{"gene":"PEX5","run_date":"2026-04-29T11:37:58","timeline":{"discoveries":[{"year":1995,"finding":"PEX5 (PXR1) functions as the cytosolic receptor for peroxisomal targeting signal type-1 (PTS1), recognizing PTS1-containing proteins in the cytosol and directing them to the peroxisome. Mutations in PEX5 define complementation group 2 of peroxisome biogenesis disorders, and PEX5 expression rescues the PTS1 import defect in patient fibroblasts.","method":"Complementation assay in patient fibroblasts, homology cloning, subcellular fractionation showing cytosolic and peroxisome-associated pools","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — complementation rescue in patient cells, replicated across multiple labs subsequently","pmids":["7719337"],"is_preprint":false},{"year":2000,"finding":"Crystal structure of the C-terminal TPR domain of human PEX5 in complex with a PTS1 pentapeptide revealed that two clusters of three TPRs almost completely surround the peptide, while a hinge region (TPR4) enables the two sets to form a single binding site, establishing the molecular basis for PTS1 recognition via a novel TPR-peptide interaction mode.","method":"X-ray crystallography of PEX5 TPR domain in complex with PTS1 peptide","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation, highly cited foundational paper","pmids":["11101887"],"is_preprint":false},{"year":1998,"finding":"The longer isoform of PEX5 (PEX5L/PTS1RL) is required for PTS2 protein import in addition to PTS1 import, whereas the shorter isoform (PEX5S) supports only PTS1 import. Mutations in the TPR domains (TPR1 and TPR6) abolish protein translocation, demonstrating the functional importance of these domains.","method":"CHO cell mutant complementation assays, reverse transcription-PCR mutation analysis, isoform-specific rescue experiments","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — multiple complementation experiments with defined mutations, replicated by other labs","pmids":["9418886"],"is_preprint":false},{"year":1999,"finding":"PEX5 possesses multiple binding sites for PEX14 distributed throughout its N-terminal half, with nanomolar affinity interaction. The N-terminal half of PEX5 also mediates oligomerization, while the C-terminal TPR domain binds PTS1 cargo. Surface plasmon resonance demonstrated that PEX5 binds PEX14-(1-78) with very high affinity in the low nanomolar range.","method":"Surface plasmon resonance, in vitro binding assays, sizing chromatography, electron microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro reconstitution with quantitative binding measurements, multiple orthogonal methods","pmids":["10026185"],"is_preprint":false},{"year":1999,"finding":"PEX12 (zinc RING domain) binds both PEX5 and PEX10 downstream of receptor docking; a patient missense mutation S320F in the PEX12 zinc-binding domain reduces binding to both PEX5 and PEX10. Overexpression of PEX5 or PEX10 suppresses this PEX12 mutation, providing genetic evidence for biologically relevant interactions. Loss of PEX12 or PEX10 does not reduce PEX5 association with peroxisomes, placing them downstream of docking.","method":"Two-hybrid studies, blot overlay assays, coimmunoprecipitation, genetic suppressor overexpression","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods plus genetic epistasis, patient mutation validation","pmids":["10562279"],"is_preprint":false},{"year":2001,"finding":"Human PEX5L amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for peroxisome targeting. A 21-amino acid motif (aa 209-229) shared with yeast Pex18p/Pex21p mediates PTS2 import and PEX7 binding; a conserved serine mutation in this motif abolishes PTS2 import and reduces PEX5L-PEX7 interaction in vitro.","method":"Domain mapping, in vitro binding assays, mutagenesis, complementation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis combined with in vitro binding and functional rescue assays","pmids":["11546814"],"is_preprint":false},{"year":2000,"finding":"Disruption of the Pex5pL-Pex7p interaction by a point mutation (S214F in the PEX5L-specific region) completely abolishes PTS2 import in mammalian cells without affecting PTS1 import or Pex14p binding, establishing that the Pex5pL-Pex7p interaction is essential and sufficient for PTS2 pathway function.","method":"CHO cell mutant isolation, point mutation analysis, co-immunoprecipitation, complementation rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — clean separation of PTS1 and PTS2 defects by single mutation with multiple functional readouts","pmids":["10767287"],"is_preprint":false},{"year":2001,"finding":"The seven conserved di-aromatic pentapeptide repeats (WX(E/D/Q/A/S)(E/D/Q)(F/Y) motifs) in the N-terminus of PEX5 each bind independently to the same site in the N-terminus of PEX14 with nanomolar affinity. The conserved aromatic residues at positions 1 and 5 of the motif are essential for high-affinity binding.","method":"Two-hybrid analysis, in vitro binding assays, surface plasmon resonance, mutational analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — quantitative in vitro binding with mutagenesis, multiple orthogonal approaches","pmids":["11438541"],"is_preprint":false},{"year":2005,"finding":"PEX5 shuttles into peroxisomes in an ATP-independent manner and is exported in an ATP-dependent manner. PEX1 and PEX6 (AAA ATPases) and their recruiter PEX26 are essential for PEX5 export. PEX14 is required as a docking site for PEX5 import. DTM-embedded PEX5 exists in two distinct membrane complexes (~500 kDa and ~800 kDa) comprising different sets of peroxins.","method":"Cell-free translocation assay with isolated peroxisomes, blue-native PAGE, multiple cell mutant lines","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — cell-free system with multiple cell mutants, biochemical fractionation, replicated findings","pmids":["16314507"],"is_preprint":false},{"year":2009,"finding":"The N-terminal domain of PEX14 adopts a three-helical fold, and PEX5 and PEX19 ligand helices bind competitively to the same surface in PEX14(N) with opposite directionality. The conserved aromatic side chains in the PEX5 WxxxF/Y motif mediate this interaction. Mutations in the PEX14 binding surface disrupt PEX5 and/or PEX19 binding in vitro and impair peroxisomal membrane localisation in vivo.","method":"NMR structure determination, competitive binding assays, mutagenesis, in vivo localization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with functional mutagenesis validation in vitro and in vivo","pmids":["19197237"],"is_preprint":false},{"year":2009,"finding":"Cargo protein translocation across the peroxisomal membrane occurs downstream of a reversible docking step and upstream of PEX5 ubiquitination (the first ATP-dependent cytosolic step), placing cargo translocation concurrent with PEX5 insertion into the docking/translocation machinery.","method":"In vitro peroxisomal import system, protease protection assays, ATP depletion experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — cell-free reconstitution system with defined biochemical steps, validated by multiple manipulations","pmids":["19632994"],"is_preprint":false},{"year":2011,"finding":"PEX5 binds monomeric catalase and potently inhibits its tetramerization; no complex forms with tetrameric catalase. The PEX5-catalase interaction requires domains in both the N- and C-terminal halves of PEX5. The N-terminal domain of PEX14 disrupts the PEX5-catalase interaction, suggesting PEX14 participates in cargo protein release at the docking/translocation machinery.","method":"In vitro binding assays, size-exclusion chromatography, PEX5 domain truncation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — reconstitution with defined protein fragments, multiple orthogonal experiments","pmids":["21976670"],"is_preprint":false},{"year":2012,"finding":"USP9X is identified as the primary deubiquitinase acting on the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) in female rat liver and HeLa cells. USP9X can hydrolyze thioester, isopeptide, and peptide bonds and is an elongated monomeric protein.","method":"Biochemical fractionation, in vitro deubiquitinase assay, mass spectrometry identification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — unbiased biochemical approach with functional validation of enzymatic activity","pmids":["22371489"],"is_preprint":false},{"year":2013,"finding":"PEX5 is monoubiquitinated at a conserved cysteine residue (Cys11 in human PEX5), which is mandatory for ATP-dependent dislocation of PEX5 back into the cytosol. This cysteine functions as a redox switch: exposure to oxidized glutathione yields a ubiquitination-deficient PEX5, and substitution of Cys11 with lysine counteracts this oxidative inactivation.","method":"Cell-free in vitro import system, mutagenesis, PEGylation assays for cysteine modification state, human fibroblast experiments","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro and cell-based systems with mutagenesis, redox manipulation","pmids":["24118911"],"is_preprint":false},{"year":2013,"finding":"A novel PEX5-PEX14 interaction site (LVAEF/LVXEF motif) was identified in PEX5 by peptide library screening. NMR structure shows this motif binds PEX14-N in an α-helical orientation similar to WxxxF/Y but with faster dissociation kinetics. Alanine substitution of LVAEF strongly impairs matrix protein import in vivo, and replacing it with the higher-affinity WxxxF/Y motif paradoxically also impairs import, suggesting distinct kinetic properties are required.","method":"Peptide library blot analysis, NMR structure determination, surface plasmon resonance, in vivo import assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — NMR structure combined with quantitative binding kinetics and in vivo functional assays","pmids":["24235149"],"is_preprint":false},{"year":2013,"finding":"Cargo translocation across the peroxisomal membrane occurs prior to PEX5 ubiquitination, and a reversible docking step precedes irreversible membrane insertion. This positions cargo release upstream of the ATP-dependent PEX5 extraction step.","method":"In vitro import system with cargo protein-centered perspective, ATP manipulation, protease protection","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — cell-free reconstitution system confirming pathway order with multiple experimental manipulations","pmids":["23963456"],"is_preprint":false},{"year":2014,"finding":"The PEX5 receptor adapts its TPR binding cavity conformation for high-affinity PTS1 binding rather than the cargo signal adapting. Upon ligand binding, the binding cavity shrinks to one-third of its original volume. A bulky side chain in the wild-type cargo blocks this compaction; a single-residue mutation removing this impediment increases peroxisomal import efficiency from 34% to 80%.","method":"High-resolution crystal structure, mutagenesis, in vivo import efficiency measurements","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and quantitative in vivo import assays","pmids":["25369882"],"is_preprint":false},{"year":2015,"finding":"Export-deficient PEX5 proteins bearing bulky C-terminal tags trigger pexophagy in an autophagy-dependent manner. Monoubiquitination of the N-terminal cysteine of peroxisome-associated PEX5 is required for this process. A C-terminal tag does not inhibit PEX5 monoubiquitination but strongly inhibits its export, suggesting that monoubiquitinated PEX5 retained at the membrane serves as a quality control signal for pexophagy.","method":"Autophagy assays, PEX5 tagging and export-block experiments, ubiquitination assays in mouse embryonic fibroblasts","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — clean mechanistic dissection with defined mutants and autophagy-dependence validation","pmids":["26086376"],"is_preprint":false},{"year":2015,"finding":"A frameshift mutation in the PEX5L-specific exon 9 (c.722dupA) causes selective loss of the PEX5L isoform, resulting in defective PTS2 protein import only (not PTS1), causing RCDP5 rather than Zellweger syndrome. PEX5L expression in patient fibroblasts restores PTS2 import, confirming the isoform-specific function.","method":"Patient genetic analysis, biochemical complementation assays in fibroblasts, isoform-specific functional rescue","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — natural human mutation with clean isoform-specific biochemical phenotype and rescue","pmids":["26220973"],"is_preprint":false},{"year":2017,"finding":"TRIM37 localizes to peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with PEX5's C-terminal 51 amino acids. This non-degradative ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import. TRIM37 depletion reduces PEX5 abundance via proteasomal degradation and impairs cargo binding and PTS protein import.","method":"Co-immunoprecipitation, ubiquitination assays, mutagenesis (K464A), siRNA knockdown, import assays in human cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods identifying E3 ligase, substrate site, and functional consequence","pmids":["28724525"],"is_preprint":false},{"year":2017,"finding":"The DTM (docking/translocation module) is a large cavity-forming protein assembly into which PEX5 enters to release its cargo. Truncated PEX5(1-125) interacts with DTM but remains accessible to exogenously added proteinase K, and multiple PEX5 truncated molecules can be accommodated simultaneously, suggesting a cavity architecture.","method":"Truncated PEX5 probing of DTM architecture, proteinase K accessibility assays, competition experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — cell-free system with defined PEX5 truncations, single lab study","pmids":["28765278"],"is_preprint":false},{"year":2018,"finding":"DTM-embedded monoubiquitinated PEX5 (Ub-PEX5) interacts directly with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event, establishing Ub-PEX5 as a bona fide substrate of the PEX1-PEX6 AAA ATPase complex.","method":"Cell-free in vitro system, photoaffinity cross-linking, protein PEGylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — cell-free reconstitution with photoaffinity crosslinking and unfolding assays","pmids":["29884772"],"is_preprint":false},{"year":2017,"finding":"Cys11 of human PEX5 functions as a redox switch modulating import receptor activity in response to oxidative stress. Oxidative stress specifically impairs catalase import more than canonical PTS1 reporters, and PEX5 does not oligomerize in cellulo even under oxidative stress. Cytosolic catalase retained when PEX5 is inactivated can protect against H2O2-mediated redox changes.","method":"Live-cell FRET-based import assay, redox manipulation, mutagenesis in human fibroblasts","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"High","confidence_rationale":"Tier 2 — novel live-cell approach combined with mutagenesis and functional readouts","pmids":["28760655"],"is_preprint":false},{"year":2020,"finding":"PEX5 escorts adipose triglyceride lipase (ATGL) to contact points between peroxisomes and lipid droplets during fasting, facilitating ATGL translocation onto lipid droplets and promoting fasting-induced lipolysis. Adipocyte-specific PEX5 knockout mice show defective ATGL recruitment to lipid droplets and attenuated fasting-induced lipolysis.","method":"Conditional knockout mice, live imaging of peroxisome-lipid droplet contacts, coimmunoprecipitation, ATGL localization assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with defined cellular phenotype plus mechanistic co-localization data","pmids":["31996685"],"is_preprint":false},{"year":2022,"finding":"PEX5 accompanies cargo completely into the peroxisome lumen in Xenopus egg extract, utilizing WxxxF/Y motifs near its N-terminus to bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix binding to the lumenal side of the ubiquitin ligase; the N-terminus then emerges in the cytosol for monoubiquitination. PEX5 is extracted from the lumen by unfolding of the receptor, resulting in cargo release.","method":"Xenopus egg extract import system, domain mapping, mutagenesis of WxxxF/Y motifs and amphipathic helix","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — reconstitution in egg extract with multiple mutagenesis experiments establishing mechanistic steps","pmids":["35931083"],"is_preprint":false},{"year":2022,"finding":"The PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and serves as the extraction initiator; the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6. An intra-molecularly cross-linked ubiquitin at position 11 blocks extraction, confirming ubiquitin unfolding is required for the extraction mechanism.","method":"Cell-free in vitro system, engineered PEX5 and ubiquitin molecules, cross-linked ubiquitin experiments","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 — cell-free reconstitution with chemically engineered substrates and rigorous controls","pmids":["36442669"],"is_preprint":false},{"year":2011,"finding":"AWP1/ZFAND6 is a cofactor of PEX6 involved in PEX5 export. AWP1 preferentially binds cysteine-ubiquitinated PEX5 via its A20 zinc-finger domain, stimulates PEX5 export in vitro, and interacts with PEX6 AAA ATPase. AWP1 knockdown reduces PTS1-protein import and decreases PEX5 stability similarly to PEX1/PEX6/PEX26 deficient cells.","method":"Biochemical fractionation, in vitro Pex5 export assay, co-immunoprecipitation, siRNA knockdown","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 — biochemical identification with functional in vitro and in vivo validation","pmids":["21980954"],"is_preprint":false},{"year":2013,"finding":"PEX5 in Pichia pastoris functions as a redox-regulated receptor: disulfide bond-linked Pex5 dimers/oligomers show highest affinity for PTS1 cargo; reduction transitions Pex5 to a noncovalent dimer with partial cargo release. A hetero-oligomeric interaction between the Pex5 N-terminal domain (aa 1-110) and the C-terminal motif of Pex8 further facilitates cargo release under reducing conditions.","method":"In vitro binding assays, disulfide cross-linking, DTT reduction experiments, Pex5-Pex8 pulldown","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro reconstitution in yeast ortholog, mechanistic details on redox control","pmids":["23902771"],"is_preprint":false},{"year":2013,"finding":"Pex5p stabilizes Pex14p; in the absence of Pex5p, Pex14p is unstable due to inefficient translocation to the peroxisomal membrane. The fifth WXXXF/Y motif of Pex5pL is an auxiliary binding site for Pex14p required for Pex14p stability. Pex5p-Pex13p interaction is essential for import of PTS1 proteins and catalase but not PTS2 proteins.","method":"CHO cell mutant isolation, complementation with Pex5p domain mutants, western blot stability assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 — clean cell mutant with domain mapping, single lab","pmids":["23009329"],"is_preprint":false},{"year":2024,"finding":"Monoubiquitination of PEX5 at cysteine 11 is noncanonical (thioester bond) and reversible; this reversibility prevents polyubiquitination of PEX5 at the peroxisomal membrane. A PEX5 variant with lysine at position 11 undergoes polyubiquitination that negatively interferes with extraction. E2-mediated deubiquitination kinetics are faster than PEX5 polyubiquitination, ensuring the transient monoubiquitinated state.","method":"Cell-free rat liver in vitro system, engineered PEX5 Cys11Lys mutant, ubiquitination kinetics analysis","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1-2 — reconstitution with defined mutants establishing mechanistic rationale for cysteine ubiquitination","pmids":["38470934"],"is_preprint":false},{"year":2018,"finding":"Chemically synthesized monoubiquitinated PEX5 binds PEX13, PEX14, and the receptor export module components PEX1, PEX6, and PEX26. Interactions with PEX13 and PEX14 are independent of PEX5 ubiquitination status, whereas interactions with PEX1, PEX6, and PEX26 are enhanced by ubiquitination. Monoubiquitinated PEX5 also binds PEX7/PTS2 complexes and restores PTS2 import in ΔPEX5 fibroblasts.","method":"Click chemistry synthesis of Ub-PEX5, in vitro pulldown assays, complementation in ΔPEX5 fibroblasts","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1-2 — chemically defined ubiquitin conjugates with in vitro binding and in vivo rescue","pmids":["30375424"],"is_preprint":false},{"year":2009,"finding":"Small angle X-ray scattering reveals that free full-length human Pex5p is monomeric in solution with an elongated, partially unfolded N-terminal domain. The Pex5p:Pex14p complex shows 1:6 stoichiometry. In the complex, the N-terminus of Pex5p remains extended, with Pex14p molecules significantly intermingled with the Pex5p moiety.","method":"Small angle X-ray scattering, static light scattering, solution structure modeling","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 — structural analysis in solution, single study","pmids":["19584060"],"is_preprint":false},{"year":2022,"finding":"PEX5L is monomeric with compact spherical conformation in solution. Labeled PEX5L accumulates ~100-fold at lipid bilayers and forms ion-conducting membrane channels in electrophysiological recordings. The truncated PEX5L(1-335) lacking the cargo-binding domain does not form channels, suggesting that PEX5L is the pore-forming component of the oligomeric peroxisomal translocon and that membrane surface binding precedes channel assembly.","method":"Horizontal lipid bilayer electrophysiology, fluorescence TCSPC, diffusion coefficient measurements","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro reconstituted membrane system with electrophysiology, single lab study","pmids":["36260915"],"is_preprint":false},{"year":2003,"finding":"Full-length tetrameric PEX5 has high intrinsic affinity for the PTS1 peptide (Kd ~35 nM) as measured by fluorescence anisotropy. PEX5-PTS1 binding kinetics are unaffected by Hsp70 (with or without ATP/ADP) or by the PEX12 zinc RING domain, indicating that initial PTS1 recognition is an autonomous step not regulated by these factors.","method":"Fluorescence anisotropy binding assay with purified recombinant proteins","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 — quantitative in vitro binding assay, single lab","pmids":["12456682"],"is_preprint":false},{"year":2003,"finding":"PTS1 variants corresponding to known functional targeting signals bind PEX5 TPR domain within 1.8 kcal/mol of the optimal -SKL sequence. A binding energy threshold determines PTS1 functionality, correlating structural features from the PEX5:PTS1 crystal structure with thermodynamic binding parameters.","method":"Fluorescence-based binding assay with synthetic peptide library, thermodynamic analysis","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 — quantitative in vitro binding with structure-activity correlation, single lab","pmids":["12578380"],"is_preprint":false},{"year":2021,"finding":"A missense mutation F218S in PEX5 (within the PEX7-interaction domain) disrupts the trimeric complex formation between PEX5, PEX7, and a PTS2 cargo protein, abolishing PTS2 protein import while leaving PTS1 import, monoubiquitination, and export intact, causing congenital cataracts in affected patients.","method":"In vitro import assays, trimeric complex pulldown, patient fibroblast studies, lens-specific mouse KO","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 2 — mechanistic dissection with in vitro assays plus patient and mouse model validation","pmids":["33389129"],"is_preprint":false},{"year":2023,"finding":"PEX13 loss causes accumulation of ubiquitinated PEX5 on peroxisomes and increased peroxisome-derived ROS, together inducing pexophagy. PEX13 protein levels are downregulated during amino acid starvation to facilitate pexophagy induction, establishing PEX13 as a gatekeeper that prevents premature pexophagy by limiting accumulation of ubiquitinated PEX5.","method":"Gene editing, quantitative fluorescence microscopy, zebrafish model, ubiquitination assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — multiple model systems with mechanistic pathway placement","pmids":["36541703"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure of yeast Pex5 in complex with cargo protein Eci1 reveals that Eci1 can bind Pex5 and reach peroxisomes in the absence of a canonical PTS1 signal, through additional binding interfaces beyond the PTS1-binding site in the TPR domain.","method":"Cryo-electron microscopy structure determination, PTS1-deletion mutant analysis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with functional validation of non-canonical binding","pmids":["40376748"],"is_preprint":false},{"year":2021,"finding":"In mammalian cells, PEX5 depletion under serum starvation leads to downregulation of TSC2, activation of mTORC1 (increased phosphorylation of 70S6K, S6K, and 4E-BP1), and suppression of TFEB nuclear localization. Pharmacological mTOR inhibition upon PEX5 depletion during starvation activates TFEB and recovers peroxisome biogenesis, placing PEX5 upstream of mTORC1-TFEB axis.","method":"siRNA knockdown, pharmacological mTOR inhibition, nuclear localization assays, peroxisome biogenesis readouts","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — pathway epistasis via pharmacological and genetic approaches, single lab","pmids":["29622767"],"is_preprint":false}],"current_model":"PEX5 is a cytosolic shuttling receptor that recognizes PTS1-containing peroxisomal matrix proteins via its C-terminal TPR domain and, in its longer isoform (PEX5L), co-receptors PTS2 cargo by binding PEX7 through an N-terminal 21-amino acid motif; cargo-loaded PEX5 docks at the peroxisomal membrane via multiple WxxxF/Y motifs binding PEX14, then enters completely into the peroxisomal lumen through a large cavity-forming translocon (DTM), releases cargo upon unfolding driven by extraction, becomes monoubiquitinated at Cys11 (via a reversible thioester bond that prevents overubiquitination) by a membrane-embedded ubiquitin ligase, and is then extracted back to the cytosol by the PEX1-PEX6 AAA ATPase complex (aided by AWP1/ZFAND6 and deubiquitinated by USP9X), with Cys11 additionally functioning as a redox sensor that inactivates PEX5 during oxidative stress; when export is blocked, monoubiquitinated membrane-retained PEX5 serves as a pexophagy signal, and TRIM37 stabilizes PEX5 via non-degradative K464 ubiquitination to maintain adequate receptor levels for efficient import."},"narrative":{"teleology":[{"year":1995,"claim":"Identification of PEX5 as the cytosolic PTS1 receptor resolved how peroxisomal matrix proteins are recognized in the cytosol and established that PEX5 mutations define complementation group 2 of peroxisome biogenesis disorders.","evidence":"Complementation cloning and rescue of PTS1 import in patient fibroblasts","pmids":["7719337"],"confidence":"High","gaps":["Mechanism of membrane engagement unknown","Structural basis for PTS1 recognition undetermined","Role in PTS2 import unclear"]},{"year":1998,"claim":"Discovery of two PEX5 isoforms (PEX5S and PEX5L) established that the longer isoform is uniquely required for PTS2 protein import, separating PTS1 and PTS2 import pathways at the receptor level.","evidence":"CHO cell mutant complementation with isoform-specific constructs and TPR mutagenesis","pmids":["9418886"],"confidence":"High","gaps":["Molecular basis of PEX5L–PEX7 interaction not mapped","Whether PEX5L acts as a co-receptor or independent receptor for PTS2 unclear"]},{"year":1999,"claim":"Mapping of multiple PEX14-binding sites in PEX5's N-terminal half and demonstration of nanomolar affinity defined the docking mechanism at the peroxisomal membrane, while interaction with PEX12 was placed downstream of docking.","evidence":"Surface plasmon resonance, two-hybrid, co-IP, and genetic suppressor experiments","pmids":["10026185","10562279"],"confidence":"High","gaps":["Nature of the translocation pore unknown","Order of PEX5 steps at the membrane unresolved"]},{"year":2000,"claim":"The crystal structure of PEX5's TPR domain bound to a PTS1 peptide revealed a novel two-cluster TPR architecture that nearly encircles the cargo signal, providing the molecular basis for PTS1 recognition specificity.","evidence":"X-ray crystallography of human PEX5 TPR–PTS1 complex","pmids":["11101887"],"confidence":"High","gaps":["How TPR domain conformational changes regulate binding/release not established","Full-length PEX5 structure unavailable"]},{"year":2001,"claim":"Identification of the conserved WxxxF/Y pentapeptide motifs as independent PEX14-binding elements and the 21-amino-acid PEX7-binding motif in PEX5L resolved the modular architecture underlying receptor docking and PTS2 co-receptor function.","evidence":"Mutagenesis, SPR quantification, in vitro binding, and complementation assays","pmids":["11438541","11546814","10767287"],"confidence":"High","gaps":["Functional non-equivalence among the seven WxxxF/Y motifs not understood","Stoichiometry of PEX5–PEX14 complex at the membrane unresolved"]},{"year":2005,"claim":"Establishment that PEX5 insertion into peroxisomes is ATP-independent whereas its export requires ATP and the PEX1–PEX6–PEX26 complex defined the energy-dependent recycling step and ordered the import cycle.","evidence":"Cell-free translocation assay with isolated peroxisomes and blue-native PAGE","pmids":["16314507"],"confidence":"High","gaps":["Whether PEX5 fully enters the lumen or remains membrane-embedded debated","Ubiquitination requirement for export not yet shown"]},{"year":2009,"claim":"NMR structures of the PEX14-N domain bound to PEX5 and PEX19 helices, and SAXS analysis showing a 1:6 PEX5:PEX14 stoichiometry, provided atomic-level understanding of docking and revealed that PEX5 remains extended in solution.","evidence":"NMR structure, SAXS, competitive binding, mutagenesis, and in vivo localization","pmids":["19197237","19584060"],"confidence":"High","gaps":["PEX5–PEX14 complex structure in a membrane context not determined","Function of the intrinsically disordered N-terminus beyond PEX14 binding unclear"]},{"year":2009,"claim":"Ordering cargo translocation as concurrent with PEX5 membrane insertion and upstream of ubiquitination clarified that cargo release precedes the ATP-dependent extraction step.","evidence":"In vitro peroxisomal import with protease protection and ATP depletion","pmids":["19632994","23963456"],"confidence":"High","gaps":["Mechanism of cargo release inside the organelle not established","Whether PEX14 directly triggers cargo release in vivo unconfirmed"]},{"year":2011,"claim":"Discovery that PEX5 binds monomeric but not tetrameric catalase, and that PEX14 disrupts the PEX5–catalase complex, suggested a cargo-release mechanism at the docking site; identification of AWP1/ZFAND6 as a PEX6 cofactor that preferentially binds Cys-ubiquitinated PEX5 added a new component to the export machinery.","evidence":"In vitro reconstitution, size-exclusion chromatography, siRNA knockdown, and export assays","pmids":["21976670","21980954"],"confidence":"High","gaps":["AWP1's exact role in the extraction mechanism unclear","How catalase monomeric state is maintained during transport unresolved"]},{"year":2012,"claim":"Identification of USP9X as the primary deubiquitinase for the Ub–PEX5 thioester conjugate established the recycling pathway's final step returning PEX5 to its import-competent state.","evidence":"Biochemical fractionation of rat liver and HeLa cytosol, in vitro DUB assay, mass spectrometry","pmids":["22371489"],"confidence":"High","gaps":["Whether USP9X acts before or after PEX5 extraction not determined","Redundancy with other DUBs not excluded"]},{"year":2013,"claim":"Demonstration that PEX5 Cys11 monoubiquitination is mandatory for export, and that this cysteine functions as a redox switch inactivated by oxidized glutathione, unified the ubiquitination and redox-sensing functions into a single regulatory residue; a novel LVAEF motif with distinct kinetics was added to the PEX14-binding repertoire.","evidence":"Cell-free import, Cys11 mutagenesis, PEGylation assays, NMR of LVAEF–PEX14 complex, SPR kinetics","pmids":["24118911","24235149"],"confidence":"High","gaps":["How redox state transitions are reversed in vivo unknown","Functional hierarchy among WxxxF/Y and LVAEF motifs in the import cycle not fully mapped"]},{"year":2014,"claim":"Crystal structures showing that PEX5's TPR cavity compacts upon PTS1 binding (volume reduced to one-third) established a receptor-adapts-to-cargo model and linked cavity plasticity to import efficiency.","evidence":"High-resolution crystal structures, mutagenesis, in vivo import quantification","pmids":["25369882"],"confidence":"High","gaps":["Whether compaction triggers downstream signaling events unknown","Structural basis for non-canonical cargo binding not defined"]},{"year":2015,"claim":"Demonstration that export-deficient monoubiquitinated PEX5 retained at the membrane triggers autophagy-dependent pexophagy established PEX5 as a quality-control signal; a frameshift mutation selectively ablating PEX5L confirmed isoform-specific causation of RCDP5.","evidence":"Export-block assays in MEFs with autophagy readouts; patient fibroblast complementation with PEX5L rescue","pmids":["26086376","26220973"],"confidence":"High","gaps":["Receptor for ubiquitinated PEX5 in pexophagy not identified","Whether RCDP5 phenotype involves additional PEX5L-specific functions beyond PEX7 binding unknown"]},{"year":2017,"claim":"TRIM37 was identified as a peroxisomal E3 ligase that stabilizes PEX5 through non-degradative K464 ubiquitination, maintaining receptor levels for efficient import; concurrent studies confirmed PEX5 Cys11 as a redox sensor in live cells and characterized the DTM as a large cavity that accommodates multiple PEX5 molecules.","evidence":"Co-IP, ubiquitination site mapping, siRNA, FRET-based import assays, proteinase K accessibility on DTM","pmids":["28724525","28760655","28765278"],"confidence":"High","gaps":["TRIM37 regulation and its coordination with Cys11 ubiquitination unexplored","DTM architecture at atomic resolution unknown"]},{"year":2018,"claim":"Direct interaction of ubiquitinated PEX5 with both PEX1 and PEX6, and demonstration that the PEX5 polypeptide is globally unfolded during extraction, established Ub-PEX5 as a bona fide substrate of the AAA-ATPase complex and linked unfolding to cargo release.","evidence":"Cell-free system, photoaffinity cross-linking, PEGylation-based unfolding assay, chemically synthesized Ub-PEX5","pmids":["29884772","30375424"],"confidence":"High","gaps":["Structure of PEX1–PEX6 engaged with Ub-PEX5 not determined","How unfolded PEX5 refolds in the cytosol unknown"]},{"year":2020,"claim":"Discovery that PEX5 escorts ATGL to peroxisome–lipid droplet contact sites during fasting expanded PEX5's role beyond matrix protein import to inter-organelle lipid metabolism.","evidence":"Adipocyte-specific PEX5 conditional knockout mice, live imaging, co-IP","pmids":["31996685"],"confidence":"High","gaps":["Whether PEX5's ATGL-escort function uses the same WxxxF/PEX14 docking or a distinct mechanism unknown","Generality to other non-peroxisomal cargoes not tested"]},{"year":2022,"claim":"Reconstitution in Xenopus egg extract proved PEX5 enters the peroxisomal lumen entirely, using WxxxF/Y motifs for lumenal docking-complex interaction and an amphipathic helix for ubiquitin-ligase engagement to initiate N-terminal extrusion; concurrent electrophysiology showed PEX5L forms ion-conducting channels in lipid bilayers.","evidence":"Xenopus egg extract import system with mutagenesis; horizontal lipid bilayer electrophysiology","pmids":["35931083","36260915"],"confidence":"High","gaps":["Physiological relevance of PEX5 channel activity not established","Identity of the lumenal PEX5 receptor unknown"]},{"year":2022,"claim":"Demonstration that the ubiquitin moiety conjugated to Cys11 must itself be unfolded before PEX1–PEX6-mediated extraction, using cross-linked ubiquitin to block threading, defined ubiquitin unfolding as the extraction-initiating event.","evidence":"Cell-free system with engineered intramolecularly cross-linked ubiquitin–PEX5 conjugates","pmids":["36442669"],"confidence":"High","gaps":["Step-by-step threading directionality not visualized","Whether ubiquitin refolding after extraction is spontaneous or chaperone-assisted unknown"]},{"year":2024,"claim":"Elucidation of why PEX5 uses a thioester (Cys11) rather than an isopeptide bond for monoubiquitination showed that rapid E2-mediated deubiquitination outcompetes polyubiquitin chain elongation, preventing premature degradation and maintaining the cycling receptor pool.","evidence":"Cell-free reconstitution with Cys11Lys mutant, ubiquitination kinetics quantification","pmids":["38470934"],"confidence":"High","gaps":["In vivo kinetics of thioester turnover not measured","Potential regulatory inputs controlling E2 deubiquitination rate unknown"]},{"year":2025,"claim":"Cryo-EM structure of yeast Pex5 with cargo Eci1 revealed non-canonical binding interfaces beyond PTS1, demonstrating that PEX5 can import proteins lacking a canonical PTS1 signal.","evidence":"Cryo-EM structure determination with PTS1-deletion mutant analysis","pmids":["40376748"],"confidence":"High","gaps":["Prevalence of non-canonical PEX5 cargoes in the human proteome unknown","Whether non-canonical binding uses the same translocation mechanism not tested"]},{"year":null,"claim":"Key unresolved questions include: the atomic structure of the full DTM translocon with PEX5 engaged, the identity and regulation of the pexophagy receptor that recognizes membrane-retained Ub-PEX5, and how PEX5 refolds in the cytosol after extraction-coupled global unfolding.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of the assembled DTM–PEX5 complex","Pexophagy receptor for Ub-PEX5 not identified","PEX5 refolding mechanism after extraction unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[0,1,2,16,34]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,6,23]},{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[24,32]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,31,33]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,20,24]},{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[8,24]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[17,36]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,8,24]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,8]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[23]}],"complexes":["DTM (docking/translocation machinery)","PEX5–PEX7 co-receptor complex"],"partners":["PEX14","PEX7","PEX12","PEX13","PEX1","PEX6","USP9X","TRIM37"],"other_free_text":[]},"mechanistic_narrative":"PEX5 is the cytosolic cycling receptor for peroxisomal matrix protein import, recognizing PTS1 cargo through its C-terminal TPR domain and, in its longer isoform (PEX5L), bridging PTS2 cargo via a conserved N-terminal motif that binds PEX7 [PMID:7719337, PMID:11101887, PMID:9418886, PMID:11546814]. Cargo-loaded PEX5 docks at the peroxisomal membrane through multiple WxxxF/Y pentapeptide motifs that bind PEX14 with nanomolar affinity, then inserts fully into the lumenal cavity of the docking/translocation machinery (DTM) where it releases cargo; PEX5 is subsequently monoubiquitinated at a conserved cysteine (Cys11) via a thioester bond whose reversibility prevents polyubiquitination, and the ubiquitin–PEX5 conjugate is extracted back to the cytosol by the PEX1–PEX6 AAA-ATPase complex through global unfolding of both ubiquitin and PEX5 [PMID:10026185, PMID:11438541, PMID:35931083, PMID:29884772, PMID:36442669, PMID:38470934]. Cys11 additionally serves as a redox sensor that inactivates PEX5 under oxidative stress, preferentially retaining catalase in the cytosol as a protective response, while monoubiquitinated PEX5 that fails to be exported acts as a pexophagy signal linking import failure to organelle quality control [PMID:24118911, PMID:28760655, PMID:26086376, PMID:36541703]. Mutations in PEX5 cause peroxisome biogenesis disorders: complete loss underlies Zellweger-spectrum disease (complementation group 2), whereas isoform-specific PEX5L mutations cause rhizomelic chondrodysplasia punctata type 5 (RCDP5), and a PEX7-interaction-domain missense variant causes congenital cataracts [PMID:7719337, PMID:26220973, PMID:33389129]."},"prefetch_data":{"uniprot":{"accession":"P50542","full_name":"Peroxisomal targeting signal 1 receptor","aliases":["PTS1-BP","Peroxin-5","Peroxisomal C-terminal targeting signal import receptor","Peroxisome receptor 1"],"length_aa":639,"mass_kda":70.9,"function":"Receptor that mediates peroxisomal import of proteins containing a C-terminal PTS1-type tripeptide peroxisomal targeting signal (SKL-type) (PubMed:11101887, PubMed:11336669, PubMed:12456682, PubMed:16314507, PubMed:17157249, PubMed:17428317, PubMed:21976670, PubMed:26344566, PubMed:7706321, PubMed:7719337, PubMed:7790377). Binds to cargo proteins containing a PTS1 peroxisomal targeting signal in the cytosol, and translocates them into the peroxisome matrix by passing through the PEX13-PEX14 docking complex along with cargo proteins (PubMed:12456682, PubMed:17157249, PubMed:21976670, PubMed:26344566). PEX5 receptor is then retrotranslocated into the cytosol, leading to release of bound cargo in the peroxisome matrix, and reset for a subsequent peroxisome import cycle (PubMed:11336669, PubMed:24662292) In addition to promoting peroxisomal translocation of proteins containing a PTS1 peroxisomal targeting signal, mediates peroxisomal import of proteins containing a C-terminal PTS2-type peroxisomal targeting signal via its interaction with PEX7 (PubMed:11336669, PubMed:11546814, PubMed:25538232, PubMed:33389129, PubMed:9668159). Interaction with PEX7 only takes place when PEX7 is associated with cargo proteins containing a PTS2 peroxisomal targeting signal (PubMed:25538232). PEX7 along with PTS2-containing cargo proteins are then translocated through the PEX13-PEX14 docking complex together with PEX5 (PubMed:25538232) Does not mediate translocation of peroxisomal import of proteins containing a C-terminal PTS2-type peroxisomal targeting signal","subcellular_location":"Cytoplasm, cytosol; Peroxisome matrix","url":"https://www.uniprot.org/uniprotkb/P50542/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PEX5","classification":"Not Classified","n_dependent_lines":57,"n_total_lines":1208,"dependency_fraction":0.04718543046357616},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PEX5","total_profiled":1310},"omim":[{"mim_id":"621410","title":"PEROXISOME BIOGENESIS FACTOR 39; PEX39","url":"https://www.omim.org/entry/621410"},{"mim_id":"616716","title":"RHIZOMELIC CHONDRODYSPLASIA PUNCTATA, TYPE 5; RCDP5","url":"https://www.omim.org/entry/616716"},{"mim_id":"616154","title":"PEROXISOMAL FATTY ACYL-CoA REDUCTASE 1 DISORDER; PFCRD","url":"https://www.omim.org/entry/616154"},{"mim_id":"611058","title":"PEROXISOME BIOGENESIS FACTOR 5-LIKE; PEX5L","url":"https://www.omim.org/entry/611058"},{"mim_id":"607622","title":"PHOSPHOMEVALONATE KINASE; PMVK","url":"https://www.omim.org/entry/607622"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PEX5"},"hgnc":{"alias_symbol":["PTS1R"],"prev_symbol":["PXR1"]},"alphafold":{"accession":"P50542","domains":[{"cath_id":"1.25.40.10","chopping":"330-399","consensus_level":"medium","plddt":95.7083,"start":330,"end":399},{"cath_id":"1.25.40.10","chopping":"417-441_459-518","consensus_level":"medium","plddt":91.6894,"start":417,"end":518},{"cath_id":"1.25.40.10","chopping":"523-589_598-639","consensus_level":"medium","plddt":94.1368,"start":523,"end":639}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P50542","model_url":"https://alphafold.ebi.ac.uk/files/AF-P50542-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P50542-F1-predicted_aligned_error_v6.png","plddt_mean":69.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PEX5","jax_strain_url":"https://www.jax.org/strain/search?query=PEX5"},"sequence":{"accession":"P50542","fasta_url":"https://rest.uniprot.org/uniprotkb/P50542.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P50542/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P50542"}},"corpus_meta":[{"pmid":"7719337","id":"PMC_7719337","title":"Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders.","date":"1995","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/7719337","citation_count":384,"is_preprint":false},{"pmid":"11101887","id":"PMC_11101887","title":"Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.","date":"2000","source":"Nature structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/11101887","citation_count":311,"is_preprint":false},{"pmid":"11583975","id":"PMC_11583975","title":"Mitochondrial alterations caused by defective peroxisomal biogenesis in a mouse model for Zellweger syndrome (PEX5 knockout mouse).","date":"2001","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/11583975","citation_count":177,"is_preprint":false},{"pmid":"9418886","id":"PMC_9418886","title":"Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.","date":"1998","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9418886","citation_count":177,"is_preprint":false},{"pmid":"16314507","id":"PMC_16314507","title":"Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16314507","citation_count":175,"is_preprint":false},{"pmid":"10026185","id":"PMC_10026185","title":"Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10026185","citation_count":154,"is_preprint":false},{"pmid":"15548601","id":"PMC_15548601","title":"The Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 is necessary for peroxisome function and dependent on PEX5.","date":"2004","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/15548601","citation_count":119,"is_preprint":false},{"pmid":"10562279","id":"PMC_10562279","title":"PEX12 interacts with PEX5 and PEX10 and acts downstream of receptor docking in peroxisomal matrix protein import.","date":"1999","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10562279","citation_count":115,"is_preprint":false},{"pmid":"11546814","id":"PMC_11546814","title":"Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11546814","citation_count":102,"is_preprint":false},{"pmid":"11438541","id":"PMC_11438541","title":"The di-aromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11438541","citation_count":100,"is_preprint":false},{"pmid":"10767287","id":"PMC_10767287","title":"Disruption of the interaction of the longer isoform of Pex5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals. 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Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40614015","citation_count":3,"is_preprint":false},{"pmid":"38470934","id":"PMC_38470934","title":"Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membrane.","date":"2024","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/38470934","citation_count":3,"is_preprint":false},{"pmid":"30378028","id":"PMC_30378028","title":"Identification of Peroxisomal Protein Complexes with PTS Receptors, Pex5 and Pex7, in Mammalian Cells.","date":"2018","source":"Sub-cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30378028","citation_count":3,"is_preprint":false},{"pmid":"33842617","id":"PMC_33842617","title":"Association of genetic variants of TMEM135 and PEX5 in the peroxisome pathway with cutaneous melanoma-specific survival.","date":"2021","source":"Annals of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33842617","citation_count":3,"is_preprint":false},{"pmid":"23266609","id":"PMC_23266609","title":"Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei.","date":"2012","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/23266609","citation_count":3,"is_preprint":false},{"pmid":"9778800","id":"PMC_9778800","title":"Cloning and sequence of a 3.835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans.","date":"1998","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/9778800","citation_count":3,"is_preprint":false},{"pmid":"39190270","id":"PMC_39190270","title":"Polymorphism in the Hsa-miR-4274 seed region influences the expression of PEX5 and enhances radiotherapy resistance in colorectal cancer.","date":"2024","source":"Frontiers of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39190270","citation_count":2,"is_preprint":false},{"pmid":"39304044","id":"PMC_39304044","title":"Structural dynamics of the TPR domain of the peroxisomal cargo receptor Pex5 in Trypanosoma.","date":"2024","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/39304044","citation_count":2,"is_preprint":false},{"pmid":"8586442","id":"PMC_8586442","title":"The gene for the peroxisomal targeting signal import receptor (PXR1) is located on human chromosome 12p13, flanked by TPI1 and D12S1089.","date":"1995","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8586442","citation_count":2,"is_preprint":false},{"pmid":"40376748","id":"PMC_40376748","title":"A cryo-electron microscopy structure of yeast Pex5 in complex with a cargo uncovers a novel binding interface.","date":"2025","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/40376748","citation_count":1,"is_preprint":false},{"pmid":"36407094","id":"PMC_36407094","title":"Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay.","date":"2022","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/36407094","citation_count":1,"is_preprint":false},{"pmid":"41134761","id":"PMC_41134761","title":"TRIM37-mediated stabilization of PEX5 via monoubiquitination attenuates oxidative stress and demyelination in multiple sclerosis insights from EAE and LPC-induced experimental models.","date":"2025","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/41134761","citation_count":1,"is_preprint":false},{"pmid":"40319819","id":"PMC_40319819","title":"PEX5 acts as a negative regulator of RANKL-induced osteoclastogenesis in vitro and inflammatory calvarial bone destruction in vivo.","date":"2025","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/40319819","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":51061,"output_tokens":9367,"usd":0.146844},"stage2":{"model":"claude-opus-4-6","input_tokens":13209,"output_tokens":4898,"usd":0.282743},"total_usd":0.429587,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"PEX5 (PXR1) functions as the cytosolic receptor for peroxisomal targeting signal type-1 (PTS1), recognizing PTS1-containing proteins in the cytosol and directing them to the peroxisome. Mutations in PEX5 define complementation group 2 of peroxisome biogenesis disorders, and PEX5 expression rescues the PTS1 import defect in patient fibroblasts.\",\n      \"method\": \"Complementation assay in patient fibroblasts, homology cloning, subcellular fractionation showing cytosolic and peroxisome-associated pools\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — complementation rescue in patient cells, replicated across multiple labs subsequently\",\n      \"pmids\": [\"7719337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Crystal structure of the C-terminal TPR domain of human PEX5 in complex with a PTS1 pentapeptide revealed that two clusters of three TPRs almost completely surround the peptide, while a hinge region (TPR4) enables the two sets to form a single binding site, establishing the molecular basis for PTS1 recognition via a novel TPR-peptide interaction mode.\",\n      \"method\": \"X-ray crystallography of PEX5 TPR domain in complex with PTS1 peptide\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation, highly cited foundational paper\",\n      \"pmids\": [\"11101887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The longer isoform of PEX5 (PEX5L/PTS1RL) is required for PTS2 protein import in addition to PTS1 import, whereas the shorter isoform (PEX5S) supports only PTS1 import. Mutations in the TPR domains (TPR1 and TPR6) abolish protein translocation, demonstrating the functional importance of these domains.\",\n      \"method\": \"CHO cell mutant complementation assays, reverse transcription-PCR mutation analysis, isoform-specific rescue experiments\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple complementation experiments with defined mutations, replicated by other labs\",\n      \"pmids\": [\"9418886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PEX5 possesses multiple binding sites for PEX14 distributed throughout its N-terminal half, with nanomolar affinity interaction. The N-terminal half of PEX5 also mediates oligomerization, while the C-terminal TPR domain binds PTS1 cargo. Surface plasmon resonance demonstrated that PEX5 binds PEX14-(1-78) with very high affinity in the low nanomolar range.\",\n      \"method\": \"Surface plasmon resonance, in vitro binding assays, sizing chromatography, electron microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution with quantitative binding measurements, multiple orthogonal methods\",\n      \"pmids\": [\"10026185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PEX12 (zinc RING domain) binds both PEX5 and PEX10 downstream of receptor docking; a patient missense mutation S320F in the PEX12 zinc-binding domain reduces binding to both PEX5 and PEX10. Overexpression of PEX5 or PEX10 suppresses this PEX12 mutation, providing genetic evidence for biologically relevant interactions. Loss of PEX12 or PEX10 does not reduce PEX5 association with peroxisomes, placing them downstream of docking.\",\n      \"method\": \"Two-hybrid studies, blot overlay assays, coimmunoprecipitation, genetic suppressor overexpression\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods plus genetic epistasis, patient mutation validation\",\n      \"pmids\": [\"10562279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Human PEX5L amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for peroxisome targeting. A 21-amino acid motif (aa 209-229) shared with yeast Pex18p/Pex21p mediates PTS2 import and PEX7 binding; a conserved serine mutation in this motif abolishes PTS2 import and reduces PEX5L-PEX7 interaction in vitro.\",\n      \"method\": \"Domain mapping, in vitro binding assays, mutagenesis, complementation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis combined with in vitro binding and functional rescue assays\",\n      \"pmids\": [\"11546814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Disruption of the Pex5pL-Pex7p interaction by a point mutation (S214F in the PEX5L-specific region) completely abolishes PTS2 import in mammalian cells without affecting PTS1 import or Pex14p binding, establishing that the Pex5pL-Pex7p interaction is essential and sufficient for PTS2 pathway function.\",\n      \"method\": \"CHO cell mutant isolation, point mutation analysis, co-immunoprecipitation, complementation rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean separation of PTS1 and PTS2 defects by single mutation with multiple functional readouts\",\n      \"pmids\": [\"10767287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The seven conserved di-aromatic pentapeptide repeats (WX(E/D/Q/A/S)(E/D/Q)(F/Y) motifs) in the N-terminus of PEX5 each bind independently to the same site in the N-terminus of PEX14 with nanomolar affinity. The conserved aromatic residues at positions 1 and 5 of the motif are essential for high-affinity binding.\",\n      \"method\": \"Two-hybrid analysis, in vitro binding assays, surface plasmon resonance, mutational analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — quantitative in vitro binding with mutagenesis, multiple orthogonal approaches\",\n      \"pmids\": [\"11438541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PEX5 shuttles into peroxisomes in an ATP-independent manner and is exported in an ATP-dependent manner. PEX1 and PEX6 (AAA ATPases) and their recruiter PEX26 are essential for PEX5 export. PEX14 is required as a docking site for PEX5 import. DTM-embedded PEX5 exists in two distinct membrane complexes (~500 kDa and ~800 kDa) comprising different sets of peroxins.\",\n      \"method\": \"Cell-free translocation assay with isolated peroxisomes, blue-native PAGE, multiple cell mutant lines\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-free system with multiple cell mutants, biochemical fractionation, replicated findings\",\n      \"pmids\": [\"16314507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The N-terminal domain of PEX14 adopts a three-helical fold, and PEX5 and PEX19 ligand helices bind competitively to the same surface in PEX14(N) with opposite directionality. The conserved aromatic side chains in the PEX5 WxxxF/Y motif mediate this interaction. Mutations in the PEX14 binding surface disrupt PEX5 and/or PEX19 binding in vitro and impair peroxisomal membrane localisation in vivo.\",\n      \"method\": \"NMR structure determination, competitive binding assays, mutagenesis, in vivo localization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with functional mutagenesis validation in vitro and in vivo\",\n      \"pmids\": [\"19197237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cargo protein translocation across the peroxisomal membrane occurs downstream of a reversible docking step and upstream of PEX5 ubiquitination (the first ATP-dependent cytosolic step), placing cargo translocation concurrent with PEX5 insertion into the docking/translocation machinery.\",\n      \"method\": \"In vitro peroxisomal import system, protease protection assays, ATP depletion experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — cell-free reconstitution system with defined biochemical steps, validated by multiple manipulations\",\n      \"pmids\": [\"19632994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PEX5 binds monomeric catalase and potently inhibits its tetramerization; no complex forms with tetrameric catalase. The PEX5-catalase interaction requires domains in both the N- and C-terminal halves of PEX5. The N-terminal domain of PEX14 disrupts the PEX5-catalase interaction, suggesting PEX14 participates in cargo protein release at the docking/translocation machinery.\",\n      \"method\": \"In vitro binding assays, size-exclusion chromatography, PEX5 domain truncation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstitution with defined protein fragments, multiple orthogonal experiments\",\n      \"pmids\": [\"21976670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"USP9X is identified as the primary deubiquitinase acting on the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) in female rat liver and HeLa cells. USP9X can hydrolyze thioester, isopeptide, and peptide bonds and is an elongated monomeric protein.\",\n      \"method\": \"Biochemical fractionation, in vitro deubiquitinase assay, mass spectrometry identification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — unbiased biochemical approach with functional validation of enzymatic activity\",\n      \"pmids\": [\"22371489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PEX5 is monoubiquitinated at a conserved cysteine residue (Cys11 in human PEX5), which is mandatory for ATP-dependent dislocation of PEX5 back into the cytosol. This cysteine functions as a redox switch: exposure to oxidized glutathione yields a ubiquitination-deficient PEX5, and substitution of Cys11 with lysine counteracts this oxidative inactivation.\",\n      \"method\": \"Cell-free in vitro import system, mutagenesis, PEGylation assays for cysteine modification state, human fibroblast experiments\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro and cell-based systems with mutagenesis, redox manipulation\",\n      \"pmids\": [\"24118911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A novel PEX5-PEX14 interaction site (LVAEF/LVXEF motif) was identified in PEX5 by peptide library screening. NMR structure shows this motif binds PEX14-N in an α-helical orientation similar to WxxxF/Y but with faster dissociation kinetics. Alanine substitution of LVAEF strongly impairs matrix protein import in vivo, and replacing it with the higher-affinity WxxxF/Y motif paradoxically also impairs import, suggesting distinct kinetic properties are required.\",\n      \"method\": \"Peptide library blot analysis, NMR structure determination, surface plasmon resonance, in vivo import assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure combined with quantitative binding kinetics and in vivo functional assays\",\n      \"pmids\": [\"24235149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cargo translocation across the peroxisomal membrane occurs prior to PEX5 ubiquitination, and a reversible docking step precedes irreversible membrane insertion. This positions cargo release upstream of the ATP-dependent PEX5 extraction step.\",\n      \"method\": \"In vitro import system with cargo protein-centered perspective, ATP manipulation, protease protection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — cell-free reconstitution system confirming pathway order with multiple experimental manipulations\",\n      \"pmids\": [\"23963456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The PEX5 receptor adapts its TPR binding cavity conformation for high-affinity PTS1 binding rather than the cargo signal adapting. Upon ligand binding, the binding cavity shrinks to one-third of its original volume. A bulky side chain in the wild-type cargo blocks this compaction; a single-residue mutation removing this impediment increases peroxisomal import efficiency from 34% to 80%.\",\n      \"method\": \"High-resolution crystal structure, mutagenesis, in vivo import efficiency measurements\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and quantitative in vivo import assays\",\n      \"pmids\": [\"25369882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Export-deficient PEX5 proteins bearing bulky C-terminal tags trigger pexophagy in an autophagy-dependent manner. Monoubiquitination of the N-terminal cysteine of peroxisome-associated PEX5 is required for this process. A C-terminal tag does not inhibit PEX5 monoubiquitination but strongly inhibits its export, suggesting that monoubiquitinated PEX5 retained at the membrane serves as a quality control signal for pexophagy.\",\n      \"method\": \"Autophagy assays, PEX5 tagging and export-block experiments, ubiquitination assays in mouse embryonic fibroblasts\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean mechanistic dissection with defined mutants and autophagy-dependence validation\",\n      \"pmids\": [\"26086376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A frameshift mutation in the PEX5L-specific exon 9 (c.722dupA) causes selective loss of the PEX5L isoform, resulting in defective PTS2 protein import only (not PTS1), causing RCDP5 rather than Zellweger syndrome. PEX5L expression in patient fibroblasts restores PTS2 import, confirming the isoform-specific function.\",\n      \"method\": \"Patient genetic analysis, biochemical complementation assays in fibroblasts, isoform-specific functional rescue\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — natural human mutation with clean isoform-specific biochemical phenotype and rescue\",\n      \"pmids\": [\"26220973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRIM37 localizes to peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with PEX5's C-terminal 51 amino acids. This non-degradative ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import. TRIM37 depletion reduces PEX5 abundance via proteasomal degradation and impairs cargo binding and PTS protein import.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, mutagenesis (K464A), siRNA knockdown, import assays in human cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods identifying E3 ligase, substrate site, and functional consequence\",\n      \"pmids\": [\"28724525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The DTM (docking/translocation module) is a large cavity-forming protein assembly into which PEX5 enters to release its cargo. Truncated PEX5(1-125) interacts with DTM but remains accessible to exogenously added proteinase K, and multiple PEX5 truncated molecules can be accommodated simultaneously, suggesting a cavity architecture.\",\n      \"method\": \"Truncated PEX5 probing of DTM architecture, proteinase K accessibility assays, competition experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-free system with defined PEX5 truncations, single lab study\",\n      \"pmids\": [\"28765278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DTM-embedded monoubiquitinated PEX5 (Ub-PEX5) interacts directly with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event, establishing Ub-PEX5 as a bona fide substrate of the PEX1-PEX6 AAA ATPase complex.\",\n      \"method\": \"Cell-free in vitro system, photoaffinity cross-linking, protein PEGylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — cell-free reconstitution with photoaffinity crosslinking and unfolding assays\",\n      \"pmids\": [\"29884772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cys11 of human PEX5 functions as a redox switch modulating import receptor activity in response to oxidative stress. Oxidative stress specifically impairs catalase import more than canonical PTS1 reporters, and PEX5 does not oligomerize in cellulo even under oxidative stress. Cytosolic catalase retained when PEX5 is inactivated can protect against H2O2-mediated redox changes.\",\n      \"method\": \"Live-cell FRET-based import assay, redox manipulation, mutagenesis in human fibroblasts\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — novel live-cell approach combined with mutagenesis and functional readouts\",\n      \"pmids\": [\"28760655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PEX5 escorts adipose triglyceride lipase (ATGL) to contact points between peroxisomes and lipid droplets during fasting, facilitating ATGL translocation onto lipid droplets and promoting fasting-induced lipolysis. Adipocyte-specific PEX5 knockout mice show defective ATGL recruitment to lipid droplets and attenuated fasting-induced lipolysis.\",\n      \"method\": \"Conditional knockout mice, live imaging of peroxisome-lipid droplet contacts, coimmunoprecipitation, ATGL localization assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with defined cellular phenotype plus mechanistic co-localization data\",\n      \"pmids\": [\"31996685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PEX5 accompanies cargo completely into the peroxisome lumen in Xenopus egg extract, utilizing WxxxF/Y motifs near its N-terminus to bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix binding to the lumenal side of the ubiquitin ligase; the N-terminus then emerges in the cytosol for monoubiquitination. PEX5 is extracted from the lumen by unfolding of the receptor, resulting in cargo release.\",\n      \"method\": \"Xenopus egg extract import system, domain mapping, mutagenesis of WxxxF/Y motifs and amphipathic helix\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstitution in egg extract with multiple mutagenesis experiments establishing mechanistic steps\",\n      \"pmids\": [\"35931083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and serves as the extraction initiator; the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6. An intra-molecularly cross-linked ubiquitin at position 11 blocks extraction, confirming ubiquitin unfolding is required for the extraction mechanism.\",\n      \"method\": \"Cell-free in vitro system, engineered PEX5 and ubiquitin molecules, cross-linked ubiquitin experiments\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — cell-free reconstitution with chemically engineered substrates and rigorous controls\",\n      \"pmids\": [\"36442669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AWP1/ZFAND6 is a cofactor of PEX6 involved in PEX5 export. AWP1 preferentially binds cysteine-ubiquitinated PEX5 via its A20 zinc-finger domain, stimulates PEX5 export in vitro, and interacts with PEX6 AAA ATPase. AWP1 knockdown reduces PTS1-protein import and decreases PEX5 stability similarly to PEX1/PEX6/PEX26 deficient cells.\",\n      \"method\": \"Biochemical fractionation, in vitro Pex5 export assay, co-immunoprecipitation, siRNA knockdown\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical identification with functional in vitro and in vivo validation\",\n      \"pmids\": [\"21980954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PEX5 in Pichia pastoris functions as a redox-regulated receptor: disulfide bond-linked Pex5 dimers/oligomers show highest affinity for PTS1 cargo; reduction transitions Pex5 to a noncovalent dimer with partial cargo release. A hetero-oligomeric interaction between the Pex5 N-terminal domain (aa 1-110) and the C-terminal motif of Pex8 further facilitates cargo release under reducing conditions.\",\n      \"method\": \"In vitro binding assays, disulfide cross-linking, DTT reduction experiments, Pex5-Pex8 pulldown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution in yeast ortholog, mechanistic details on redox control\",\n      \"pmids\": [\"23902771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Pex5p stabilizes Pex14p; in the absence of Pex5p, Pex14p is unstable due to inefficient translocation to the peroxisomal membrane. The fifth WXXXF/Y motif of Pex5pL is an auxiliary binding site for Pex14p required for Pex14p stability. Pex5p-Pex13p interaction is essential for import of PTS1 proteins and catalase but not PTS2 proteins.\",\n      \"method\": \"CHO cell mutant isolation, complementation with Pex5p domain mutants, western blot stability assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean cell mutant with domain mapping, single lab\",\n      \"pmids\": [\"23009329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Monoubiquitination of PEX5 at cysteine 11 is noncanonical (thioester bond) and reversible; this reversibility prevents polyubiquitination of PEX5 at the peroxisomal membrane. A PEX5 variant with lysine at position 11 undergoes polyubiquitination that negatively interferes with extraction. E2-mediated deubiquitination kinetics are faster than PEX5 polyubiquitination, ensuring the transient monoubiquitinated state.\",\n      \"method\": \"Cell-free rat liver in vitro system, engineered PEX5 Cys11Lys mutant, ubiquitination kinetics analysis\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstitution with defined mutants establishing mechanistic rationale for cysteine ubiquitination\",\n      \"pmids\": [\"38470934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Chemically synthesized monoubiquitinated PEX5 binds PEX13, PEX14, and the receptor export module components PEX1, PEX6, and PEX26. Interactions with PEX13 and PEX14 are independent of PEX5 ubiquitination status, whereas interactions with PEX1, PEX6, and PEX26 are enhanced by ubiquitination. Monoubiquitinated PEX5 also binds PEX7/PTS2 complexes and restores PTS2 import in ΔPEX5 fibroblasts.\",\n      \"method\": \"Click chemistry synthesis of Ub-PEX5, in vitro pulldown assays, complementation in ΔPEX5 fibroblasts\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — chemically defined ubiquitin conjugates with in vitro binding and in vivo rescue\",\n      \"pmids\": [\"30375424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Small angle X-ray scattering reveals that free full-length human Pex5p is monomeric in solution with an elongated, partially unfolded N-terminal domain. The Pex5p:Pex14p complex shows 1:6 stoichiometry. In the complex, the N-terminus of Pex5p remains extended, with Pex14p molecules significantly intermingled with the Pex5p moiety.\",\n      \"method\": \"Small angle X-ray scattering, static light scattering, solution structure modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — structural analysis in solution, single study\",\n      \"pmids\": [\"19584060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PEX5L is monomeric with compact spherical conformation in solution. Labeled PEX5L accumulates ~100-fold at lipid bilayers and forms ion-conducting membrane channels in electrophysiological recordings. The truncated PEX5L(1-335) lacking the cargo-binding domain does not form channels, suggesting that PEX5L is the pore-forming component of the oligomeric peroxisomal translocon and that membrane surface binding precedes channel assembly.\",\n      \"method\": \"Horizontal lipid bilayer electrophysiology, fluorescence TCSPC, diffusion coefficient measurements\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstituted membrane system with electrophysiology, single lab study\",\n      \"pmids\": [\"36260915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Full-length tetrameric PEX5 has high intrinsic affinity for the PTS1 peptide (Kd ~35 nM) as measured by fluorescence anisotropy. PEX5-PTS1 binding kinetics are unaffected by Hsp70 (with or without ATP/ADP) or by the PEX12 zinc RING domain, indicating that initial PTS1 recognition is an autonomous step not regulated by these factors.\",\n      \"method\": \"Fluorescence anisotropy binding assay with purified recombinant proteins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — quantitative in vitro binding assay, single lab\",\n      \"pmids\": [\"12456682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PTS1 variants corresponding to known functional targeting signals bind PEX5 TPR domain within 1.8 kcal/mol of the optimal -SKL sequence. A binding energy threshold determines PTS1 functionality, correlating structural features from the PEX5:PTS1 crystal structure with thermodynamic binding parameters.\",\n      \"method\": \"Fluorescence-based binding assay with synthetic peptide library, thermodynamic analysis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — quantitative in vitro binding with structure-activity correlation, single lab\",\n      \"pmids\": [\"12578380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A missense mutation F218S in PEX5 (within the PEX7-interaction domain) disrupts the trimeric complex formation between PEX5, PEX7, and a PTS2 cargo protein, abolishing PTS2 protein import while leaving PTS1 import, monoubiquitination, and export intact, causing congenital cataracts in affected patients.\",\n      \"method\": \"In vitro import assays, trimeric complex pulldown, patient fibroblast studies, lens-specific mouse KO\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic dissection with in vitro assays plus patient and mouse model validation\",\n      \"pmids\": [\"33389129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PEX13 loss causes accumulation of ubiquitinated PEX5 on peroxisomes and increased peroxisome-derived ROS, together inducing pexophagy. PEX13 protein levels are downregulated during amino acid starvation to facilitate pexophagy induction, establishing PEX13 as a gatekeeper that prevents premature pexophagy by limiting accumulation of ubiquitinated PEX5.\",\n      \"method\": \"Gene editing, quantitative fluorescence microscopy, zebrafish model, ubiquitination assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple model systems with mechanistic pathway placement\",\n      \"pmids\": [\"36541703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structure of yeast Pex5 in complex with cargo protein Eci1 reveals that Eci1 can bind Pex5 and reach peroxisomes in the absence of a canonical PTS1 signal, through additional binding interfaces beyond the PTS1-binding site in the TPR domain.\",\n      \"method\": \"Cryo-electron microscopy structure determination, PTS1-deletion mutant analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with functional validation of non-canonical binding\",\n      \"pmids\": [\"40376748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In mammalian cells, PEX5 depletion under serum starvation leads to downregulation of TSC2, activation of mTORC1 (increased phosphorylation of 70S6K, S6K, and 4E-BP1), and suppression of TFEB nuclear localization. Pharmacological mTOR inhibition upon PEX5 depletion during starvation activates TFEB and recovers peroxisome biogenesis, placing PEX5 upstream of mTORC1-TFEB axis.\",\n      \"method\": \"siRNA knockdown, pharmacological mTOR inhibition, nuclear localization assays, peroxisome biogenesis readouts\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pathway epistasis via pharmacological and genetic approaches, single lab\",\n      \"pmids\": [\"29622767\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PEX5 is a cytosolic shuttling receptor that recognizes PTS1-containing peroxisomal matrix proteins via its C-terminal TPR domain and, in its longer isoform (PEX5L), co-receptors PTS2 cargo by binding PEX7 through an N-terminal 21-amino acid motif; cargo-loaded PEX5 docks at the peroxisomal membrane via multiple WxxxF/Y motifs binding PEX14, then enters completely into the peroxisomal lumen through a large cavity-forming translocon (DTM), releases cargo upon unfolding driven by extraction, becomes monoubiquitinated at Cys11 (via a reversible thioester bond that prevents overubiquitination) by a membrane-embedded ubiquitin ligase, and is then extracted back to the cytosol by the PEX1-PEX6 AAA ATPase complex (aided by AWP1/ZFAND6 and deubiquitinated by USP9X), with Cys11 additionally functioning as a redox sensor that inactivates PEX5 during oxidative stress; when export is blocked, monoubiquitinated membrane-retained PEX5 serves as a pexophagy signal, and TRIM37 stabilizes PEX5 via non-degradative K464 ubiquitination to maintain adequate receptor levels for efficient import.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PEX5 is the cytosolic cycling receptor for peroxisomal matrix protein import, recognizing PTS1 cargo through its C-terminal TPR domain and, in its longer isoform (PEX5L), bridging PTS2 cargo via a conserved N-terminal motif that binds PEX7 [PMID:7719337, PMID:11101887, PMID:9418886, PMID:11546814]. Cargo-loaded PEX5 docks at the peroxisomal membrane through multiple WxxxF/Y pentapeptide motifs that bind PEX14 with nanomolar affinity, then inserts fully into the lumenal cavity of the docking/translocation machinery (DTM) where it releases cargo; PEX5 is subsequently monoubiquitinated at a conserved cysteine (Cys11) via a thioester bond whose reversibility prevents polyubiquitination, and the ubiquitin–PEX5 conjugate is extracted back to the cytosol by the PEX1–PEX6 AAA-ATPase complex through global unfolding of both ubiquitin and PEX5 [PMID:10026185, PMID:11438541, PMID:35931083, PMID:29884772, PMID:36442669, PMID:38470934]. Cys11 additionally serves as a redox sensor that inactivates PEX5 under oxidative stress, preferentially retaining catalase in the cytosol as a protective response, while monoubiquitinated PEX5 that fails to be exported acts as a pexophagy signal linking import failure to organelle quality control [PMID:24118911, PMID:28760655, PMID:26086376, PMID:36541703]. Mutations in PEX5 cause peroxisome biogenesis disorders: complete loss underlies Zellweger-spectrum disease (complementation group 2), whereas isoform-specific PEX5L mutations cause rhizomelic chondrodysplasia punctata type 5 (RCDP5), and a PEX7-interaction-domain missense variant causes congenital cataracts [PMID:7719337, PMID:26220973, PMID:33389129].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of PEX5 as the cytosolic PTS1 receptor resolved how peroxisomal matrix proteins are recognized in the cytosol and established that PEX5 mutations define complementation group 2 of peroxisome biogenesis disorders.\",\n      \"evidence\": \"Complementation cloning and rescue of PTS1 import in patient fibroblasts\",\n      \"pmids\": [\"7719337\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of membrane engagement unknown\", \"Structural basis for PTS1 recognition undetermined\", \"Role in PTS2 import unclear\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Discovery of two PEX5 isoforms (PEX5S and PEX5L) established that the longer isoform is uniquely required for PTS2 protein import, separating PTS1 and PTS2 import pathways at the receptor level.\",\n      \"evidence\": \"CHO cell mutant complementation with isoform-specific constructs and TPR mutagenesis\",\n      \"pmids\": [\"9418886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of PEX5L–PEX7 interaction not mapped\", \"Whether PEX5L acts as a co-receptor or independent receptor for PTS2 unclear\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapping of multiple PEX14-binding sites in PEX5's N-terminal half and demonstration of nanomolar affinity defined the docking mechanism at the peroxisomal membrane, while interaction with PEX12 was placed downstream of docking.\",\n      \"evidence\": \"Surface plasmon resonance, two-hybrid, co-IP, and genetic suppressor experiments\",\n      \"pmids\": [\"10026185\", \"10562279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nature of the translocation pore unknown\", \"Order of PEX5 steps at the membrane unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"The crystal structure of PEX5's TPR domain bound to a PTS1 peptide revealed a novel two-cluster TPR architecture that nearly encircles the cargo signal, providing the molecular basis for PTS1 recognition specificity.\",\n      \"evidence\": \"X-ray crystallography of human PEX5 TPR–PTS1 complex\",\n      \"pmids\": [\"11101887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TPR domain conformational changes regulate binding/release not established\", \"Full-length PEX5 structure unavailable\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of the conserved WxxxF/Y pentapeptide motifs as independent PEX14-binding elements and the 21-amino-acid PEX7-binding motif in PEX5L resolved the modular architecture underlying receptor docking and PTS2 co-receptor function.\",\n      \"evidence\": \"Mutagenesis, SPR quantification, in vitro binding, and complementation assays\",\n      \"pmids\": [\"11438541\", \"11546814\", \"10767287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional non-equivalence among the seven WxxxF/Y motifs not understood\", \"Stoichiometry of PEX5–PEX14 complex at the membrane unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishment that PEX5 insertion into peroxisomes is ATP-independent whereas its export requires ATP and the PEX1–PEX6–PEX26 complex defined the energy-dependent recycling step and ordered the import cycle.\",\n      \"evidence\": \"Cell-free translocation assay with isolated peroxisomes and blue-native PAGE\",\n      \"pmids\": [\"16314507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PEX5 fully enters the lumen or remains membrane-embedded debated\", \"Ubiquitination requirement for export not yet shown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"NMR structures of the PEX14-N domain bound to PEX5 and PEX19 helices, and SAXS analysis showing a 1:6 PEX5:PEX14 stoichiometry, provided atomic-level understanding of docking and revealed that PEX5 remains extended in solution.\",\n      \"evidence\": \"NMR structure, SAXS, competitive binding, mutagenesis, and in vivo localization\",\n      \"pmids\": [\"19197237\", \"19584060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PEX5–PEX14 complex structure in a membrane context not determined\", \"Function of the intrinsically disordered N-terminus beyond PEX14 binding unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Ordering cargo translocation as concurrent with PEX5 membrane insertion and upstream of ubiquitination clarified that cargo release precedes the ATP-dependent extraction step.\",\n      \"evidence\": \"In vitro peroxisomal import with protease protection and ATP depletion\",\n      \"pmids\": [\"19632994\", \"23963456\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of cargo release inside the organelle not established\", \"Whether PEX14 directly triggers cargo release in vivo unconfirmed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Discovery that PEX5 binds monomeric but not tetrameric catalase, and that PEX14 disrupts the PEX5–catalase complex, suggested a cargo-release mechanism at the docking site; identification of AWP1/ZFAND6 as a PEX6 cofactor that preferentially binds Cys-ubiquitinated PEX5 added a new component to the export machinery.\",\n      \"evidence\": \"In vitro reconstitution, size-exclusion chromatography, siRNA knockdown, and export assays\",\n      \"pmids\": [\"21976670\", \"21980954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"AWP1's exact role in the extraction mechanism unclear\", \"How catalase monomeric state is maintained during transport unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identification of USP9X as the primary deubiquitinase for the Ub–PEX5 thioester conjugate established the recycling pathway's final step returning PEX5 to its import-competent state.\",\n      \"evidence\": \"Biochemical fractionation of rat liver and HeLa cytosol, in vitro DUB assay, mass spectrometry\",\n      \"pmids\": [\"22371489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether USP9X acts before or after PEX5 extraction not determined\", \"Redundancy with other DUBs not excluded\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstration that PEX5 Cys11 monoubiquitination is mandatory for export, and that this cysteine functions as a redox switch inactivated by oxidized glutathione, unified the ubiquitination and redox-sensing functions into a single regulatory residue; a novel LVAEF motif with distinct kinetics was added to the PEX14-binding repertoire.\",\n      \"evidence\": \"Cell-free import, Cys11 mutagenesis, PEGylation assays, NMR of LVAEF–PEX14 complex, SPR kinetics\",\n      \"pmids\": [\"24118911\", \"24235149\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How redox state transitions are reversed in vivo unknown\", \"Functional hierarchy among WxxxF/Y and LVAEF motifs in the import cycle not fully mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Crystal structures showing that PEX5's TPR cavity compacts upon PTS1 binding (volume reduced to one-third) established a receptor-adapts-to-cargo model and linked cavity plasticity to import efficiency.\",\n      \"evidence\": \"High-resolution crystal structures, mutagenesis, in vivo import quantification\",\n      \"pmids\": [\"25369882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether compaction triggers downstream signaling events unknown\", \"Structural basis for non-canonical cargo binding not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstration that export-deficient monoubiquitinated PEX5 retained at the membrane triggers autophagy-dependent pexophagy established PEX5 as a quality-control signal; a frameshift mutation selectively ablating PEX5L confirmed isoform-specific causation of RCDP5.\",\n      \"evidence\": \"Export-block assays in MEFs with autophagy readouts; patient fibroblast complementation with PEX5L rescue\",\n      \"pmids\": [\"26086376\", \"26220973\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor for ubiquitinated PEX5 in pexophagy not identified\", \"Whether RCDP5 phenotype involves additional PEX5L-specific functions beyond PEX7 binding unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"TRIM37 was identified as a peroxisomal E3 ligase that stabilizes PEX5 through non-degradative K464 ubiquitination, maintaining receptor levels for efficient import; concurrent studies confirmed PEX5 Cys11 as a redox sensor in live cells and characterized the DTM as a large cavity that accommodates multiple PEX5 molecules.\",\n      \"evidence\": \"Co-IP, ubiquitination site mapping, siRNA, FRET-based import assays, proteinase K accessibility on DTM\",\n      \"pmids\": [\"28724525\", \"28760655\", \"28765278\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"TRIM37 regulation and its coordination with Cys11 ubiquitination unexplored\", \"DTM architecture at atomic resolution unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Direct interaction of ubiquitinated PEX5 with both PEX1 and PEX6, and demonstration that the PEX5 polypeptide is globally unfolded during extraction, established Ub-PEX5 as a bona fide substrate of the AAA-ATPase complex and linked unfolding to cargo release.\",\n      \"evidence\": \"Cell-free system, photoaffinity cross-linking, PEGylation-based unfolding assay, chemically synthesized Ub-PEX5\",\n      \"pmids\": [\"29884772\", \"30375424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of PEX1–PEX6 engaged with Ub-PEX5 not determined\", \"How unfolded PEX5 refolds in the cytosol unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery that PEX5 escorts ATGL to peroxisome–lipid droplet contact sites during fasting expanded PEX5's role beyond matrix protein import to inter-organelle lipid metabolism.\",\n      \"evidence\": \"Adipocyte-specific PEX5 conditional knockout mice, live imaging, co-IP\",\n      \"pmids\": [\"31996685\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PEX5's ATGL-escort function uses the same WxxxF/PEX14 docking or a distinct mechanism unknown\", \"Generality to other non-peroxisomal cargoes not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Reconstitution in Xenopus egg extract proved PEX5 enters the peroxisomal lumen entirely, using WxxxF/Y motifs for lumenal docking-complex interaction and an amphipathic helix for ubiquitin-ligase engagement to initiate N-terminal extrusion; concurrent electrophysiology showed PEX5L forms ion-conducting channels in lipid bilayers.\",\n      \"evidence\": \"Xenopus egg extract import system with mutagenesis; horizontal lipid bilayer electrophysiology\",\n      \"pmids\": [\"35931083\", \"36260915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of PEX5 channel activity not established\", \"Identity of the lumenal PEX5 receptor unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstration that the ubiquitin moiety conjugated to Cys11 must itself be unfolded before PEX1–PEX6-mediated extraction, using cross-linked ubiquitin to block threading, defined ubiquitin unfolding as the extraction-initiating event.\",\n      \"evidence\": \"Cell-free system with engineered intramolecularly cross-linked ubiquitin–PEX5 conjugates\",\n      \"pmids\": [\"36442669\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Step-by-step threading directionality not visualized\", \"Whether ubiquitin refolding after extraction is spontaneous or chaperone-assisted unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Elucidation of why PEX5 uses a thioester (Cys11) rather than an isopeptide bond for monoubiquitination showed that rapid E2-mediated deubiquitination outcompetes polyubiquitin chain elongation, preventing premature degradation and maintaining the cycling receptor pool.\",\n      \"evidence\": \"Cell-free reconstitution with Cys11Lys mutant, ubiquitination kinetics quantification\",\n      \"pmids\": [\"38470934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo kinetics of thioester turnover not measured\", \"Potential regulatory inputs controlling E2 deubiquitination rate unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM structure of yeast Pex5 with cargo Eci1 revealed non-canonical binding interfaces beyond PTS1, demonstrating that PEX5 can import proteins lacking a canonical PTS1 signal.\",\n      \"evidence\": \"Cryo-EM structure determination with PTS1-deletion mutant analysis\",\n      \"pmids\": [\"40376748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Prevalence of non-canonical PEX5 cargoes in the human proteome unknown\", \"Whether non-canonical binding uses the same translocation mechanism not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the atomic structure of the full DTM translocon with PEX5 engaged, the identity and regulation of the pexophagy receptor that recognizes membrane-retained Ub-PEX5, and how PEX5 refolds in the cytosol after extraction-coupled global unfolding.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of the assembled DTM–PEX5 complex\", \"Pexophagy receptor for Ub-PEX5 not identified\", \"PEX5 refolding mechanism after extraction unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [0, 1, 2, 16, 34]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 6, 23]},\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [24, 32]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 31, 33]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 20, 24]},\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [8, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [17, 36]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 8, 24]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [23]}\n    ],\n    \"complexes\": [\n      \"DTM (docking/translocation machinery)\",\n      \"PEX5–PEX7 co-receptor complex\"\n    ],\n    \"partners\": [\n      \"PEX14\",\n      \"PEX7\",\n      \"PEX12\",\n      \"PEX13\",\n      \"PEX1\",\n      \"PEX6\",\n      \"USP9X\",\n      \"TRIM37\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}