Affinage

DNPEP

Aspartyl aminopeptidase · UniProt Q9ULA0

Length
485 aa
Mass
53.4 kDa
Annotated
2026-06-09
21 papers in source corpus 12 papers cited in narrative 13 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

DNPEP is a cytosolic M18-family aspartyl aminopeptidase that cleaves N-terminal acidic residues from short peptides, including angiotensins, and assembles into a tetrahedral dodecameric complex built from domain-swapped dimers, a self-compartmentalization strategy that restricts substrate access and explains its preference for short substrates (PMID:22720794, PMID:22356908). Catalysis proceeds at a binuclear zinc active site in which one zinc is exchangeable with manganese to strongly stimulate activity, and the substrate-specificity pocket is shaped for acidic amino acids; small-molecule inhibitors selective over the related aminopeptidase ENPEP engage this metal-dependent active site (PMID:22356908, PMID:24913940). Through its proteolytic and hydrolase activity DNPEP acts on several substrates and pathways: it can generate inhibitory SPAK fragments that suppress the NKCC2 cotransporter, although knockout kidneys retain SPAK-cleaving activity, indicating DNPEP is not the sole kidney protease performing this cleavage (PMID:25164821, PMID:29122955). In cancer cells DNPEP functions as an intermediary in signaling control, being phosphorylated at serine 119 and destabilized by PAK5 via the ubiquitin-proteasome pathway, and in turn promoting downregulation of USP4 in the PAK5–DNPEP–USP4 axis (PMID:31219614). DNPEP also acts on the BMP pathway downstream of Smad activation as a target of miR-140 in chondrocytes (PMID:21576357), and physically binds the chloride channel ClC-5 to modulate its surface levels and albumin endocytosis in renal proximal tubule cells (PMID:25587118).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2011 Medium

    Established a regulatory and pathway context for DNPEP by identifying it as a miR-140 target that antagonizes BMP signaling downstream of Smad activation.

    Evidence Ago2-associated RNA profiling with gain/loss-of-function in chondrocytes and Mir140-null mouse, BMP reporter assay

    PMID:21576357

    Open questions at the time
    • Molecular mechanism by which DNPEP dampens BMP signaling is undefined
    • No demonstration that DNPEP's peptidase activity is required for the BMP effect
  2. 2012 High

    Resolved the catalytic architecture of DNPEP, defining a dodecameric self-compartmentalized complex with a binuclear zinc active site and an acidic-residue specificity pocket.

    Evidence X-ray crystallography with substrate analogue, EM, XAS, and in vitro metal-substitution enzymatic assays

    PMID:22356908 PMID:22720794

    Open questions at the time
    • In vivo physiological substrates beyond angiotensins not defined by structure
    • Role of the manganese-exchangeable zinc site in cellular regulation unknown
  3. 2014 Medium

    Identified DNPEP as a protease generating inhibitory SPAK fragments, linking it to ion cotransporter regulation in kidney.

    Evidence Chromatographic purification of kidney lysate activity, MS protease/cleavage-site identification, recombinant reconstitution, NKCC2 functional assay

    PMID:25164821

    Open questions at the time
    • Did not establish DNPEP as the sole or dominant kidney SPAK protease
    • Physiological relevance of SPAK cleavage not tested in vivo
  4. 2014 Medium

    Demonstrated DNPEP is a druggable enzyme with selective active-site inhibitors, validating angiotensin II as a substrate and mapping pharmacophore features.

    Evidence High-throughput enzymatic screen of ~25,000 compounds, ENPEP counter-screen, SAR and molecular modeling

    PMID:24913940

    Open questions at the time
    • Inhibitor efficacy in cells or in vivo not shown
    • No co-crystal structure confirming binding mode
  5. 2015 Medium

    Placed DNPEP at the endocytic machinery of renal proximal tubule by showing direct binding to ClC-5 and modulation of albumin uptake.

    Evidence GST pulldown/MS, co-IP, purified-protein binding, confocal colocalization, albumin uptake assay; plus low-confidence actin co-IP/G-actin assay

    PMID:25587118

    Open questions at the time
    • Whether the ClC-5 effect requires DNPEP peptidase activity is unresolved
    • Cytoskeletal stabilization role rests on a single co-IP and assay
  6. 2017 Medium

    Challenged the SPAK-protease model by showing DNPEP-null kidneys retain SPAK-cleaving activity, indicating redundancy or misidentification.

    Evidence Two independent knockout mouse models (EUCOMM and CRISPR/Cas9), in vitro kidney lysate proteolysis

    PMID:29122955

    Open questions at the time
    • Identity of the additional/alternative SPAK protease(s) not determined
    • Does not address DNPEP's contribution under physiological conditions
  7. 2019 Medium

    Defined DNPEP as both a regulated node and an effector in cancer signaling: a PAK5 substrate (S119) that is proteasomally destabilized and that drives USP4 and CD44 downregulation.

    Evidence Co-IP, in vitro/in vivo kinase assays, proteasome inhibition, hydrolase-mutant analysis, proliferation/invasion and xenograft assays

    PMID:31219614 PMID:31228326

    Open questions at the time
    • Direct proteolytic cleavage of USP4/CD44 by DNPEP not biochemically reconstituted
    • CD44 degradation mechanism beyond hydrolase-activity dependence undefined
  8. 2023 Low

    Extended the DNPEP-USP4 axis by showing FBXO3 shields USP4 from DNPEP, reinforcing DNPEP as a USP4-destabilizing protease.

    Evidence Co-IP, overexpression/knockdown, protein stability assays

    PMID:38134227

    Open questions at the time
    • Direct DNPEP-mediated USP4 degradation not reconstituted biochemically
    • Single lab; mechanism of cleavage versus regulated turnover unclear
  9. 2026 Low

    Implicated DNPEP in a further oncogenic interaction by reporting direct binding to RACK1 and ERK pathway activation in tongue carcinoma.

    Evidence Co-IP/interaction assay, in vitro and in vivo functional assays, signaling analysis

    PMID:42180904

    Open questions at the time
    • Limited methodological detail; no reconstitution or structural validation
    • Whether peptidase activity is required for RACK1/ERK effect unknown

Open questions

Synthesis pass · forward-looking unresolved questions
  • The endogenous physiological substrate repertoire of DNPEP and how its peptidase activity mechanistically connects to its diverse reported roles (BMP, ClC-5, USP4/CD44, RACK1) remain unresolved.
  • No unifying in vivo substrate identified across pathways
  • Unclear which interactions depend on catalysis versus scaffolding
  • Tissue-specific functions not integrated into one mechanistic model

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016787 hydrolase activity 4 GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0005829 cytosol 2 GO:0031410 cytoplasmic vesicle 2
Pathway
R-HSA-162582 Signal Transduction 3 R-HSA-392499 Metabolism of proteins 3
Partners
ACTBCLCN5FBXO3PAK5RACK1USP4
Complex memberships
DNPEP homododecamer

Evidence

Reading pass · 13 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2012 Crystal structure of human DNPEP complexed with zinc and substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers. The active site contains a binuclear zinc centre, and a substrate specificity pocket for acidic amino acids is defined. Active site loop swapping mediates catalysis, a mechanism shared with other M18/M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. X-ray crystallography with substrate analogue, electron microscopy BMC structural biology High 22720794
2012 DNPEP possesses a binuclear zinc active site in which one zinc ion is readily exchangeable with manganese, which strongly stimulates enzymatic activity. DNPEP assembles into a tetrahedral (dodecameric) complex that restricts substrate access to the active site, explaining preference for short peptide substrates with N-terminal acidic residues. DNPEP cleaves angiotensins and other physiologically relevant peptide substrates in vitro. X-ray crystallography, X-ray absorption spectroscopy, single-particle electron microscopy, in vitro enzymatic assay with metal substitution The Journal of biological chemistry High 22356908
2014 DNPEP is the protease responsible for generating short inhibitory forms of SPAK kinase in kidney. Kidney lysate proteolytic activity toward SPAK was purified by ion exchange and size exclusion chromatography; mass spectrometry identified DNPEP as the protease. Recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Mass spectrometry identified specific cleavage sites, and the resulting SPAK fragments were shown to inhibit the Na+-K+-2Cl- cotransporter NKCC2. Ion exchange chromatography, size exclusion chromatography, mass spectrometry, recombinant protein reconstitution, functional cotransporter assay The Journal of biological chemistry Medium 25164821
2017 DNPEP knockout mouse kidney lysates (generated by EUCOMM mutation and CRISPR/Cas9) retain proteolytic activity toward SPAK, indicating that DNPEP is not the sole protease responsible for generating SPAK fragments in kidney, and that DNPEP may have been misidentified as the primary kidney lysate protease or is not the only one cleaving SPAK. CRISPR/Cas9 knockout mouse model, EUCOMM mutant mouse, in vitro proteolytic assay with kidney lysate Physiological reports Medium 29122955
2011 DNPEP is a target of miR-140 in chondrocytes (identified by Ago2-associated RNA profiling). DNPEP overexpression exerts a mild antagonistic effect on BMP signaling at a position downstream of Smad activation. In Mir140-null chondrocytes, elevated DNPEP reduces basal BMP signaling, and DNPEP knockdown reverses this reduction, placing DNPEP downstream of Smad activation in the BMP pathway. Ago2-associated RNA profiling, overexpression and knockdown in chondrocytes, BMP signaling reporter assay, Mir140-null mouse model Molecular and cellular biology Medium 21576357
2015 DNPEP directly binds to the C-terminus of the chloride channel ClC-5. This interaction was identified by GST-pulldown/MS, confirmed by co-immunoprecipitation in cells, and further validated by direct binding of purified GST-ClC-5 and His-DNPEP proteins. DNPEP colocalizes with albumin-containing endocytic vesicles in renal proximal tubule cells, and DNPEP overexpression increases cell-surface ClC-5 levels and albumin uptake. GST pulldown, mass spectrometry, co-immunoprecipitation, purified protein binding assay, confocal immunofluorescence, albumin uptake assay American journal of physiology. Renal physiology Medium 25587118
2015 DNPEP overexpression in renal proximal tubule cells causes a significant decrease in G-actin as measured by DNase I inhibition assay, and DNPEP co-immunoprecipitates with β-actin and tubulin from kidney lysate, suggesting DNPEP stabilizes the actin cytoskeleton. Co-immunoprecipitation from kidney lysate, DNase I inhibition assay for G-actin quantification, overexpression in OK cells American journal of physiology. Renal physiology Low 25587118
2019 PAK5 interacts with and phosphorylates DNPEP at serine 119. PAK5 also decreases DNPEP protein abundance via the ubiquitin-proteasome pathway. DNPEP in turn mediates downregulation of USP4, placing DNPEP as an intermediary in the PAK5-DNPEP-USP4 signaling axis that controls breast cancer cell proliferation and invasion. Co-immunoprecipitation, in vitro/in vivo kinase assay (phosphorylation at S119), overexpression and knockdown with proliferation/invasion assays, proteasome inhibitor treatment, mouse xenograft model International journal of cancer Medium 31219614
2019 DNPEP promotes degradation of CD44 protein through a mechanism dependent on DNPEP's hydrolase activity and independent of the ubiquitin-proteasome pathway. Overexpression of DNPEP reduces CD44 levels and suppresses breast cancer cell stemness, while DNPEP knockdown elevates CD44. Overexpression and knockdown with CD44 protein quantification, proteasome inhibitor treatment (pathway exclusion), hydrolase-activity mutant analysis Anatomical record Low 31228326
2023 FBXO3 disrupts the interaction between USP4 and DNPEP, thereby protecting USP4 from DNPEP-mediated degradation. This places DNPEP as a protease that degrades USP4 when not blocked by FBXO3. Co-immunoprecipitation, overexpression/knockdown assays, protein stability assays PLoS biology Low 38134227
2010 DNPEP (DAP) protein is localized to secretory granules and lysosomal-like structures in pancreatic islet alpha cells, as demonstrated by immunofluorescence and electron microscopy, consistent with a role in post-translational processing of hormones. Immunofluorescence, electron microscopy, cell-type-specific staining Bioscience, biotechnology, and biochemistry Low 20944418
2014 High-throughput screening of ~25,000 small molecules identified 23 DNPEP inhibitors that block DNPEP-catalyzed hydrolysis of angiotensin II with micromolar potency. Eight DNPEP-selective compounds were identified over the related glutamyl aminopeptidase ENPEP. Structure-activity relationships identified a metal-chelating group and charged/polar moieties as key pharmacophore features for active-site engagement. High-throughput enzymatic screen, counter-screen against ENPEP, SAR analysis, molecular modeling Molecular pharmacology Medium 24913940
2026 DNPEP directly binds to RACK1 protein in tongue squamous cell carcinoma cells, activating the ERK signaling pathway, promoting EMT, proliferation, migration, invasion, and cisplatin resistance. Co-immunoprecipitation/protein interaction assay, in vitro and in vivo functional assays, signaling pathway analysis Translational cancer research Low 42180904

Source papers

Stage 0 corpus · 21 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2011 Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling. Molecular and cellular biology 147 21576357
2009 Protein profilings in mouse liver regeneration after partial hepatectomy using iTRAQ technology. Journal of proteome research 47 19099420
2012 Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family. BMC structural biology 30 22720794
2012 Insights into substrate specificity and metal activation of mammalian tetrahedral aspartyl aminopeptidase. The Journal of biological chemistry 29 22356908
2019 A PAK5-DNPEP-USP4 axis dictates breast cancer growth and metastasis. International journal of cancer 28 31219614
2023 FBXO3 stabilizes USP4 and Twist1 to promote PI3K-mediated breast cancer metastasis. PLoS biology 22 38134227
2014 STE20/SPS1-related proline/alanine-rich kinase (SPAK) is critical for sodium reabsorption in isolated, perfused thick ascending limb. American journal of physiology. Renal physiology 21 25477470
2014 Short forms of Ste20-related proline/alanine-rich kinase (SPAK) in the kidney are created by aspartyl aminopeptidase (Dnpep)-mediated proteolytic cleavage. The Journal of biological chemistry 16 25164821
2022 Cancer genes disfavoring T cell immunity identified via integrated systems approach. Cell reports 12 35926468
2010 A locus for juvenile myoclonic epilepsy maps to 2q33-q36. Human genetics 12 20467754
2014 Identification and characterization of novel inhibitors of Mammalian aspartyl aminopeptidase. Molecular pharmacology 11 24913940
2015 Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis. American journal of physiology. Renal physiology 8 25587118
2010 Aspartyl aminopeptidase, encoded by an evolutionarily conserved syntenic gene, is colocalized with its cluster in secretory granules of pancreatic islet cells. Bioscience, biotechnology, and biochemistry 8 20944418
2022 MicroRNA and mRNA Expression Changes in Glioblastoma Cells Cultivated under Conditions of Neurosphere Formation. Current issues in molecular biology 6 36354672
2021 Molecular characterization of a novel aspartyl aminopeptidase that contributes to the increase in glutamic acid content in chicken meat during cooking. Food chemistry. Molecular sciences 5 35415631
2019 Aspartyl Aminopeptidase Suppresses Proliferation, Invasion, and Stemness of Breast Cancer Cells via Targeting CD44. Anatomical record (Hoboken, N.J. : 2007) 5 31228326
2020 Aspartic Aminopeptidase Is a Novel Biomarker of Aggressive Chronic Lymphocytic Leukemia. Cancers 4 32664705
2019 Profiling microRNA expression in murine bone healing and non-union formation: Role of miR-140 during the early stage of bone healing. PloS one 3 31323027
2017 DNPEP is not the only peptidase that produces SPAK fragments in kidney. Physiological reports 2 29122955
2026 DNPEP promotes the growth, metastasis, and cisplatin resistance of tongue squamous cell carcinoma through RACK1/ERK signaling pathway. Translational cancer research 0 42180904
2025 Detection of aspartyl aminopeptidase in atherosclerosis mice and clinical sample using an optical probe. Materials today. Bio 0 41142432

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