{"gene":"PSMF1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2000,"finding":"PI31 is a proline-rich inhibitor of the 20S proteasome; its C-terminal proline-rich domain confers inhibition by forming a proteasome-PI31 complex and blocking hydrolysis of both protein and peptide substrates. PI31 also inhibits activation of the proteasome by regulatory proteins PA700 and PA28.","method":"Recombinant protein expression in E. coli, truncation mutant analysis, in vitro proteasome activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with defined truncation mutants and multiple orthogonal assays (protein and peptide substrate hydrolysis, complex formation, inhibition of two distinct activators)","pmids":["10764772"],"is_preprint":false},{"year":2008,"finding":"PI31 interacts with Fbxo7·Skp1 through a conserved N-terminal FP (Fbxo7/PI31) domain that mediates both homodimerization of PI31 and heterodimerization with Fbxo7. The crystal structure of the PI31 FP domain reveals a novel α/β-fold. Knockdown of Fbxo7 does not affect PI31 levels, arguing against PI31 being an SCF(Fbxo7) substrate.","method":"Crystal structure determination, biophysical analysis (SEC, ITC), site-directed mutagenesis, co-immunoprecipitation, RNAi knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and binding assays; multiple orthogonal methods in one study","pmids":["18495667"],"is_preprint":false},{"year":2014,"finding":"PI31 contains a C-terminal HbYX motif; peptides corresponding to this motif bind to and activate the 20S proteasome in an HbYX-dependent manner, but intact PI31 inhibits 20S activity. PI31 blocks ATP-dependent in vitro assembly of 26S proteasome from 20S and PA700 subcomplexes but has no effect on activity of intact 26S proteasome. Ectopic overexpression or RNAi knockdown of PI31 in cells produced no detectable change in overall cellular proteasome content or function.","method":"In vitro proteasome activity assays with truncation and point mutants, 26S assembly assay, RNAi knockdown, ectopic overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple in vitro reconstitution experiments plus cellular RNAi/overexpression; single lab but orthogonal methods","pmids":["24770418"],"is_preprint":false},{"year":2014,"finding":"In yeast (S. cerevisiae), the PI31 ortholog Fub1 is essential when the CP assembly chaperone Pba4 is deleted. Deletion of the N-terminus of α7 (α7ΔN), but not α3ΔN, suppresses the lethality of Δfub1 Δpba4, indicating that Fub1 functionally antagonizes a specific gate-opening role of the α7 N-terminus in CP activation.","method":"Yeast genetic epistasis (double-mutant analysis), suppressor screen with α-subunit N-terminal deletions","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in yeast with multiple suppressor alleles tested; single lab","pmids":["25332237"],"is_preprint":false},{"year":2015,"finding":"VCP (type II AAA-ATPase) directly interacts with PSMF1/PI31 and the two proteins antagonistically regulate proteasomal activity.","method":"Direct interaction assay (co-immunoprecipitation/pull-down implied), proteasome activity measurements","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, limited methodological detail in abstract; interaction and functional antagonism reported but methods not fully described","pmids":["26086101"],"is_preprint":false},{"year":2019,"finding":"PI31 serves as an adaptor to couple proteasomes with dynein light chain proteins DYNLL1/2, enabling microtubule-dependent transport of proteasomes in axons. Phosphorylation of PI31 by p38 MAPK enhances PI31 binding to DYNLL1/2 and promotes directional movement of proteasomes in axons. Inactivation of PI31 inhibited proteasome motility in axons and disrupted synaptic proteostasis, structure, and function.","method":"Co-immunoprecipitation, live-cell axonal transport imaging, phosphorylation assays, PI31 inactivation in Drosophila and mouse neurons, synaptic structure/function readouts","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, live imaging of motility, genetic inactivation in two organisms, p38 MAPK phosphorylation assay, multiple orthogonal methods","pmids":["31327739"],"is_preprint":false},{"year":2019,"finding":"PI31 interacts with TBK1 and Sec16A at endoplasmic reticulum exit sites (ERES); NOD2/TLR2 signaling causes TBK1 to phosphorylate PI31 in dendritic cells, which positively regulates MHC class I peptide loading and immunoproteasome stability. Depletion of PI31 impairs DC cross-presentation and CD8+ T cell activation.","method":"Co-immunoprecipitation of PI31 with TBK1 and Sec16A, phosphorylation assay, PI31 depletion in dendritic cells, cross-presentation assay, in vivo CD8+ T cell activation","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — PI31–TBK1 interaction and phosphorylation demonstrated; functional consequences shown in primary DCs and in vivo, single lab","pmids":["31114588"],"is_preprint":false},{"year":2019,"finding":"Conditional knockout of PI31 in mouse spinal motor neurons and cerebellar Purkinje cells causes markers of proteotoxic stress followed by axon degeneration, neuronal loss, and progressive motor dysfunction, establishing PI31 as essential for neuronal protein homeostasis in vivo.","method":"Conditional knockout mouse model, histological and behavioral analysis, proteotoxic stress markers","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO in two distinct neuron types with temporal dissection of proteotoxic stress preceding degeneration; replicated across cell types","pmids":["31754024"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structure of S. cerevisiae PI31 (Fub1) inside the proteasome core particle shows that the conserved C-terminal domain simultaneously interacts with all six active sites. Targeted mutations disrupt inhibition at individual active sites independently. Fub1 evades degradation through distinct mechanisms at each active site. The proteasome gate is constitutively closed when Fub1 is bound, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting Fub1 neutralizes aberrant proteasomes.","method":"Cryo-EM structure determination, site-directed mutagenesis of active-site contacts, biochemical proteasome inhibition assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure at high resolution combined with targeted mutagenesis and functional assays; multiple orthogonal methods","pmids":["35927584"],"is_preprint":false},{"year":2023,"finding":"High-resolution cryo-EM structure of the mammalian 20S proteasome–PI31 complex shows that two copies of the intrinsically disordered C-terminus of PI31 enter the central cavity from opposite ends of the 20S cylinder and interact with catalytic sites in a closed-gate conformation, blocking proteolysis while resisting their own degradation. PI31 can inhibit proteasome activity in mammalian cells.","method":"Cryo-EM structure determination, cellular proteasome activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with cellular functional validation; complements yeast structural data","pmids":["37236357"],"is_preprint":false},{"year":2024,"finding":"PI31 inhibits the constitutive 20S proteasome (20Sc) more strongly than the immunoproteasome (20Si). Unlike 20Sc, 20Si hydrolyzes the C-terminus of PI31, contributing to reduced PI31 inhibitory activity toward 20Si. PI31 point mutants that lose inhibition of 20Sc are degraded by 20Sc.","method":"In vitro proteasome activity assays with purified 20Sc and 20Si, PI31 point mutant analysis, proteolytic degradation assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis comparing two proteasome isoforms; single lab with multiple orthogonal assays","pmids":["38577872"],"is_preprint":false},{"year":2024,"finding":"An FBXO7 L250P patient mutation selectively ablates the Fbxo7–PI31 interaction and causes reduced Fbxo7 and PI31 levels in patient fibroblasts, reduced proteasome activity and proteasome subunit levels. PI31 interacts with mitochondrial fission adaptors MiD49/51 and facilitates SCF(Fbxo7)-mediated ubiquitination of MiD49.","method":"Patient fibroblast analysis, co-immunoprecipitation, proteasome activity assay, ubiquitination assay, L250P mutant structural validation","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP of PI31–MiD49/51, ubiquitination assay, patient mutation functional dissection; single lab","pmids":["38466799"],"is_preprint":false},{"year":2024,"finding":"The INF2 R218Q mutation disrupts sequestration of Dynll1 by INF2, freeing Dynll1 to interact with PI31 and promoting dynein-mediated transport of nephrin to the proteasome for degradation. Knockdown of Dynll1 or PI31, dynein inactivation, or proteasome inhibition each restores nephrin proteostasis in R218Q podocytes.","method":"Genetic knockdown (Dynll1, PI31), dynein inactivation, proteasome inhibition, nephrin stability assays in R218Q KI mouse podocytes, in vivo mouse model","journal":"Kidney360","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic interventions in cell and mouse model; mechanistic placement of PI31 in dynein-proteasome coupling pathway; single lab","pmids":["39621430"],"is_preprint":false},{"year":2025,"finding":"Genetic ablation of PI31 in mammalian cells had no effect on constitutive proteasome content or activity but reduced the cellular content and activity of interferon-γ-induced immunoproteasomes (20Si) due to impaired 20Si assembly, evidenced by accumulation of 20Si assembly intermediates. PI31 thus plays a chaperone-like role specifically in 20Si assembly.","method":"CRISPR/Cas9 genetic ablation in mammalian cells, immunoproteasome induction by IFN-γ, proteasome activity assays, analysis of assembly intermediates","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple functional readouts (activity, assembly intermediates), selective effect on immunoproteasome replicated in peer-reviewed publication following preprint","pmids":["40337847","39868238"],"is_preprint":false},{"year":2025,"finding":"Restoring PI31 levels in Fbxo7 mutant flies and mice prevents neuronal degeneration and improves neuronal function and lifespan. Fbxo7 inactivation in mouse neurons causes tau hyperphosphorylation, which is suppressed by transgenic PI31 expression, establishing PI31 as the key downstream effector of Fbxo7 in neuroprotection.","method":"Transgenic PI31 expression in Fbxo7 mutant Drosophila and mouse models, neuronal survival assays, tau phosphorylation analysis, lifespan and locomotor assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic rescue in two organisms with multiple phenotypic readouts including tau hyperphosphorylation suppression; positions PI31 downstream of Fbxo7","pmids":["40956890"],"is_preprint":false},{"year":2025,"finding":"Biallelic PSMF1 loss-of-function variants in patients impair mitochondrial membrane potential, dynamics, and mitophagy, and reduce proteasomal abundance and assembly in patient-derived fibroblasts. PI31 loss-of-function in Drosophila causes dopaminergic neurodegeneration and mitochondrial depolarization.","method":"Patient-derived fibroblast functional assays (mitochondrial membrane potential, dynamics, mitophagy, proteasome assembly), Drosophila and mouse loss-of-function models","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays in patient cells plus two model organisms; single consortium but multiple orthogonal methods","pmids":["41986367","39148840"],"is_preprint":false}],"current_model":"PSMF1/PI31 is a multifunctional proteasome regulator whose intrinsically disordered, proline-rich C-terminus physically enters the 20S proteasome barrel and simultaneously engages all catalytic active sites to inhibit proteolysis while resisting its own degradation; its N-terminal FP domain mediates homodimerization and heterodimerization with Fbxo7 (an interaction required for normal proteasome activity and mitochondrial homeostasis); PI31 also acts as an adaptor coupling proteasomes to dynein light chains DYNLL1/2 for p38 MAPK-regulated axonal transport of proteasomes to synapses; it plays a chaperone-like role in 20S immunoproteasome assembly; and it is phosphorylated by TBK1 downstream of NOD2/TLR2 signaling to regulate MHC class I cross-presentation, with loss of PI31 in neurons causing proteotoxic stress, axon degeneration, and neurodegeneration in mice."},"narrative":{"mechanistic_narrative":"PSMF1/PI31 is a multifunctional proteasome regulator that controls 20S proteasome activity, proteasome assembly, and the spatial delivery of proteasomes within neurons [PMID:10764772, PMID:31327739, PMID:40337847, PMID:39868238]. Its intrinsically disordered, proline-rich C-terminus physically enters the 20S core particle: two copies thread into the central cavity from opposite ends of the cylinder and simultaneously engage all six catalytic sites in a closed-gate conformation, blocking proteolysis of both peptide and protein substrates while resisting its own degradation [PMID:10764772, PMID:35927584, PMID:37236357]. PI31 inhibits the constitutive 20S proteasome more potently than the immunoproteasome, which can cleave the PI31 C-terminus and thereby escape inhibition [PMID:38577872], and structural data indicate PI31 is enriched on aberrant core particles with abnormally open gates, consistent with a role in neutralizing defective proteasomes [PMID:35927584]. Distinct from this inhibitory function, PI31 acts as a chaperone-like factor required specifically for assembly of the interferon-γ-induced immunoproteasome [PMID:40337847, PMID:39868238], and serves as an adaptor that couples proteasomes to dynein light chains DYNLL1/2 for microtubule-dependent axonal transport, an interaction enhanced by p38 MAPK phosphorylation [PMID:31327739]. Through its conserved N-terminal FP domain PI31 homodimerizes and heterodimerizes with Fbxo7·Skp1, an interaction required for normal proteasome activity and mitochondrial homeostasis [PMID:18495667, PMID:38466799], and PI31 is the key downstream effector of Fbxo7 in neuroprotection [PMID:40956890]. Loss of PI31 in neurons produces proteotoxic stress, axon degeneration, and progressive neurodegeneration in mice, and biallelic PSMF1 loss-of-function variants in patients impair mitochondrial function and reduce proteasome assembly [PMID:31754024, PMID:41986367, PMID:39148840].","teleology":[{"year":2000,"claim":"Established PI31 as a direct proteasome inhibitor and localized the inhibitory activity to its proline-rich C-terminus, defining its founding biochemical function.","evidence":"Recombinant protein, truncation mutants, in vitro proteasome activity assays","pmids":["10764772"],"confidence":"High","gaps":["Structural basis of inhibition unresolved","Physiological cellular consequence of inhibition not addressed"]},{"year":2008,"claim":"Defined the N-terminal FP domain as the structural module mediating PI31 homodimerization and heterodimerization with Fbxo7·Skp1, and showed PI31 is not an SCF(Fbxo7) substrate.","evidence":"Crystal structure, SEC/ITC, mutagenesis, co-IP, RNAi in cells","pmids":["18495667"],"confidence":"High","gaps":["Functional consequence of the Fbxo7 interaction not defined","Cellular role of dimerization unknown"]},{"year":2014,"claim":"Clarified that PI31 carries an HbYX motif yet acts as an inhibitor, blocking 26S assembly in vitro, while revealing no detectable effect on bulk cellular proteasome activity upon perturbation.","evidence":"In vitro activity/assembly assays with mutants, RNAi and overexpression in cells","pmids":["24770418"],"confidence":"High","gaps":["Discrepancy between in vitro inhibition and absent cellular phenotype unexplained","In vivo substrate context unknown"]},{"year":2014,"claim":"Genetic epistasis in yeast positioned the PI31 ortholog Fub1 as a functional antagonist of α7-mediated proteasome gate opening, connecting PI31 to core-particle activation control.","evidence":"Yeast double-mutant epistasis and α-subunit N-terminal deletion suppressor analysis","pmids":["25332237"],"confidence":"Medium","gaps":["Direct molecular interaction with α7 not shown structurally","Relevance to mammalian PI31 not established here"]},{"year":2015,"claim":"Identified VCP as a direct PI31 interactor that antagonistically co-regulates proteasome activity, adding an AAA-ATPase node to PI31 regulation.","evidence":"Interaction assay and proteasome activity measurements","pmids":["26086101"],"confidence":"Medium","gaps":["Limited methodological detail; interaction not reciprocally validated","Mechanism of antagonism undefined"]},{"year":2019,"claim":"Revealed a transport function distinct from inhibition: PI31 adapts proteasomes to dynein light chains DYNLL1/2 for axonal transport, regulated by p38 MAPK phosphorylation, linking PI31 to synaptic proteostasis.","evidence":"Reciprocal co-IP, live axonal transport imaging, phosphorylation assays, genetic inactivation in Drosophila and mouse neurons","pmids":["31327739"],"confidence":"High","gaps":["How transport and inhibitory functions are coordinated unclear","Structural basis of DYNLL1/2 coupling not resolved"]},{"year":2019,"claim":"Connected PI31 to innate-immune signaling and antigen presentation, showing TBK1 phosphorylates PI31 downstream of NOD2/TLR2 to regulate MHC class I cross-presentation and immunoproteasome stability.","evidence":"Co-IP with TBK1 and Sec16A, phosphorylation assay, PI31 depletion in dendritic cells, cross-presentation and in vivo CD8+ T cell assays","pmids":["31114588"],"confidence":"Medium","gaps":["Phosphosite-resolved mechanism not defined","Direct structural link to immunoproteasome unestablished here"]},{"year":2019,"claim":"Demonstrated in vivo that PI31 is essential for neuronal protein homeostasis, with proteotoxic stress preceding axon degeneration and neuronal loss.","evidence":"Conditional knockout in mouse motor neurons and Purkinje cells, histology, behavior, proteotoxic stress markers","pmids":["31754024"],"confidence":"High","gaps":["Which PI31 function (inhibition vs transport) drives neuroprotection not separated","Molecular trigger of proteotoxic stress unresolved"]},{"year":2022,"claim":"Provided the structural mechanism of inhibition: the conserved C-terminal domain of the yeast ortholog simultaneously contacts all six active sites and evades degradation by distinct mechanisms, while keeping the gate closed and accumulating on aberrant proteasomes.","evidence":"Cryo-EM of Fub1 inside the core particle, active-site contact mutagenesis, inhibition assays","pmids":["35927584"],"confidence":"High","gaps":["Whether the aberrant-proteasome-neutralizing role operates in mammals not tested","Regulation of entry into the barrel unknown"]},{"year":2023,"claim":"Confirmed the conserved inhibitory architecture in mammals, showing two PI31 C-termini enter the 20S cavity from opposite ends in a closed-gate state, and that PI31 inhibits proteasomes in mammalian cells.","evidence":"High-resolution cryo-EM of mammalian 20S–PI31 complex, cellular proteasome activity assays","pmids":["37236357"],"confidence":"High","gaps":["Stoichiometry and dynamics of binding in vivo unresolved","How inhibition is relieved physiologically unknown"]},{"year":2024,"claim":"Distinguished PI31 action on the two proteasome isoforms, showing it inhibits the constitutive 20S more strongly because the immunoproteasome cleaves its C-terminus, with inhibition and degradation-resistance being coupled.","evidence":"In vitro assays with purified 20Sc and 20Si, point-mutant and degradation analyses","pmids":["38577872"],"confidence":"High","gaps":["Cellular consequence of differential isoform inhibition not addressed here","Cleavage site mapping incomplete"]},{"year":2024,"claim":"Tied the Fbxo7–PI31 axis to human disease and mitochondrial regulation, showing a patient FBXO7 L250P mutation ablates the interaction, lowers PI31 and proteasome activity, and that PI31 facilitates SCF(Fbxo7) ubiquitination of fission adaptor MiD49.","evidence":"Patient fibroblasts, co-IP, proteasome activity and ubiquitination assays, mutant structural validation","pmids":["38466799"],"confidence":"Medium","gaps":["Direct PI31–MiD49/51 interaction not reciprocally confirmed","Mechanism by which PI31 promotes MiD49 ubiquitination undefined"]},{"year":2024,"claim":"Placed PI31 within a disease-relevant dynein-proteasome degradation pathway, where freed Dynll1 in INF2 R218Q podocytes engages PI31 to deliver nephrin for proteasomal degradation.","evidence":"Knockdown of Dynll1/PI31, dynein inactivation, proteasome inhibition, nephrin stability assays in R218Q knock-in mouse podocytes","pmids":["39621430"],"confidence":"Medium","gaps":["Direct PI31–nephrin physical link not shown","Generality beyond podocyte context unknown"]},{"year":2025,"claim":"Defined a chaperone-like assembly function specific to the immunoproteasome, showing PI31 ablation impairs 20Si assembly without affecting constitutive proteasome content or activity.","evidence":"CRISPR/Cas9 ablation in mammalian cells, IFN-γ induction, activity assays, assembly intermediate analysis","pmids":["40337847","39868238"],"confidence":"High","gaps":["Molecular step in 20Si assembly catalyzed by PI31 unresolved","Relationship to its inhibitory function unclear"]},{"year":2025,"claim":"Established PI31 as the key downstream effector of Fbxo7 neuroprotection, with PI31 restoration rescuing neurodegeneration, lifespan, and suppressing tau hyperphosphorylation in Fbxo7 mutants.","evidence":"Transgenic PI31 rescue in Fbxo7 mutant Drosophila and mouse, neuronal survival, tau phosphorylation, lifespan/locomotor assays","pmids":["40956890"],"confidence":"High","gaps":["Mechanism linking PI31 to tau phosphorylation undefined","Which PI31 molecular activity mediates rescue not isolated"]},{"year":2025,"claim":"Linked biallelic PSMF1 loss-of-function to a human disorder with mitochondrial and proteasomal defects, recapitulated by dopaminergic neurodegeneration in flies.","evidence":"Patient fibroblast assays (membrane potential, dynamics, mitophagy, assembly), Drosophila and mouse loss-of-function models","pmids":["41986367","39148840"],"confidence":"Medium","gaps":["Causal chain from PI31 loss to mitochondrial depolarization unresolved","Genotype-phenotype spectrum not fully defined"]},{"year":null,"claim":"How PI31's multiple functions — active-site inhibition, immunoproteasome assembly, dynein-mediated transport, and Fbxo7-dependent mitochondrial regulation — are coordinated and which is decisive for neuroprotection remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coupling inhibitory and adaptor functions","Regulatory switch governing barrel entry vs transport unknown","Direct mechanistic basis for mitochondrial phenotypes undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,8,9,10]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,12]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[9]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,8,9,13]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,13]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[5]}],"complexes":["20S proteasome core particle","immunoproteasome (20Si)","SCF(Fbxo7)"],"partners":["FBXO7","DYNLL1","DYNLL2","TBK1","SEC16A","VCP","MIEF2","MIEF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q92530","full_name":"Proteasome inhibitor PI31 subunit","aliases":[],"length_aa":271,"mass_kda":29.8,"function":"Plays an important role in control of proteasome function. Inhibits the hydrolysis of protein and peptide substrates by the 20S proteasome. Also inhibits the activation of the proteasome by the proteasome regulatory proteins PA700 and PA28","subcellular_location":"Cytoplasm; Endoplasmic reticulum","url":"https://www.uniprot.org/uniprotkb/Q92530/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PSMF1","classification":"Not Classified","n_dependent_lines":228,"n_total_lines":1208,"dependency_fraction":0.18874172185430463},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000125818","cell_line_id":"CID000114","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"PSMA7","stoichiometry":10.0},{"gene":"PSMD4","stoichiometry":4.0},{"gene":"PSMA2","stoichiometry":4.0},{"gene":"PSMA5","stoichiometry":4.0},{"gene":"CBX1","stoichiometry":0.2},{"gene":"PSMA6","stoichiometry":0.2},{"gene":"PSMB1","stoichiometry":0.2},{"gene":"PSMC1","stoichiometry":0.2},{"gene":"PSMD6","stoichiometry":0.2},{"gene":"UCHL5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000114","total_profiled":1310},"omim":[{"mim_id":"617858","title":"PROTEASOME INHIBITOR SUBUNIT 1; PSMF1","url":"https://www.omim.org/entry/617858"},{"mim_id":"617676","title":"PROTEASOME 26S SUBUNIT, NON-ATPase, 3; PSMD3","url":"https://www.omim.org/entry/617676"},{"mim_id":"312180","title":"UBIQUITIN-CONJUGATING ENZYME E2 A; UBE2A","url":"https://www.omim.org/entry/312180"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Cytosol","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PSMF1"},"hgnc":{"alias_symbol":["PI31"],"prev_symbol":[]},"alphafold":{"accession":"Q92530","domains":[{"cath_id":"3.40.1000.30","chopping":"3-150","consensus_level":"high","plddt":93.831,"start":3,"end":150}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92530","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q92530-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q92530-F1-predicted_aligned_error_v6.png","plddt_mean":74.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PSMF1","jax_strain_url":"https://www.jax.org/strain/search?query=PSMF1"},"sequence":{"accession":"Q92530","fasta_url":"https://rest.uniprot.org/uniprotkb/Q92530.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q92530/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92530"}},"corpus_meta":[{"pmid":"10764772","id":"PMC_10764772","title":"cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10764772","citation_count":110,"is_preprint":false},{"pmid":"18495667","id":"PMC_18495667","title":"Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18495667","citation_count":76,"is_preprint":false},{"pmid":"24770418","id":"PMC_24770418","title":"Molecular and cellular roles of PI31 (PSMF1) protein in regulation of proteasome function.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24770418","citation_count":55,"is_preprint":false},{"pmid":"31327739","id":"PMC_31327739","title":"PI31 Is an Adaptor Protein for Proteasome Transport in Axons and Required for Synaptic 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function.","date":"2025","source":"medRxiv : the preprint server for health sciences","url":"https://pubmed.ncbi.nlm.nih.gov/39148840","citation_count":5,"is_preprint":false},{"pmid":"27936908","id":"PMC_27936908","title":"The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson's disease.","date":"2016","source":"Genome","url":"https://pubmed.ncbi.nlm.nih.gov/27936908","citation_count":5,"is_preprint":false},{"pmid":"38577872","id":"PMC_38577872","title":"Differential Interactions of the Proteasome Inhibitor PI31 with Constitutive and Immuno-20S Proteasomes.","date":"2024","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38577872","citation_count":4,"is_preprint":false},{"pmid":"40337847","id":"PMC_40337847","title":"PI31 is a positive regulator of 20S immunoproteasome assembly.","date":"2025","source":"Journal of cell 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interactions","date":"2025-09-21","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.20.677505","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13871,"output_tokens":4262,"usd":0.052771,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12249,"output_tokens":5018,"usd":0.093347,"stage2_stop_reason":"end_turn"},"total_usd":0.146118,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"PI31 is a proline-rich inhibitor of the 20S proteasome; its C-terminal proline-rich domain confers inhibition by forming a proteasome-PI31 complex and blocking hydrolysis of both protein and peptide substrates. PI31 also inhibits activation of the proteasome by regulatory proteins PA700 and PA28.\",\n      \"method\": \"Recombinant protein expression in E. coli, truncation mutant analysis, in vitro proteasome activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with defined truncation mutants and multiple orthogonal assays (protein and peptide substrate hydrolysis, complex formation, inhibition of two distinct activators)\",\n      \"pmids\": [\"10764772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PI31 interacts with Fbxo7·Skp1 through a conserved N-terminal FP (Fbxo7/PI31) domain that mediates both homodimerization of PI31 and heterodimerization with Fbxo7. The crystal structure of the PI31 FP domain reveals a novel α/β-fold. Knockdown of Fbxo7 does not affect PI31 levels, arguing against PI31 being an SCF(Fbxo7) substrate.\",\n      \"method\": \"Crystal structure determination, biophysical analysis (SEC, ITC), site-directed mutagenesis, co-immunoprecipitation, RNAi knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and binding assays; multiple orthogonal methods in one study\",\n      \"pmids\": [\"18495667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PI31 contains a C-terminal HbYX motif; peptides corresponding to this motif bind to and activate the 20S proteasome in an HbYX-dependent manner, but intact PI31 inhibits 20S activity. PI31 blocks ATP-dependent in vitro assembly of 26S proteasome from 20S and PA700 subcomplexes but has no effect on activity of intact 26S proteasome. Ectopic overexpression or RNAi knockdown of PI31 in cells produced no detectable change in overall cellular proteasome content or function.\",\n      \"method\": \"In vitro proteasome activity assays with truncation and point mutants, 26S assembly assay, RNAi knockdown, ectopic overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple in vitro reconstitution experiments plus cellular RNAi/overexpression; single lab but orthogonal methods\",\n      \"pmids\": [\"24770418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In yeast (S. cerevisiae), the PI31 ortholog Fub1 is essential when the CP assembly chaperone Pba4 is deleted. Deletion of the N-terminus of α7 (α7ΔN), but not α3ΔN, suppresses the lethality of Δfub1 Δpba4, indicating that Fub1 functionally antagonizes a specific gate-opening role of the α7 N-terminus in CP activation.\",\n      \"method\": \"Yeast genetic epistasis (double-mutant analysis), suppressor screen with α-subunit N-terminal deletions\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in yeast with multiple suppressor alleles tested; single lab\",\n      \"pmids\": [\"25332237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"VCP (type II AAA-ATPase) directly interacts with PSMF1/PI31 and the two proteins antagonistically regulate proteasomal activity.\",\n      \"method\": \"Direct interaction assay (co-immunoprecipitation/pull-down implied), proteasome activity measurements\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited methodological detail in abstract; interaction and functional antagonism reported but methods not fully described\",\n      \"pmids\": [\"26086101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PI31 serves as an adaptor to couple proteasomes with dynein light chain proteins DYNLL1/2, enabling microtubule-dependent transport of proteasomes in axons. Phosphorylation of PI31 by p38 MAPK enhances PI31 binding to DYNLL1/2 and promotes directional movement of proteasomes in axons. Inactivation of PI31 inhibited proteasome motility in axons and disrupted synaptic proteostasis, structure, and function.\",\n      \"method\": \"Co-immunoprecipitation, live-cell axonal transport imaging, phosphorylation assays, PI31 inactivation in Drosophila and mouse neurons, synaptic structure/function readouts\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, live imaging of motility, genetic inactivation in two organisms, p38 MAPK phosphorylation assay, multiple orthogonal methods\",\n      \"pmids\": [\"31327739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PI31 interacts with TBK1 and Sec16A at endoplasmic reticulum exit sites (ERES); NOD2/TLR2 signaling causes TBK1 to phosphorylate PI31 in dendritic cells, which positively regulates MHC class I peptide loading and immunoproteasome stability. Depletion of PI31 impairs DC cross-presentation and CD8+ T cell activation.\",\n      \"method\": \"Co-immunoprecipitation of PI31 with TBK1 and Sec16A, phosphorylation assay, PI31 depletion in dendritic cells, cross-presentation assay, in vivo CD8+ T cell activation\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — PI31–TBK1 interaction and phosphorylation demonstrated; functional consequences shown in primary DCs and in vivo, single lab\",\n      \"pmids\": [\"31114588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Conditional knockout of PI31 in mouse spinal motor neurons and cerebellar Purkinje cells causes markers of proteotoxic stress followed by axon degeneration, neuronal loss, and progressive motor dysfunction, establishing PI31 as essential for neuronal protein homeostasis in vivo.\",\n      \"method\": \"Conditional knockout mouse model, histological and behavioral analysis, proteotoxic stress markers\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO in two distinct neuron types with temporal dissection of proteotoxic stress preceding degeneration; replicated across cell types\",\n      \"pmids\": [\"31754024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structure of S. cerevisiae PI31 (Fub1) inside the proteasome core particle shows that the conserved C-terminal domain simultaneously interacts with all six active sites. Targeted mutations disrupt inhibition at individual active sites independently. Fub1 evades degradation through distinct mechanisms at each active site. The proteasome gate is constitutively closed when Fub1 is bound, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting Fub1 neutralizes aberrant proteasomes.\",\n      \"method\": \"Cryo-EM structure determination, site-directed mutagenesis of active-site contacts, biochemical proteasome inhibition assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure at high resolution combined with targeted mutagenesis and functional assays; multiple orthogonal methods\",\n      \"pmids\": [\"35927584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"High-resolution cryo-EM structure of the mammalian 20S proteasome–PI31 complex shows that two copies of the intrinsically disordered C-terminus of PI31 enter the central cavity from opposite ends of the 20S cylinder and interact with catalytic sites in a closed-gate conformation, blocking proteolysis while resisting their own degradation. PI31 can inhibit proteasome activity in mammalian cells.\",\n      \"method\": \"Cryo-EM structure determination, cellular proteasome activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with cellular functional validation; complements yeast structural data\",\n      \"pmids\": [\"37236357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PI31 inhibits the constitutive 20S proteasome (20Sc) more strongly than the immunoproteasome (20Si). Unlike 20Sc, 20Si hydrolyzes the C-terminus of PI31, contributing to reduced PI31 inhibitory activity toward 20Si. PI31 point mutants that lose inhibition of 20Sc are degraded by 20Sc.\",\n      \"method\": \"In vitro proteasome activity assays with purified 20Sc and 20Si, PI31 point mutant analysis, proteolytic degradation assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis comparing two proteasome isoforms; single lab with multiple orthogonal assays\",\n      \"pmids\": [\"38577872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"An FBXO7 L250P patient mutation selectively ablates the Fbxo7–PI31 interaction and causes reduced Fbxo7 and PI31 levels in patient fibroblasts, reduced proteasome activity and proteasome subunit levels. PI31 interacts with mitochondrial fission adaptors MiD49/51 and facilitates SCF(Fbxo7)-mediated ubiquitination of MiD49.\",\n      \"method\": \"Patient fibroblast analysis, co-immunoprecipitation, proteasome activity assay, ubiquitination assay, L250P mutant structural validation\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP of PI31–MiD49/51, ubiquitination assay, patient mutation functional dissection; single lab\",\n      \"pmids\": [\"38466799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The INF2 R218Q mutation disrupts sequestration of Dynll1 by INF2, freeing Dynll1 to interact with PI31 and promoting dynein-mediated transport of nephrin to the proteasome for degradation. Knockdown of Dynll1 or PI31, dynein inactivation, or proteasome inhibition each restores nephrin proteostasis in R218Q podocytes.\",\n      \"method\": \"Genetic knockdown (Dynll1, PI31), dynein inactivation, proteasome inhibition, nephrin stability assays in R218Q KI mouse podocytes, in vivo mouse model\",\n      \"journal\": \"Kidney360\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic interventions in cell and mouse model; mechanistic placement of PI31 in dynein-proteasome coupling pathway; single lab\",\n      \"pmids\": [\"39621430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Genetic ablation of PI31 in mammalian cells had no effect on constitutive proteasome content or activity but reduced the cellular content and activity of interferon-γ-induced immunoproteasomes (20Si) due to impaired 20Si assembly, evidenced by accumulation of 20Si assembly intermediates. PI31 thus plays a chaperone-like role specifically in 20Si assembly.\",\n      \"method\": \"CRISPR/Cas9 genetic ablation in mammalian cells, immunoproteasome induction by IFN-γ, proteasome activity assays, analysis of assembly intermediates\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple functional readouts (activity, assembly intermediates), selective effect on immunoproteasome replicated in peer-reviewed publication following preprint\",\n      \"pmids\": [\"40337847\", \"39868238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Restoring PI31 levels in Fbxo7 mutant flies and mice prevents neuronal degeneration and improves neuronal function and lifespan. Fbxo7 inactivation in mouse neurons causes tau hyperphosphorylation, which is suppressed by transgenic PI31 expression, establishing PI31 as the key downstream effector of Fbxo7 in neuroprotection.\",\n      \"method\": \"Transgenic PI31 expression in Fbxo7 mutant Drosophila and mouse models, neuronal survival assays, tau phosphorylation analysis, lifespan and locomotor assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic rescue in two organisms with multiple phenotypic readouts including tau hyperphosphorylation suppression; positions PI31 downstream of Fbxo7\",\n      \"pmids\": [\"40956890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Biallelic PSMF1 loss-of-function variants in patients impair mitochondrial membrane potential, dynamics, and mitophagy, and reduce proteasomal abundance and assembly in patient-derived fibroblasts. PI31 loss-of-function in Drosophila causes dopaminergic neurodegeneration and mitochondrial depolarization.\",\n      \"method\": \"Patient-derived fibroblast functional assays (mitochondrial membrane potential, dynamics, mitophagy, proteasome assembly), Drosophila and mouse loss-of-function models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays in patient cells plus two model organisms; single consortium but multiple orthogonal methods\",\n      \"pmids\": [\"41986367\", \"39148840\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PSMF1/PI31 is a multifunctional proteasome regulator whose intrinsically disordered, proline-rich C-terminus physically enters the 20S proteasome barrel and simultaneously engages all catalytic active sites to inhibit proteolysis while resisting its own degradation; its N-terminal FP domain mediates homodimerization and heterodimerization with Fbxo7 (an interaction required for normal proteasome activity and mitochondrial homeostasis); PI31 also acts as an adaptor coupling proteasomes to dynein light chains DYNLL1/2 for p38 MAPK-regulated axonal transport of proteasomes to synapses; it plays a chaperone-like role in 20S immunoproteasome assembly; and it is phosphorylated by TBK1 downstream of NOD2/TLR2 signaling to regulate MHC class I cross-presentation, with loss of PI31 in neurons causing proteotoxic stress, axon degeneration, and neurodegeneration in mice.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PSMF1/PI31 is a multifunctional proteasome regulator that controls 20S proteasome activity, proteasome assembly, and the spatial delivery of proteasomes within neurons [#0, #5, #13]. Its intrinsically disordered, proline-rich C-terminus physically enters the 20S core particle: two copies thread into the central cavity from opposite ends of the cylinder and simultaneously engage all six catalytic sites in a closed-gate conformation, blocking proteolysis of both peptide and protein substrates while resisting its own degradation [#0, #8, #9]. PI31 inhibits the constitutive 20S proteasome more potently than the immunoproteasome, which can cleave the PI31 C-terminus and thereby escape inhibition [#10], and structural data indicate PI31 is enriched on aberrant core particles with abnormally open gates, consistent with a role in neutralizing defective proteasomes [#8]. Distinct from this inhibitory function, PI31 acts as a chaperone-like factor required specifically for assembly of the interferon-\\u03b3-induced immunoproteasome [#13], and serves as an adaptor that couples proteasomes to dynein light chains DYNLL1/2 for microtubule-dependent axonal transport, an interaction enhanced by p38 MAPK phosphorylation [#5]. Through its conserved N-terminal FP domain PI31 homodimerizes and heterodimerizes with Fbxo7\\u00b7Skp1, an interaction required for normal proteasome activity and mitochondrial homeostasis [#1, #11], and PI31 is the key downstream effector of Fbxo7 in neuroprotection [#14]. Loss of PI31 in neurons produces proteotoxic stress, axon degeneration, and progressive neurodegeneration in mice, and biallelic PSMF1 loss-of-function variants in patients impair mitochondrial function and reduce proteasome assembly [#7, #15].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established PI31 as a direct proteasome inhibitor and localized the inhibitory activity to its proline-rich C-terminus, defining its founding biochemical function.\",\n      \"evidence\": \"Recombinant protein, truncation mutants, in vitro proteasome activity assays\",\n      \"pmids\": [\"10764772\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of inhibition unresolved\", \"Physiological cellular consequence of inhibition not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the N-terminal FP domain as the structural module mediating PI31 homodimerization and heterodimerization with Fbxo7\\u00b7Skp1, and showed PI31 is not an SCF(Fbxo7) substrate.\",\n      \"evidence\": \"Crystal structure, SEC/ITC, mutagenesis, co-IP, RNAi in cells\",\n      \"pmids\": [\"18495667\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the Fbxo7 interaction not defined\", \"Cellular role of dimerization unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Clarified that PI31 carries an HbYX motif yet acts as an inhibitor, blocking 26S assembly in vitro, while revealing no detectable effect on bulk cellular proteasome activity upon perturbation.\",\n      \"evidence\": \"In vitro activity/assembly assays with mutants, RNAi and overexpression in cells\",\n      \"pmids\": [\"24770418\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Discrepancy between in vitro inhibition and absent cellular phenotype unexplained\", \"In vivo substrate context unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Genetic epistasis in yeast positioned the PI31 ortholog Fub1 as a functional antagonist of \\u03b17-mediated proteasome gate opening, connecting PI31 to core-particle activation control.\",\n      \"evidence\": \"Yeast double-mutant epistasis and \\u03b1-subunit N-terminal deletion suppressor analysis\",\n      \"pmids\": [\"25332237\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular interaction with \\u03b17 not shown structurally\", \"Relevance to mammalian PI31 not established here\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified VCP as a direct PI31 interactor that antagonistically co-regulates proteasome activity, adding an AAA-ATPase node to PI31 regulation.\",\n      \"evidence\": \"Interaction assay and proteasome activity measurements\",\n      \"pmids\": [\"26086101\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Limited methodological detail; interaction not reciprocally validated\", \"Mechanism of antagonism undefined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a transport function distinct from inhibition: PI31 adapts proteasomes to dynein light chains DYNLL1/2 for axonal transport, regulated by p38 MAPK phosphorylation, linking PI31 to synaptic proteostasis.\",\n      \"evidence\": \"Reciprocal co-IP, live axonal transport imaging, phosphorylation assays, genetic inactivation in Drosophila and mouse neurons\",\n      \"pmids\": [\"31327739\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How transport and inhibitory functions are coordinated unclear\", \"Structural basis of DYNLL1/2 coupling not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected PI31 to innate-immune signaling and antigen presentation, showing TBK1 phosphorylates PI31 downstream of NOD2/TLR2 to regulate MHC class I cross-presentation and immunoproteasome stability.\",\n      \"evidence\": \"Co-IP with TBK1 and Sec16A, phosphorylation assay, PI31 depletion in dendritic cells, cross-presentation and in vivo CD8+ T cell assays\",\n      \"pmids\": [\"31114588\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosite-resolved mechanism not defined\", \"Direct structural link to immunoproteasome unestablished here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated in vivo that PI31 is essential for neuronal protein homeostasis, with proteotoxic stress preceding axon degeneration and neuronal loss.\",\n      \"evidence\": \"Conditional knockout in mouse motor neurons and Purkinje cells, histology, behavior, proteotoxic stress markers\",\n      \"pmids\": [\"31754024\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which PI31 function (inhibition vs transport) drives neuroprotection not separated\", \"Molecular trigger of proteotoxic stress unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided the structural mechanism of inhibition: the conserved C-terminal domain of the yeast ortholog simultaneously contacts all six active sites and evades degradation by distinct mechanisms, while keeping the gate closed and accumulating on aberrant proteasomes.\",\n      \"evidence\": \"Cryo-EM of Fub1 inside the core particle, active-site contact mutagenesis, inhibition assays\",\n      \"pmids\": [\"35927584\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the aberrant-proteasome-neutralizing role operates in mammals not tested\", \"Regulation of entry into the barrel unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Confirmed the conserved inhibitory architecture in mammals, showing two PI31 C-termini enter the 20S cavity from opposite ends in a closed-gate state, and that PI31 inhibits proteasomes in mammalian cells.\",\n      \"evidence\": \"High-resolution cryo-EM of mammalian 20S\\u2013PI31 complex, cellular proteasome activity assays\",\n      \"pmids\": [\"37236357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and dynamics of binding in vivo unresolved\", \"How inhibition is relieved physiologically unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Distinguished PI31 action on the two proteasome isoforms, showing it inhibits the constitutive 20S more strongly because the immunoproteasome cleaves its C-terminus, with inhibition and degradation-resistance being coupled.\",\n      \"evidence\": \"In vitro assays with purified 20Sc and 20Si, point-mutant and degradation analyses\",\n      \"pmids\": [\"38577872\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular consequence of differential isoform inhibition not addressed here\", \"Cleavage site mapping incomplete\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Tied the Fbxo7\\u2013PI31 axis to human disease and mitochondrial regulation, showing a patient FBXO7 L250P mutation ablates the interaction, lowers PI31 and proteasome activity, and that PI31 facilitates SCF(Fbxo7) ubiquitination of fission adaptor MiD49.\",\n      \"evidence\": \"Patient fibroblasts, co-IP, proteasome activity and ubiquitination assays, mutant structural validation\",\n      \"pmids\": [\"38466799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PI31\\u2013MiD49/51 interaction not reciprocally confirmed\", \"Mechanism by which PI31 promotes MiD49 ubiquitination undefined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed PI31 within a disease-relevant dynein-proteasome degradation pathway, where freed Dynll1 in INF2 R218Q podocytes engages PI31 to deliver nephrin for proteasomal degradation.\",\n      \"evidence\": \"Knockdown of Dynll1/PI31, dynein inactivation, proteasome inhibition, nephrin stability assays in R218Q knock-in mouse podocytes\",\n      \"pmids\": [\"39621430\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PI31\\u2013nephrin physical link not shown\", \"Generality beyond podocyte context unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a chaperone-like assembly function specific to the immunoproteasome, showing PI31 ablation impairs 20Si assembly without affecting constitutive proteasome content or activity.\",\n      \"evidence\": \"CRISPR/Cas9 ablation in mammalian cells, IFN-\\u03b3 induction, activity assays, assembly intermediate analysis\",\n      \"pmids\": [\"40337847\", \"39868238\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular step in 20Si assembly catalyzed by PI31 unresolved\", \"Relationship to its inhibitory function unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established PI31 as the key downstream effector of Fbxo7 neuroprotection, with PI31 restoration rescuing neurodegeneration, lifespan, and suppressing tau hyperphosphorylation in Fbxo7 mutants.\",\n      \"evidence\": \"Transgenic PI31 rescue in Fbxo7 mutant Drosophila and mouse, neuronal survival, tau phosphorylation, lifespan/locomotor assays\",\n      \"pmids\": [\"40956890\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking PI31 to tau phosphorylation undefined\", \"Which PI31 molecular activity mediates rescue not isolated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked biallelic PSMF1 loss-of-function to a human disorder with mitochondrial and proteasomal defects, recapitulated by dopaminergic neurodegeneration in flies.\",\n      \"evidence\": \"Patient fibroblast assays (membrane potential, dynamics, mitophagy, assembly), Drosophila and mouse loss-of-function models\",\n      \"pmids\": [\"41986367\", \"39148840\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from PI31 loss to mitochondrial depolarization unresolved\", \"Genotype-phenotype spectrum not fully defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PI31's multiple functions \\u2014 active-site inhibition, immunoproteasome assembly, dynein-mediated transport, and Fbxo7-dependent mitochondrial regulation \\u2014 are coordinated and which is decisive for neuroprotection remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model coupling inhibitory and adaptor functions\", \"Regulatory switch governing barrel entry vs transport unknown\", \"Direct mechanistic basis for mitochondrial phenotypes undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 8, 9, 10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 12]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 8, 9, 13]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 13]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"20S proteasome core particle\", \"immunoproteasome (20Si)\", \"SCF(Fbxo7)\"],\n    \"partners\": [\"FBXO7\", \"DYNLL1\", \"DYNLL2\", \"TBK1\", \"SEC16A\", \"VCP\", \"MIEF2\", \"MIEF1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}