Affinage

PREPL

Prolyl endopeptidase-like · UniProt Q4J6C6

Length
727 aa
Mass
83.9 kDa
Annotated
2026-04-28
20 papers in source corpus 9 papers cited in narrative 9 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 7 steps
  1. 2005 High

    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

    PMID:16143824 PMID:16385448

    Open questions at the time
    • 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
  2. 2009 Medium

    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

    PMID:19575798

    Open questions at the time
    • 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
  3. 2011 Medium

    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

    PMID:21692504

    Open questions at the time
    • Phenotypic consequences of pharmacological PREPL inhibition in vivo have not been reported
    • Selectivity across the broader serine hydrolase family beyond initial profiling is incomplete
  4. 2013 High

    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

    PMID:23321636

    Open questions at the time
    • 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
  5. 2014 High

    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

    PMID:24586561 PMID:24610330

    Open questions at the time
    • 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
  6. 2024 High

    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

    PMID:39078710

    Open questions at the time
    • 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
  7. 2025 Medium

    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)

    PMID:bio_10.1101_2025.10.28.685080

    Open questions at the time
    • 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

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016787 hydrolase activity 4 GO:0098772 molecular function regulator activity 2
Localization
GO:0005794 Golgi apparatus 1 GO:0005829 cytosol 1
Pathway
R-HSA-1643685 Disease 2 R-HSA-5653656 Vesicle-mediated transport 2
Partners
AP1M1NRF2YY1

Evidence

Reading pass · 9 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2005 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. Activity-based probe labeling with active-site mutagenesis (Ser470A, Asp556A, His601A); subcellular fractionation for cytosolic localization American journal of human genetics High 16385448
2005 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. Recombinant protein expression in E. coli, peptide hydrolysis assays, ester hydrolysis assay, DFP reactivity, differential scanning calorimetry, secondary structure analysis Cellular and molecular life sciences : CMLS High 16143824
2013 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. Yeast two-hybrid library screen; PREPL overexpression and knockdown with AP-1 membrane fractionation; rescue experiments; fluorescence co-localization; patient cell lines Journal of cell science High 23321636
2014 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). Immunoblot confirmation of absent PREPL expression; in vitro electrophysiology of neuromuscular junction (quantal content, MEPP amplitude); edrophonium test; histochemical and ultrastructural studies Neurology High 24610330
2014 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. Conditional knockout mouse model (exon 11 deletion); immunoblot; righting reflex behavioral assay; body measurement PloS one High 24586561
2011 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. Fluorescence polarization activity-based protein profiling (fluopol-ABPP) screen of >300,000 compounds; cell-based activity assays; mouse in vivo pharmacokinetics Journal of the American Chemical Society Medium 21692504
2009 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. Reporter gene assays; transcription factor binding site identification; co-transfection of NRF-2 and YY-1 with promoter constructs; gel shift/ChIP-like analyses BMC molecular biology Medium 19575798
2024 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. 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 JCI insight High 39078710
2025 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. 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 bioRxivpreprint Medium bio_10.1101_2025.10.28.685080

Source papers

Stage 0 corpus · 20 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2005 Deletion of PREPL, a gene encoding a putative serine oligopeptidase, in patients with hypotonia-cystinuria syndrome. American journal of human genetics 69 16385448
2014 PREPL deficiency with or without cystinuria causes a novel myasthenic syndrome. Neurology 48 24610330
2008 Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome. Journal of medical genetics 37 18234729
2006 PREPL: a putative novel oligopeptidase propelled into the limelight. Biological chemistry 30 16913837
2005 The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity. Cellular and molecular life sciences : CMLS 29 16143824
2017 PREPL deficiency: delineation of the phenotype and development of a functional blood assay. Genetics in medicine : official journal of the American College of Medical Genetics 28 28726805
2011 A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors. Journal of the American Chemical Society 23 21692504
2013 Trans-Golgi network morphology and sorting is regulated by prolyl-oligopeptidase-like protein PREPL and the AP-1 complex subunit μ1A. Journal of cell science 22 23321636
2011 PREPL, a prolyl endopeptidase-like enzyme by name only?--Lessons from patients. CNS & neurological disorders drug targets 19 21222627
2014 Deletion of PREPl causes growth impairment and hypotonia in mice. PloS one 17 24586561
2009 Cooperation between NRF-2 and YY-1 transcription factors is essential for triggering the expression of the PREPL-C2ORF34 bidirectional gene pair. BMC molecular biology 14 19575798
2020 PREPL Deficiency: A Homozygous Splice Site PREPL Mutation in a Patient With Congenital Myasthenic Syndrome and Absence of Ovaries and Hypoplasia of Uterus. Frontiers in genetics 11 32218803
2018 The second point mutation in PREPL: a case report and literature review. Journal of human genetics 11 29483676
2009 Deletion of C2orf34, PREPL and SLC3A1 causes atypical hypotonia-cystinuria syndrome. BMJ case reports 9 21686663
2020 First maternal uniparental disomy for chromosome 2 with PREPL novel frameshift mutation of congenital myasthenic syndrome 22 in an infant. Molecular genetics & genomic medicine 8 31985178
2021 Cleavage of PrePL by Lon promotes growth and pathogenesis in Magnaporthe oryzae. Environmental microbiology 6 33225564
2024 Missense variants in CMS22 patients reveal that PREPL has both enzymatic and nonenzymatic functions. JCI insight 2 39078710
2025 Uncovering the mechanism of Buyang Huanwu Decoction in regulating mitochondrial dysfunction to alleviate atherosclerosis: BTK, PREPL, and P2RX7 proteins play key roles. International journal of biological macromolecules 1 40494461
1991 Semianalytical expressions for (L/rho)(air)med and P(repl) for electron beams. Medical physics 1 1908048
1995 Proposed semi-analytical formulae for the determination of (L/rho)medair and Prepl for electron beams as used in radiotherapy. Medical dosimetry : official journal of the American Association of Medical Dosimetrists 0 7632347