurement plus genetic epistasis, two orthogonal methods, single study\",\n      \"pmids\": [\"40702097\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DDI1/Ddi1 is a multidomain ubiquitin receptor and ubiquitin-dependent aspartyl protease containing an N-terminal UBL domain (which unusually binds ubiquitin directly and also contacts the proteasome subunit Rpn1), a central helical domain (HDD) essential for protease activity, a retroviral-like protease (RVP) domain that is inactive in isolation but active together with HDD, and a C-terminal UBA domain that binds polyubiquitin chains; Ddi1 functions as a proteasome shuttle factor that delivers specific ubiquitylated substrates (e.g., Ho endonuclease, Ufo1/SCF complexes) to the 26S proteasome via Rpn1, and also acts as an autonomous ubiquitin-dependent endoprotease that cleaves substrates bearing long K48-linked polyubiquitin chains, contributes to DNA-protein crosslink repair (targeting stalled RNA Pol II and other DPC moieties in an S-phase-dependent manner), and in higher eukaryotes cleaves the ER-associated Nrf1/SKN-1 transcription factor to activate proteasome subunit gene transcription in response to proteasome stress.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DDI1/Ddi1 is a multidomain ubiquitin receptor and ubiquitin-dependent aspartyl protease that couples ubiquitin recognition to substrate proteolysis and proteasomal delivery [#0, #10]. Its central RVP domain adopts a retroviral protease-like fold, dimerizing with paired Asp-Thr-Gly-Ala active sites arranged like HIV protease, establishing the catalytic core flanked by UBL and UBA ubiquitin-binding modules [#0]. The RVP domain is inactive in isolation and requires the preceding helical domain (HDD) for proteolytic activity, while the atypical UBL domain—which binds ubiquitin directly through a non-canonical interface rather than acting as a conventional proteasome-targeting tag—and the UBA domain together confer high-affinity binding to long K48-linked polyubiquitin chains, restricting cleavage to substrates bearing such chains [#6, #8, #10]. As a proteasome shuttle factor, Ddi1 docks on the proteasome subunit Rpn1 (at residue D517) via its UBL domain and captures ubiquitylated substrates such as the Ho endonuclease and the F-box protein Ufo1 via its UBA domain, simultaneously bridging substrate and proteasome in a ternary transfer complex to drive SCF recycling and cell-cycle progression [#1, #2, #4, #5]. Independently of the proteasome, Ddi1 functions in DNA-protein crosslink repair, being recruited to persistent crosslinks in S phase and degrading trapped RNA Pol II in a manner that requires its protease activity [#9, #11, #16]. In higher eukaryotes the protease cleaves and activates the ER-associated transcription factor SKN-1A/Nrf1 in response to proteasome dysfunction, linking Ddi1 to a feedback program that restores proteasome capacity [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established Ddi1 as a substrate-selective proteasomal shuttle, answering whether dedicated UBL-UBA proteins deliver specific ubiquitylated clients to the proteasome.\",\n      \"evidence\": \"Co-immunoprecipitation and genetic epistasis dissecting Ho endonuclease degradation in yeast\",\n      \"pmids\": [\"15964793\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of UBL-proteasome docking\", \"Did not test whether Ddi1 has intrinsic catalytic activity on substrates\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the catalytic core's architecture, showing Ddi1's central domain is a dimeric retroviral protease-like aspartyl protease fold.\",\n      \"evidence\": \"X-ray crystallography of the S. cerevisiae central domain\",\n      \"pmids\": [\"17010377\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic activity on substrates not demonstrated structurally\", \"Role of flanking UBL/UBA domains in activity not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended the shuttle role to SCF complex turnover, linking Ddi1 to cell-cycle control via Ufo1 degradation.\",\n      \"evidence\": \"Co-IP and cell cycle analysis in ddi1\\u0394 yeast\",\n      \"pmids\": [\"16478980\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Co-IP plus phenotype without reciprocal validation\", \"Mechanism of Ufo1 capture not biochemically reconstituted\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mapped domain-specific functions, implicating the putative protease active site in checkpoint rescue and a separate C-terminal region in t-SNARE binding.\",\n      \"evidence\": \"GFP imaging, domain-deletion mutagenesis and genetic rescue in yeast\",\n      \"pmids\": [\"18562697\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Protease activity inferred from active-site mutant, not measured directly\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified the precise proteasome docking site, showing distinct receptors use distinct Rpn1 surfaces.\",\n      \"evidence\": \"Site-directed mutagenesis of Rpn1 (D517A) with binding and substrate degradation assays\",\n      \"pmids\": [\"21627799\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural details of the Ddi1-UBL:Rpn1 interface not resolved here\", \"Did not address catalytic versus shuttle contributions\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated simultaneous substrate-and-proteasome bridging, defining a ternary transfer complex with a substrate-shield mechanism.\",\n      \"evidence\": \"In vitro reconstitution and pull-down assays with Ho, Ufo1 and Rpn1\",\n      \"pmids\": [\"22815701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single in vitro study\", \"Kinetics of substrate handoff not quantified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed the atypical UBL domain binds ubiquitin directly, establishing a dual ubiquitin-binding mode distinct from canonical UBL receptors.\",\n      \"evidence\": \"NMR structure determination and ITC of yeast UBL and UBA domains\",\n      \"pmids\": [\"25703377\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of UBL-ubiquitin binding for catalysis not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended the protease function to higher eukaryotes, showing DDI-1 cleaves and activates SKN-1A/Nrf1 during proteasome stress.\",\n      \"evidence\": \"C. elegans genetic epistasis and immunoblotting for SKN-1 cleavage products\",\n      \"pmids\": [\"27528192\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzyme-substrate cleavage not reconstituted in vitro here\", \"Cleavage site on SKN-1A not mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided full-length structural context showing K48-diubiquitin preference and a putative substrate-recognition loop in the RVP domain.\",\n      \"evidence\": \"X-ray crystallography, NMR, SAXS and ITC of yeast Ddi1 domains\",\n      \"pmids\": [\"27646017\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic mechanism on physiological substrates not shown\", \"HDD similarity to DNA-binding folds not functionally tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined Ddi1 as an autonomous ubiquitin-activated protease requiring long polyubiquitin chains and the HDD for activity, resolving how chain length gates catalysis.\",\n      \"evidence\": \"In vitro protease assays with ubiquitinated substrates, domain mutagenesis and yeast loss-of-function\",\n      \"pmids\": [\"32193351\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous physiological substrate repertoire not defined\", \"Cleavage specificity rules within substrates not determined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established a proteasome-independent role in DNA-protein crosslink repair, identifying RNA Pol II as an S-phase Ddi1 target.\",\n      \"evidence\": \"Genetic screen, ChIP, epistasis and Pol II degradation immunoblots in yeast\",\n      \"pmids\": [\"31902667\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct cleavage of crosslinked Pol II not reconstituted\", \"Recruitment mechanism to crosslink lesions unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the protease function to integral membrane proteins, showing cleavage of K48-ubiquitinated substrates at post-ER compartments by the HDD-RVP core.\",\n      \"evidence\": \"In vitro protease, domain truncation and cell-based cleavage assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.04.13.648637\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"Membrane substrate range and physiological role not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected Ddi1-mediated DPC repair to oxidative-stress survival and host defense across yeast species.\",\n      \"evidence\": \"DPC precipitation, ddi1\\u0394/wss1\\u0394 genetics, complementation and macrophage killing assays\",\n      \"pmids\": [\"40702097\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular targets in oxidative DPC repair not identified\", \"Single study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full endogenous substrate spectrum of Ddi1's protease activity and the rules governing its cleavage-site selection within ubiquitin-tagged substrates remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No global substrate census across organisms\", \"Cleavage-site determinants beyond chain-length requirement unknown\", \"Relative contributions of shuttle versus protease functions in vivo unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [10, 11, 15]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 10, 14]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 4, 10]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [11, 16]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"26S proteasome (Rpn1-docked receptor)\"],\n    \"partners\": [\"RPN1\", \"HO\", \"UFO1\", \"SKN-1A/Nrf1\", \"UBE3A\", \"WSS1\", \"SSO1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}