{"gene":"PREPL","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2005,"finding":"PREPL is localized in the cytosol and contains a catalytic triad (Ser470, Asp556, His601); substitution of these predicted catalytic residues by alanines resulted in loss of reactivity with a serine hydrolase-specific activity-based probe, confirming an intact but unique serine hydrolase catalytic machinery. Unlike prolyl oligopeptidase and oligopeptidase B, PREPL activity depends only on the carboxyterminal domain.","method":"Activity-based probe labeling with active-site mutagenesis (Ser470A, Asp556A, His601A); subcellular fractionation for cytosolic localization","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1 — direct mutagenesis of catalytic residues with activity-based probe readout, replicated in subsequent studies","pmids":["16385448"],"is_preprint":false},{"year":2005,"finding":"PREPL A (638-residue splice variant) does not cleave peptide substrates with P1 basic residues (arginine/lysine), but slowly hydrolyses an activated ester substrate and reacts with diisopropyl fluorophosphate, indicating a reactive catalytic serine but negligible physiological peptidase activity. PREPL A forms dimers, which may account for its higher conformational stability compared to oligopeptidase B.","method":"Recombinant protein expression in E. coli, peptide hydrolysis assays, ester hydrolysis assay, DFP reactivity, differential scanning calorimetry, secondary structure analysis","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assays with purified recombinant protein and multiple substrates/inhibitors","pmids":["16143824"],"is_preprint":false},{"year":2013,"finding":"PREPL interacts directly with the N-terminal 70 amino acids of the AP-1 adaptor complex subunit μ1A. PREPL overexpression reduces AP-1 membrane binding, while reduced PREPL expression increases AP-1 membrane binding and impairs AP-1 recycling. PREPL deficiency causes an expanded trans-Golgi network morphology, which is rescued by PREPL re-expression. PREPL co-localizes with residual membrane-bound AP-1.","method":"Yeast two-hybrid library screen; PREPL overexpression and knockdown with AP-1 membrane fractionation; rescue experiments; fluorescence co-localization; patient cell lines","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — yeast two-hybrid identification plus multiple orthogonal cellular assays (OE, KD, rescue, co-localization, patient lines)","pmids":["23321636"],"is_preprint":false},{"year":2014,"finding":"Isolated PREPL deficiency causes a congenital myasthenic syndrome with decreased quantal content of the endplate potential and reduced amplitude of the miniature endplate potential, without endplate acetylcholine receptor deficiency or altered endplate geometry, indicating pre- and postsynaptic neuromuscular transmission defects. The myasthenia is attributed to abrogated interaction of PREPL with adaptor protein 1 (AP-1).","method":"Immunoblot confirmation of absent PREPL expression; in vitro electrophysiology of neuromuscular junction (quantal content, MEPP amplitude); edrophonium test; histochemical and ultrastructural studies","journal":"Neurology","confidence":"High","confidence_rationale":"Tier 2 — direct electrophysiological measurement in patient tissue with absent PREPL expression confirmed, multiple orthogonal methods","pmids":["24610330"],"is_preprint":false},{"year":2014,"finding":"Deletion of exon 11 of murine Prepl (encoding key catalytic amino acids) leads to loss of PREPL protein and lower Prepl mRNA. PREPL-null mice display significant growth impairment (shorter and lighter) and neonatal hypotonia assessed by righting reflex assay, establishing PREPL as required for normal growth and muscle tone in vivo.","method":"Conditional knockout mouse model (exon 11 deletion); immunoblot; righting reflex behavioral assay; body measurement","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — clean KO mouse with defined growth and hypotonia phenotypes and confirmed protein absence","pmids":["24586561"],"is_preprint":false},{"year":2011,"finding":"Selective small-molecule inhibitors of PREPL serine hydrolase activity were identified by fluopol-ABPP high-throughput screening and confirmed to block PREPL activity in cells. One compound (1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile) distributes to the mouse brain after systemic administration.","method":"Fluorescence polarization activity-based protein profiling (fluopol-ABPP) screen of >300,000 compounds; cell-based activity assays; mouse in vivo pharmacokinetics","journal":"Journal of the American Chemical Society","confidence":"Medium","confidence_rationale":"Tier 1/2 — in vitro and cell-based enzyme inhibition assays, single study","pmids":["21692504"],"is_preprint":false},{"year":2009,"finding":"Transcription of PREPL is driven by a 243-bp GC-rich bidirectional minimal promoter in the 405-bp intergenic region shared with C2ORF34. Two transcription factors, NRF-2 and YY-1, cooperatively and additively activate PREPL (and C2ORF34) transcription by binding to this shared promoter region.","method":"Reporter gene assays; transcription factor binding site identification; co-transfection of NRF-2 and YY-1 with promoter constructs; gel shift/ChIP-like analyses","journal":"BMC molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 — functional promoter dissection with reporter assays and identified TF binding sites, single lab","pmids":["19575798"],"is_preprint":false},{"year":2024,"finding":"Missense variants in PREPL causing CMS22 do not impair hydrolase activity but reduce binding to known interactors (including AP-1 components), demonstrating that PREPL has both enzymatic and non-enzymatic (protein-interaction) functions. Catalytically inactive PREPL p.Ser559Ala cell lines showed that hydrolytic activity is required for normal mitochondrial function but not for regulating AP-1-mediated trans-Golgi network transport.","method":"Biochemical hydrolase activity assays of patient missense variants; structural analysis; protein-protein interaction assays; CRISPR-generated catalytically inactive PREPL p.Ser559Ala cell lines with mitochondrial function assays and TGN transport assays","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 1/2 — multiple orthogonal methods: enzymatic assays, structural data, interaction assays, and engineered catalytic mutant cell lines in one study","pmids":["39078710"],"is_preprint":false},{"year":2025,"finding":"PREPL KO cells accumulate triacylglycerols and show increased lipid droplet number, and exhibit elongated peroxisomes, but PREPL does not localize to peroxisomes and global phospholipid composition is largely unchanged. The lipid storage phenotype is attributed to mitochondrial dysfunction caused by PREPL loss (impaired respiratory chain/oxidative phosphorylation), which secondarily impairs fatty acid β-oxidation and promotes TAG synthesis, rather than a direct lipase role for PREPL.","method":"CRISPR-Cas9 KO cell lines; unbiased lipidomics (Prepl KO mouse brains and KO HEK293T cells); lipid droplet imaging; peroxisome number/morphology/protein-level analysis; PREPL localization studies","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (lipidomics, imaging, localization) but preprint, not yet peer reviewed","pmids":["bio_10.1101_2025.10.28.685080"],"is_preprint":true}],"current_model":"PREPL is a cytosolic serine hydrolase of the prolyl oligopeptidase family with a reactive catalytic triad (Ser470/Ser559, Asp556, His601) that confers esterase but negligible peptidase activity; it functions both enzymatically (supporting mitochondrial respiratory chain activity) and non-enzymatically by binding the AP-1 adaptor complex subunit μ1A to regulate AP-1 membrane cycling and trans-Golgi network morphology, with loss of PREPL causing a congenital myasthenic syndrome through impaired neuromuscular transmission, growth deficiency, and secondary lipid metabolic dysregulation due to mitochondrial dysfunction."},"narrative":{"teleology":[{"year":2005,"claim":"Establishing PREPL as a genuine serine hydrolase resolved a key question about whether this prolyl oligopeptidase family member was catalytically active, and revealed that its activity resides in the C-terminal domain with an unusual substrate profile (esterase rather than peptidase).","evidence":"Activity-based probe labeling with catalytic triad mutagenesis (Ser470A, Asp556A, His601A) and recombinant protein enzymatic assays with peptide and ester substrates","pmids":["16385448","16143824"],"confidence":"High","gaps":["Physiological substrate(s) of PREPL esterase activity remain unidentified","Structural basis for lack of peptidase activity despite conservation of the prolyl oligopeptidase fold is unresolved"]},{"year":2009,"claim":"Identification of NRF-2 and YY-1 as cooperative transcriptional activators of PREPL through a shared bidirectional promoter with C2ORF34 established the regulatory logic controlling PREPL expression.","evidence":"Reporter gene assays and transcription factor co-transfection with promoter constructs","pmids":["19575798"],"confidence":"Medium","gaps":["Endogenous chromatin occupancy of NRF-2 and YY-1 at the PREPL promoter has not been validated by ChIP in physiological settings","Tissue-specific regulation of PREPL expression is unexplored"]},{"year":2011,"claim":"Discovery of selective small-molecule PREPL inhibitors provided chemical tools to probe PREPL function in cells and demonstrated brain penetrance in vivo.","evidence":"Fluopol-ABPP high-throughput screen of >300,000 compounds with cell-based validation and mouse pharmacokinetics","pmids":["21692504"],"confidence":"Medium","gaps":["Phenotypic consequences of pharmacological PREPL inhibition in vivo have not been reported","Selectivity across the broader serine hydrolase family beyond initial profiling is incomplete"]},{"year":2013,"claim":"Discovery that PREPL directly binds the μ1A subunit of AP-1 and regulates AP-1 membrane cycling revealed a non-enzymatic function in vesicle trafficking, explaining the expanded trans-Golgi network morphology in PREPL-deficient cells.","evidence":"Yeast two-hybrid screen, overexpression/knockdown with AP-1 membrane fractionation, rescue experiments, and co-localization in patient cell lines","pmids":["23321636"],"confidence":"High","gaps":["Whether PREPL binding to μ1A requires a specific conformational state or post-translational modification is unknown","Cargo specificity of PREPL-regulated AP-1 trafficking is not defined"]},{"year":2014,"claim":"Demonstration that isolated PREPL deficiency causes congenital myasthenic syndrome (CMS22) with pre- and postsynaptic neuromuscular transmission defects, and that PREPL-null mice recapitulate growth impairment and hypotonia, established the disease mechanism and in vivo requirement.","evidence":"Patient neuromuscular junction electrophysiology (quantal content, MEPP amplitude) and Prepl exon 11 knockout mouse model with righting reflex and growth assays","pmids":["24610330","24586561"],"confidence":"High","gaps":["The specific vesicle trafficking step at the neuromuscular junction that fails in PREPL deficiency is not delineated","Whether myasthenia results from the AP-1 interaction defect, loss of catalytic activity, or both was unresolved at this stage"]},{"year":2024,"claim":"Separation of PREPL's enzymatic and non-enzymatic functions revealed that hydrolase activity is required for mitochondrial function but dispensable for AP-1-mediated TGN transport, while CMS22 missense variants specifically impair protein-protein interactions rather than catalysis.","evidence":"Biochemical assays of patient missense variants, CRISPR-generated catalytically inactive Ser559Ala cell lines, mitochondrial function assays, and TGN transport assays","pmids":["39078710"],"confidence":"High","gaps":["The mitochondrial substrate or process directly regulated by PREPL catalytic activity is unknown","How PREPL missense variants structurally impair protein interactions without affecting the active site has not been resolved at atomic resolution"]},{"year":2025,"claim":"Lipid accumulation in PREPL-deficient cells was shown to be a secondary consequence of mitochondrial respiratory chain dysfunction rather than a direct lipase function, reframing the metabolic phenotype as downstream of impaired oxidative phosphorylation.","evidence":"CRISPR KO cell lines with unbiased lipidomics, lipid droplet imaging, peroxisome morphology analysis, and PREPL localization studies (preprint)","pmids":["bio_10.1101_2025.10.28.685080"],"confidence":"Medium","gaps":["Not yet peer reviewed","The specific respiratory chain complex or assembly step affected by PREPL loss is not identified","Whether lipid dysregulation contributes to the neuromuscular phenotype in vivo is untested"]},{"year":null,"claim":"The identity of PREPL's physiological enzymatic substrate(s) and the precise mechanism by which its catalytic activity supports mitochondrial function remain the central open questions.","evidence":"","pmids":[],"confidence":"High","gaps":["No endogenous substrate for PREPL esterase activity has been identified","Whether PREPL acts inside mitochondria or on a cytosolic target that feeds into mitochondrial pathways is unresolved","The relative contribution of enzymatic vs. AP-1-regulatory functions to CMS22 pathology is not quantified in vivo"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,5,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,7]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,7]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,4]}],"complexes":[],"partners":["AP1M1","NRF2","YY1"],"other_free_text":[]},"mechanistic_narrative":"PREPL is a cytosolic serine hydrolase of the prolyl oligopeptidase family that possesses both enzymatic and non-enzymatic functions critical for neuromuscular transmission, growth, and organelle homeostasis. Its catalytic triad (Ser470/Ser559, Asp556, His601) confers esterase but negligible peptidase activity, and hydrolytic function is specifically required for normal mitochondrial respiratory chain activity, with PREPL loss causing secondary triacylglycerol accumulation through impaired fatty acid β-oxidation [PMID:16385448, PMID:16143824, PMID:39078710]. Independent of catalytic activity, PREPL binds the μ1A subunit of the AP-1 adaptor complex to regulate AP-1 membrane cycling and trans-Golgi network morphology; disease-causing missense variants disrupt this interaction without impairing hydrolase activity [PMID:23321636, PMID:39078710]. Loss of PREPL causes congenital myasthenic syndrome (CMS22) with decreased neuromuscular quantal content and reduced miniature endplate potential amplitude, as well as growth deficiency and neonatal hypotonia in knockout mice [PMID:24610330, PMID:24586561]."},"prefetch_data":{"uniprot":{"accession":"Q4J6C6","full_name":"Prolyl endopeptidase-like","aliases":["Prolylendopeptidase-like"],"length_aa":727,"mass_kda":83.9,"function":"Serine peptidase whose precise substrate specificity remains unclear (PubMed:16143824, PubMed:16385448, PubMed:28726805). Does not cleave peptides after a arginine or lysine residue (PubMed:16143824). Regulates trans-Golgi network morphology and sorting by regulating the membrane binding of the AP-1 complex (PubMed:23321636). May play a role in the regulation of synaptic vesicle exocytosis (PubMed:24610330)","subcellular_location":"Cytoplasm, cytosol; Golgi apparatus, trans-Golgi network; Cytoplasm, cytoskeleton; Golgi apparatus; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q4J6C6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PREPL","classification":"Not Classified","n_dependent_lines":19,"n_total_lines":1208,"dependency_fraction":0.015728476821192054},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PREPL","total_profiled":1310},"omim":[{"mim_id":"616224","title":"MYASTHENIC SYNDROME, CONGENITAL, 22; CMS22","url":"https://www.omim.org/entry/616224"},{"mim_id":"609557","title":"PROLYL ENDOPEPTIDASE-LIKE; PREPL","url":"https://www.omim.org/entry/609557"},{"mim_id":"606407","title":"HYPOTONIA-CYSTINURIA SYNDROME","url":"https://www.omim.org/entry/606407"},{"mim_id":"601462","title":"MYASTHENIC SYNDROME, CONGENITAL, 1A, SLOW-CHANNEL; CMS1A","url":"https://www.omim.org/entry/601462"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PREPL"},"hgnc":{"alias_symbol":["KIAA0436"],"prev_symbol":[]},"alphafold":{"accession":"Q4J6C6","domains":[{"cath_id":"3.40.50.1820","chopping":"441-725","consensus_level":"high","plddt":94.2346,"start":441,"end":725},{"cath_id":"2.20.25","chopping":"112-201","consensus_level":"medium","plddt":88.3093,"start":112,"end":201}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q4J6C6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q4J6C6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q4J6C6-F1-predicted_aligned_error_v6.png","plddt_mean":82.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PREPL","jax_strain_url":"https://www.jax.org/strain/search?query=PREPL"},"sequence":{"accession":"Q4J6C6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q4J6C6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q4J6C6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q4J6C6"}},"corpus_meta":[{"pmid":"16385448","id":"PMC_16385448","title":"Deletion of PREPL, a gene encoding a putative serine oligopeptidase, in patients with hypotonia-cystinuria syndrome.","date":"2005","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/16385448","citation_count":69,"is_preprint":false},{"pmid":"24610330","id":"PMC_24610330","title":"PREPL deficiency with or without cystinuria causes a novel myasthenic syndrome.","date":"2014","source":"Neurology","url":"https://pubmed.ncbi.nlm.nih.gov/24610330","citation_count":48,"is_preprint":false},{"pmid":"18234729","id":"PMC_18234729","title":"Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome.","date":"2008","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18234729","citation_count":37,"is_preprint":false},{"pmid":"16913837","id":"PMC_16913837","title":"PREPL: a putative novel oligopeptidase propelled into the limelight.","date":"2006","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16913837","citation_count":30,"is_preprint":false},{"pmid":"16143824","id":"PMC_16143824","title":"The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity.","date":"2005","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/16143824","citation_count":29,"is_preprint":false},{"pmid":"28726805","id":"PMC_28726805","title":"PREPL deficiency: delineation of the phenotype and development of a functional blood assay.","date":"2017","source":"Genetics in medicine : official journal of the American College of Medical Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28726805","citation_count":28,"is_preprint":false},{"pmid":"21692504","id":"PMC_21692504","title":"A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors.","date":"2011","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/21692504","citation_count":23,"is_preprint":false},{"pmid":"23321636","id":"PMC_23321636","title":"Trans-Golgi network morphology and sorting is regulated by prolyl-oligopeptidase-like protein PREPL and the AP-1 complex subunit μ1A.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23321636","citation_count":22,"is_preprint":false},{"pmid":"21222627","id":"PMC_21222627","title":"PREPL, a prolyl endopeptidase-like enzyme by name only?--Lessons from patients.","date":"2011","source":"CNS & neurological disorders drug targets","url":"https://pubmed.ncbi.nlm.nih.gov/21222627","citation_count":19,"is_preprint":false},{"pmid":"24586561","id":"PMC_24586561","title":"Deletion of PREPl causes growth impairment and hypotonia in mice.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24586561","citation_count":17,"is_preprint":false},{"pmid":"19575798","id":"PMC_19575798","title":"Cooperation between NRF-2 and YY-1 transcription factors is essential for triggering the expression of the PREPL-C2ORF34 bidirectional gene pair.","date":"2009","source":"BMC molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19575798","citation_count":14,"is_preprint":false},{"pmid":"29483676","id":"PMC_29483676","title":"The second point mutation in PREPL: a case report and literature review.","date":"2018","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29483676","citation_count":11,"is_preprint":false},{"pmid":"32218803","id":"PMC_32218803","title":"PREPL Deficiency: A Homozygous Splice Site PREPL Mutation in a Patient With Congenital Myasthenic Syndrome and Absence of Ovaries and Hypoplasia of Uterus.","date":"2020","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32218803","citation_count":11,"is_preprint":false},{"pmid":"21686663","id":"PMC_21686663","title":"Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome.","date":"2009","source":"BMJ case reports","url":"https://pubmed.ncbi.nlm.nih.gov/21686663","citation_count":9,"is_preprint":false},{"pmid":"31985178","id":"PMC_31985178","title":"First maternal uniparental disomy for chromosome 2 with PREPL novel frameshift mutation of congenital myasthenic syndrome 22 in an infant.","date":"2020","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31985178","citation_count":8,"is_preprint":false},{"pmid":"33225564","id":"PMC_33225564","title":"Cleavage of PrePL by Lon promotes growth and pathogenesis in Magnaporthe oryzae.","date":"2021","source":"Environmental microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/33225564","citation_count":6,"is_preprint":false},{"pmid":"39078710","id":"PMC_39078710","title":"Missense variants in CMS22 patients reveal that PREPL has both enzymatic and nonenzymatic functions.","date":"2024","source":"JCI insight","url":"https://pubmed.ncbi.nlm.nih.gov/39078710","citation_count":2,"is_preprint":false},{"pmid":"40494461","id":"PMC_40494461","title":"Uncovering the mechanism of Buyang Huanwu Decoction in regulating mitochondrial dysfunction to alleviate atherosclerosis: BTK, PREPL, and P2RX7 proteins play key roles.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40494461","citation_count":1,"is_preprint":false},{"pmid":"1908048","id":"PMC_1908048","title":"Semianalytical expressions for (L/rho)(air)med and P(repl) for electron beams.","date":"1991","source":"Medical physics","url":"https://pubmed.ncbi.nlm.nih.gov/1908048","citation_count":1,"is_preprint":false},{"pmid":"7632347","id":"PMC_7632347","title":"Proposed semi-analytical formulae for the determination of (L/rho)medair and Prepl for electron beams as used in radiotherapy.","date":"1995","source":"Medical dosimetry : official journal of the American Association of Medical Dosimetrists","url":"https://pubmed.ncbi.nlm.nih.gov/7632347","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.28.685080","title":"Loss of PREPL alters lipid homeostasis due to mitochondrial defects","date":"2025-10-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.28.685080","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11075,"output_tokens":2509,"usd":0.03543},"stage2":{"model":"claude-opus-4-6","input_tokens":5805,"output_tokens":2207,"usd":0.1263},"total_usd":0.16173,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"PREPL is localized in the cytosol and contains a catalytic triad (Ser470, Asp556, His601); substitution of these predicted catalytic residues by alanines resulted in loss of reactivity with a serine hydrolase-specific activity-based probe, confirming an intact but unique serine hydrolase catalytic machinery. Unlike prolyl oligopeptidase and oligopeptidase B, PREPL activity depends only on the carboxyterminal domain.\",\n      \"method\": \"Activity-based probe labeling with active-site mutagenesis (Ser470A, Asp556A, His601A); subcellular fractionation for cytosolic localization\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct mutagenesis of catalytic residues with activity-based probe readout, replicated in subsequent studies\",\n      \"pmids\": [\"16385448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PREPL A (638-residue splice variant) does not cleave peptide substrates with P1 basic residues (arginine/lysine), but slowly hydrolyses an activated ester substrate and reacts with diisopropyl fluorophosphate, indicating a reactive catalytic serine but negligible physiological peptidase activity. PREPL A forms dimers, which may account for its higher conformational stability compared to oligopeptidase B.\",\n      \"method\": \"Recombinant protein expression in E. coli, peptide hydrolysis assays, ester hydrolysis assay, DFP reactivity, differential scanning calorimetry, secondary structure analysis\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assays with purified recombinant protein and multiple substrates/inhibitors\",\n      \"pmids\": [\"16143824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PREPL interacts directly with the N-terminal 70 amino acids of the AP-1 adaptor complex subunit μ1A. PREPL overexpression reduces AP-1 membrane binding, while reduced PREPL expression increases AP-1 membrane binding and impairs AP-1 recycling. PREPL deficiency causes an expanded trans-Golgi network morphology, which is rescued by PREPL re-expression. PREPL co-localizes with residual membrane-bound AP-1.\",\n      \"method\": \"Yeast two-hybrid library screen; PREPL overexpression and knockdown with AP-1 membrane fractionation; rescue experiments; fluorescence co-localization; patient cell lines\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid identification plus multiple orthogonal cellular assays (OE, KD, rescue, co-localization, patient lines)\",\n      \"pmids\": [\"23321636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Isolated PREPL deficiency causes a congenital myasthenic syndrome with decreased quantal content of the endplate potential and reduced amplitude of the miniature endplate potential, without endplate acetylcholine receptor deficiency or altered endplate geometry, indicating pre- and postsynaptic neuromuscular transmission defects. The myasthenia is attributed to abrogated interaction of PREPL with adaptor protein 1 (AP-1).\",\n      \"method\": \"Immunoblot confirmation of absent PREPL expression; in vitro electrophysiology of neuromuscular junction (quantal content, MEPP amplitude); edrophonium test; histochemical and ultrastructural studies\",\n      \"journal\": \"Neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct electrophysiological measurement in patient tissue with absent PREPL expression confirmed, multiple orthogonal methods\",\n      \"pmids\": [\"24610330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Deletion of exon 11 of murine Prepl (encoding key catalytic amino acids) leads to loss of PREPL protein and lower Prepl mRNA. PREPL-null mice display significant growth impairment (shorter and lighter) and neonatal hypotonia assessed by righting reflex assay, establishing PREPL as required for normal growth and muscle tone in vivo.\",\n      \"method\": \"Conditional knockout mouse model (exon 11 deletion); immunoblot; righting reflex behavioral assay; body measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse with defined growth and hypotonia phenotypes and confirmed protein absence\",\n      \"pmids\": [\"24586561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Selective small-molecule inhibitors of PREPL serine hydrolase activity were identified by fluopol-ABPP high-throughput screening and confirmed to block PREPL activity in cells. One compound (1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile) distributes to the mouse brain after systemic administration.\",\n      \"method\": \"Fluorescence polarization activity-based protein profiling (fluopol-ABPP) screen of >300,000 compounds; cell-based activity assays; mouse in vivo pharmacokinetics\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro and cell-based enzyme inhibition assays, single study\",\n      \"pmids\": [\"21692504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Transcription of PREPL is driven by a 243-bp GC-rich bidirectional minimal promoter in the 405-bp intergenic region shared with C2ORF34. Two transcription factors, NRF-2 and YY-1, cooperatively and additively activate PREPL (and C2ORF34) transcription by binding to this shared promoter region.\",\n      \"method\": \"Reporter gene assays; transcription factor binding site identification; co-transfection of NRF-2 and YY-1 with promoter constructs; gel shift/ChIP-like analyses\",\n      \"journal\": \"BMC molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional promoter dissection with reporter assays and identified TF binding sites, single lab\",\n      \"pmids\": [\"19575798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Missense variants in PREPL causing CMS22 do not impair hydrolase activity but reduce binding to known interactors (including AP-1 components), demonstrating that PREPL has both enzymatic and non-enzymatic (protein-interaction) functions. Catalytically inactive PREPL p.Ser559Ala cell lines showed that hydrolytic activity is required for normal mitochondrial function but not for regulating AP-1-mediated trans-Golgi network transport.\",\n      \"method\": \"Biochemical hydrolase activity assays of patient missense variants; structural analysis; protein-protein interaction assays; CRISPR-generated catalytically inactive PREPL p.Ser559Ala cell lines with mitochondrial function assays and TGN transport assays\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — multiple orthogonal methods: enzymatic assays, structural data, interaction assays, and engineered catalytic mutant cell lines in one study\",\n      \"pmids\": [\"39078710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PREPL KO cells accumulate triacylglycerols and show increased lipid droplet number, and exhibit elongated peroxisomes, but PREPL does not localize to peroxisomes and global phospholipid composition is largely unchanged. The lipid storage phenotype is attributed to mitochondrial dysfunction caused by PREPL loss (impaired respiratory chain/oxidative phosphorylation), which secondarily impairs fatty acid β-oxidation and promotes TAG synthesis, rather than a direct lipase role for PREPL.\",\n      \"method\": \"CRISPR-Cas9 KO cell lines; unbiased lipidomics (Prepl KO mouse brains and KO HEK293T cells); lipid droplet imaging; peroxisome number/morphology/protein-level analysis; PREPL localization studies\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (lipidomics, imaging, localization) but preprint, not yet peer reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.10.28.685080\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PREPL is a cytosolic serine hydrolase of the prolyl oligopeptidase family with a reactive catalytic triad (Ser470/Ser559, Asp556, His601) that confers esterase but negligible peptidase activity; it functions both enzymatically (supporting mitochondrial respiratory chain activity) and non-enzymatically by binding the AP-1 adaptor complex subunit μ1A to regulate AP-1 membrane cycling and trans-Golgi network morphology, with loss of PREPL causing a congenital myasthenic syndrome through impaired neuromuscular transmission, growth deficiency, and secondary lipid metabolic dysregulation due to mitochondrial dysfunction.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PREPL is a cytosolic serine hydrolase of the prolyl oligopeptidase family that possesses both enzymatic and non-enzymatic functions critical for neuromuscular transmission, growth, and organelle homeostasis. Its catalytic triad (Ser470/Ser559, Asp556, His601) confers esterase but negligible peptidase activity, and hydrolytic function is specifically required for normal mitochondrial respiratory chain activity, with PREPL loss causing secondary triacylglycerol accumulation through impaired fatty acid β-oxidation [PMID:16385448, PMID:16143824, PMID:39078710]. Independent of catalytic activity, PREPL binds the μ1A subunit of the AP-1 adaptor complex to regulate AP-1 membrane cycling and trans-Golgi network morphology; disease-causing missense variants disrupt this interaction without impairing hydrolase activity [PMID:23321636, PMID:39078710]. Loss of PREPL causes congenital myasthenic syndrome (CMS22) with decreased neuromuscular quantal content and reduced miniature endplate potential amplitude, as well as growth deficiency and neonatal hypotonia in knockout mice [PMID:24610330, PMID:24586561].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing PREPL as a genuine serine hydrolase resolved a key question about whether this prolyl oligopeptidase family member was catalytically active, and revealed that its activity resides in the C-terminal domain with an unusual substrate profile (esterase rather than peptidase).\",\n      \"evidence\": \"Activity-based probe labeling with catalytic triad mutagenesis (Ser470A, Asp556A, His601A) and recombinant protein enzymatic assays with peptide and ester substrates\",\n      \"pmids\": [\"16385448\", \"16143824\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological substrate(s) of PREPL esterase activity remain unidentified\",\n        \"Structural basis for lack of peptidase activity despite conservation of the prolyl oligopeptidase fold is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of NRF-2 and YY-1 as cooperative transcriptional activators of PREPL through a shared bidirectional promoter with C2ORF34 established the regulatory logic controlling PREPL expression.\",\n      \"evidence\": \"Reporter gene assays and transcription factor co-transfection with promoter constructs\",\n      \"pmids\": [\"19575798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Endogenous chromatin occupancy of NRF-2 and YY-1 at the PREPL promoter has not been validated by ChIP in physiological settings\",\n        \"Tissue-specific regulation of PREPL expression is unexplored\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Discovery of selective small-molecule PREPL inhibitors provided chemical tools to probe PREPL function in cells and demonstrated brain penetrance in vivo.\",\n      \"evidence\": \"Fluopol-ABPP high-throughput screen of >300,000 compounds with cell-based validation and mouse pharmacokinetics\",\n      \"pmids\": [\"21692504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Phenotypic consequences of pharmacological PREPL inhibition in vivo have not been reported\",\n        \"Selectivity across the broader serine hydrolase family beyond initial profiling is incomplete\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Discovery that PREPL directly binds the μ1A subunit of AP-1 and regulates AP-1 membrane cycling revealed a non-enzymatic function in vesicle trafficking, explaining the expanded trans-Golgi network morphology in PREPL-deficient cells.\",\n      \"evidence\": \"Yeast two-hybrid screen, overexpression/knockdown with AP-1 membrane fractionation, rescue experiments, and co-localization in patient cell lines\",\n      \"pmids\": [\"23321636\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether PREPL binding to μ1A requires a specific conformational state or post-translational modification is unknown\",\n        \"Cargo specificity of PREPL-regulated AP-1 trafficking is not defined\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstration that isolated PREPL deficiency causes congenital myasthenic syndrome (CMS22) with pre- and postsynaptic neuromuscular transmission defects, and that PREPL-null mice recapitulate growth impairment and hypotonia, established the disease mechanism and in vivo requirement.\",\n      \"evidence\": \"Patient neuromuscular junction electrophysiology (quantal content, MEPP amplitude) and Prepl exon 11 knockout mouse model with righting reflex and growth assays\",\n      \"pmids\": [\"24610330\", \"24586561\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The specific vesicle trafficking step at the neuromuscular junction that fails in PREPL deficiency is not delineated\",\n        \"Whether myasthenia results from the AP-1 interaction defect, loss of catalytic activity, or both was unresolved at this stage\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Separation of PREPL's enzymatic and non-enzymatic functions revealed that hydrolase activity is required for mitochondrial function but dispensable for AP-1-mediated TGN transport, while CMS22 missense variants specifically impair protein-protein interactions rather than catalysis.\",\n      \"evidence\": \"Biochemical assays of patient missense variants, CRISPR-generated catalytically inactive Ser559Ala cell lines, mitochondrial function assays, and TGN transport assays\",\n      \"pmids\": [\"39078710\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The mitochondrial substrate or process directly regulated by PREPL catalytic activity is unknown\",\n        \"How PREPL missense variants structurally impair protein interactions without affecting the active site has not been resolved at atomic resolution\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Lipid accumulation in PREPL-deficient cells was shown to be a secondary consequence of mitochondrial respiratory chain dysfunction rather than a direct lipase function, reframing the metabolic phenotype as downstream of impaired oxidative phosphorylation.\",\n      \"evidence\": \"CRISPR KO cell lines with unbiased lipidomics, lipid droplet imaging, peroxisome morphology analysis, and PREPL localization studies (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.10.28.685080\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Not yet peer reviewed\",\n        \"The specific respiratory chain complex or assembly step affected by PREPL loss is not identified\",\n        \"Whether lipid dysregulation contributes to the neuromuscular phenotype in vivo is untested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of PREPL's physiological enzymatic substrate(s) and the precise mechanism by which its catalytic activity supports mitochondrial function remain the central open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No endogenous substrate for PREPL esterase activity has been identified\",\n        \"Whether PREPL acts inside mitochondria or on a cytosolic target that feeds into mitochondrial pathways is unresolved\",\n        \"The relative contribution of enzymatic vs. AP-1-regulatory functions to CMS22 pathology is not quantified in vivo\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 5, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"AP1M1\", \"NRF2\", \"YY1\"],\n    \"other_free_text\": []\n  }\n}\n```"}