{"gene":"DPP9","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2002,"finding":"DPP9 is a cytosolic serine protease belonging to the DPP IV gene family, containing a catalytic triad (Ser, Asp, His) and the GWSYG serine protease motif identical to DPP IV. It lacks transmembrane domains and a signal sequence, consistent with its cytosolic localization, and migrates at ~98 kDa on SDS-PAGE.","method":"In silico identification, in vitro translation, SDS-PAGE, subcellular fractionation","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (in vitro translation, fractionation) in a single characterization study; foundational identification paper","pmids":["12459266"],"is_preprint":false},{"year":2009,"finding":"DPP9 is rate-limiting for degradation of proline-containing peptides in the cytoplasm. Its first identified natural substrate is the RU1(34-42) antigenic peptide (VPYGSFKHV); DPP9 degrades this peptide in vitro, and DPP9 knockdown in intact cells increases antigen presentation of RU1(34-42).","method":"In vitro peptidase assay with cell extracts; siRNA knockdown with antigen presentation readout","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro biochemical assay plus orthogonal cell-based functional readout (antigen presentation), demonstrating substrate identity and physiological relevance","pmids":["19667070"],"is_preprint":false},{"year":2012,"finding":"DPP9 binds SUMO1 (but not SUMO2) via a novel SUMO-interacting motif in an extended arm structure flanking the substrate entry site, independent of the canonical SIM. SUMO1 binding stimulates DPP9 enzymatic activity; mutations in the SUMO1-binding arm reduce DPP9 activity, and silencing SUMO1 decreases cytosolic prolyl-peptidase activity.","method":"Pulldown/co-IP, mutagenesis, enzymatic activity assays, siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays, mutagenesis of binding arm, enzymatic activity measurements, and cellular silencing providing multiple orthogonal lines of evidence in one study","pmids":["23152501"],"is_preprint":false},{"year":2016,"finding":"DPP8 and DPP9 inhibition (by Val-boroPro/Talabostat) triggers pyroptosis in monocytes and macrophages by activating pro-caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 cleaves gasdermin D to induce pyroptosis but does not efficiently process itself or IL-1β. Caspase-1 knockout mice do not show immune stimulation after Val-boroPro treatment.","method":"Small-molecule inhibition, genetic knockout (caspase-1 KO mice), caspase-1 activity assays, gasdermin D cleavage assay","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological inhibition corroborated by genetic KO, multiple downstream effector assays, mechanistic pathway dissection","pmids":["27820798"],"is_preprint":false},{"year":2016,"finding":"DPP9 interacts with Filamin A, which recruits DPP9 to the tyrosine kinase Syk. DPP9 cleaves Syk to expose a neo-N-terminus with serine at position 1 (N-end rule substrate), which strongly influences Syk stability. DPP9 processing is a prerequisite for Syk ubiquitination by the E3 ligase Cbl, and DPP9 inhibition stabilizes Syk and modulates B-cell signaling.","method":"Co-IP (DPP9-Filamin A-Syk complex), N-terminal sequencing, pulse-chase/mutagenesis, siRNA knockdown, ubiquitination assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — complex identified by co-IP, substrate cleavage site defined biochemically, N-end rule mechanism validated by mutagenesis and pulse-chase, functional consequences confirmed by siRNA and signaling readouts","pmids":["27614019"],"is_preprint":false},{"year":2018,"finding":"CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. DPP8/9 inhibitors induce pyroptosis in the majority of human AML cell lines and primary AML samples but not in cells from many other lineages.","method":"Genetic knockdown/knockout, reconstituted inflammasome assays, primary patient samples, mouse xenograft models","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function (CARD8 KO/KD) combined with primary human samples and in vivo mouse models; mechanism pathway placed","pmids":["29967349"],"is_preprint":false},{"year":2018,"finding":"DPP9 directly interacts with human NLRP1 and CARD8 via their FIIND (Function-to-Find) autoproteolytic domains and acts as an endogenous inhibitor of the NLRP1 inflammasome. Both DPP9's scaffolding/binding function and its catalytic activity act synergistically to maintain NLRP1 in its inactive state. A patient-derived germline missense mutation in NLRP1's FIIND domain abrogates DPP9 binding and causes inflammasome hyperactivation.","method":"Proteomics screen, co-IP, CRISPR/Cas9 deletion, small-molecule inhibitors, ASC speck formation assay, IL-1β secretion, pyroptosis assays, patient mutation functional analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — interaction confirmed by proteomics and co-IP, genetic deletion by CRISPR, pharmacological inhibition, patient mutation validation; multiple orthogonal methods across multiple labs","pmids":["30291141"],"is_preprint":false},{"year":2017,"finding":"DPP9 enzyme activity is required for survival of migratory tongue muscle progenitors in vivo. Catalytically inactive DPP9 knock-in mice (S729A) die within 12–18 h of birth due to microglossia caused by increased apoptosis of occipital somite-derived migratory muscle progenitors forming distal tongue intrinsic muscles.","method":"Catalytic knock-in mouse (S729A), histology, apoptosis quantification, phenotypic rescue by manual feeding","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — defined catalytic-inactive knock-in with specific cell-type apoptosis phenotype and mechanistic rescue experiment; single lab but rigorous in vivo genetics","pmids":["28887018"],"is_preprint":false},{"year":2018,"finding":"DPP9's enzymatic catalytic activity, but not its binding to CARD8, restrains the CARD8 inflammasome. Wild-type but not catalytically inactive DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. The DPP9-CARD8 interaction (unlike the DPP9-NLRP1 interaction) is not disrupted by DPP9 inhibitors or CARD8 mutations blocking autoproteolysis.","method":"Activity-based probes, reconstituted inflammasome assays, mass spectrometry-based proteomics, DPP9 knockout cell lines, catalytic mutant rescue","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted inflammasome assay, catalytic mutant rescue, activity-based probes; multiple orthogonal methods definitively separating catalytic vs. scaffolding functions for CARD8","pmids":["31525884"],"is_preprint":false},{"year":2020,"finding":"DPP8/9 mediate N-terminal processing of adenylate kinase 2 (AK2) in the cytoplasm. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Over 100 mitochondrial proteins with putative DPP8/9 recognition sites were identified, and DPP8/9 influence cellular levels of a subset of these.","method":"Biochemical fractionation, DPP8/9 inhibition, proteasome inhibition, AK2 substrate validation, MS-based proteomics","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — biochemical substrate identification with functional consequences (proteasomal degradation), proteomics of additional substrates, multiple inhibitor and genetic approaches","pmids":["32815200"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structures of human NLRP1-DPP9 complex (alone and with Val-boroPro) reveal a ternary complex comprising DPP9, full-length NLRP1, and the NLRP1 C-terminal fragment (CT). The N-terminus of NLRP1 CT inserts into the DPP9 active site; Val-boroPro disrupts this interaction. Binding of NLRP1 CT to DPP9 requires full-length NLRP1. VbP weakens the NLRP1-DPP9 interaction and accelerates degradation of the N-terminal fragment to induce inflammasome activation.","method":"Cryo-EM structure determination, biochemical binding assays, functional inflammasome assays, ectopic expression rescue experiments","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures plus orthogonal functional assays in a single rigorous study; replicated by parallel paper (PMID 33731929)","pmids":["33731932"],"is_preprint":false},{"year":2021,"finding":"Structural and biochemical analysis of rat NLRP1-DPP9 shows a 2:1 complex with one autoinhibited full-length NLRP1 and one active UPA-CARD fragment. The ZU5 domain is required for both NLRP1 autoinhibition and 2:1 complex assembly. Complex formation prevents UPA-mediated higher-order oligomerization and strengthens ZU5-mediated autoinhibition. Both NLRP1 binding and DPP9 enzymatic activity are required to suppress NLRP1 in human cells.","method":"Cryo-EM/crystal structure, biochemical reconstitution, structure-guided mutagenesis, functional inflammasome assays in human cells","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural determination plus reconstitution, mutagenesis, and cell-based functional validation; replicated by parallel paper (PMID 33731932)","pmids":["33731929"],"is_preprint":false},{"year":2018,"finding":"Saxagliptin (but not sitagliptin) inhibits DPP9 in cardiomyocytes and impairs CaMKII phosphorylation of phospholamban and PKC activity. DPP9 knockdown (but not DPP8 knockdown) and pharmacological DPP8/9 inhibition recapitulate these effects, implicating DPP9 specifically in CaMKII-PLB and PKC signaling in cardiomyocytes.","method":"siRNA knockdown of DPP9 vs DPP8, pharmacological inhibition (TC-E-5007, saxagliptin, sitagliptin), CaMKII/PLB phosphorylation assays, PKC activity assays, Ca2+ transient measurements, action potential duration recording in cardiomyocytes","journal":"Frontiers in physiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gene-specific knockdown with pharmacological corroboration and multiple downstream readouts, single lab but two orthogonal approaches","pmids":["30487758"],"is_preprint":false},{"year":2023,"finding":"DPP9 binds KEAP1 via a conserved ESGE motif and disrupts KEAP1-NRF2 binding by competing with NRF2 for KEAP1 binding in an enzyme-independent (non-catalytic) manner. DPP9 overexpression stabilizes NRF2, drives NRF2-dependent transcription, decreases cellular ROS, suppresses ferroptosis, and induces sorafenib resistance in ccRCC cells, largely dependent on the NRF2 target SLC7A11.","method":"Protein affinity purification, co-IP competition assays, ESGE motif mutagenesis (enzyme-independent binding), NRF2 stability assays, ROS measurement, ferroptosis assays, drug resistance assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity purification plus competition co-IP and motif mutagenesis showing non-catalytic mechanism; single lab with multiple orthogonal cellular readouts","pmids":["37713596"],"is_preprint":false},{"year":2022,"finding":"The NLRP1 variant M1184V stabilizes the FIIND domain in a monomeric conformation, promotes DPP9 binding (confirmed by surface plasmon resonance and co-IP), and enhances formation of the autoinhibited NLRP1-DPP9 complex, leading to reduced inflammasome activation.","method":"Size-exclusion chromatography, molecular dynamics simulation, surface plasmon resonance, immunoprecipitation, inflammasome activity assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SPR binding measurement plus co-IP and functional assays confirm enhanced DPP9 interaction; single lab, multiple methods","pmids":["36309085"],"is_preprint":false},{"year":2023,"finding":"In addition to Syk and AK2, BRCA2 (tumor suppressor) was identified as a DPP9 substrate; N-terminal processing of BRCA2 by DPP9 triggers its rapid proteasomal turnover, placing DPP9 as an upstream component of the N-degron pathway.","method":"Enzymatic assays, substrate identification (described in review/methods context referencing experimental data)","journal":"Methods in enzymology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — substrate mention in a methods review chapter with reference to experimental work; not independently detailed in an original research abstract","pmids":["37230592"],"is_preprint":false},{"year":2023,"finding":"A de novo DPP9 mutation (p.Arg252Pro) destabilizes the DPP9 protein and causes it to fail to restrain the NLRP1 and CARD8 inflammasomes, resulting in constitutive inflammasome activation and severe infancy-onset autoinflammation with HLH-like features.","method":"Patient genetic analysis, transfection in HEK293T cells and patient iPSCs, functional inflammasome assays (caspase-1 activation, IL-1β/IL-18 secretion), protein stability assessment","journal":"The Journal of allergy and clinical immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — disease variant functionally validated in heterologous cells and patient-derived iPSCs with inflammasome assays; single lab, two orthogonal cell models","pmids":["37544411"],"is_preprint":false},{"year":2020,"finding":"FAP (Fibroblast Activation Protein) interacts intracellularly with DPP9 (identified by IP-MS), and FAP overexpression leads to reduction in DPP9 expression, promoting EMT in oral squamous cell carcinoma. DPP9 overexpression reverses FAP-induced proliferation, migration, invasion, and EMT, acting in a non-enzymatic manner.","method":"IP-mass spectrometry, DPP9 knockdown/overexpression, cell proliferation, migration and invasion assays, EMT marker analysis, in vivo xenograft","journal":"OncoTargets and therapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP/MS interaction identification; functional assays are phenotypic with limited mechanistic dissection of DPP9's molecular role","pmids":["32273729"],"is_preprint":false},{"year":2024,"finding":"KEAP1 binds DPP9 in an inactive (non-native) conformation and stabilizes this non-dimeric fold. Reciprocally, this inactive form of DPP9 inhibits KEAP1 from binding and degrading NRF2, thereby inducing an antioxidant response. This reveals an endogenous DPP9 inhibition mechanism and links DPP9, like thioredoxin-1, to the intracellular redox potential.","method":"Co-IP, biochemical characterization of DPP9 conformational states, NRF2 stability assays, binding competition assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal inhibition demonstrated by co-IP and functional NRF2 stability assays; single lab, multiple orthogonal readouts but conformational trigger not yet identified","pmids":["39615677"],"is_preprint":false},{"year":2025,"finding":"SFTSV non-structural protein NSs activates the NLRP1 and CARD8 inflammasomes by disrupting the DPP9-mediated inhibitory ternary complex: NSs binds NLRP1 and CARD8 via their FIIND domains (competing with DPP8/9 for binding) and promotes degradation of DPP8 and DPP9, releasing activated C-terminal fragments.","method":"Viral infection of primary keratinocytes and macrophages, co-IP (NSs-NLRP1/CARD8 interaction), DPP9 protein level measurement, CARD8 deletion functional assay","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of NSs-NLRP1/CARD8 interaction, genetic deletion validation, and functional inflammasome readouts; single lab with multiple approaches","pmids":["40608794"],"is_preprint":false},{"year":2026,"finding":"DPP9 interacts with the BRISC deubiquitinase complex (BRCC36/BRCC3 and ABRO1/ABRAXAS2) and CYLD-SPATA2 complex. DPP9 disrupts BRISC-SHMT2 complex formation, reducing BRISC-mediated deubiquitination and stabilization of IFNAR1, thereby suppressing JAK/STAT-driven PD-L1 transcription in ccRCC. DPP9 inhibition restores T cell cytotoxicity and enhances checkpoint blockade.","method":"TurboID proximity labeling, NanoBRET in living cells, pharmacological DPP9 inhibition (1G244), IFNAR1 ubiquitination assays, PD-L1 expression assays, T cell cytotoxicity assays, co-IP","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling plus NanoBRET competition assays and functional ubiquitination/signaling readouts; single lab, multiple orthogonal methods","pmids":["41826729"],"is_preprint":false},{"year":2026,"finding":"DPP9 interactome mapped by TurboID proximity labeling reveals DPP8, the E3 ligase CBL, the deubiquitinase complex CYLD-SPATA2, and BRISC complex components (BRCC36/BRCC3, ABRO1/ABRAXAS2) as DPP9 interactors. NanoBRET assays show DPP9 disrupts BRCC36-ABRO1 binding and CYLD-SPATA2 interaction, revealing non-catalytic scaffolding functions of DPP9 in ubiquitin signaling.","method":"TurboID proximity labeling in DPP9-KO HEK293 cells, NanoBRET in living cells, validation co-IPs","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling plus NanoBRET in living cells for interaction disruption; single lab, two orthogonal methods with inducible physiological expression controls","pmids":["41636814"],"is_preprint":false}],"current_model":"DPP9 is an intracellular serine dipeptidyl peptidase that removes N-terminal Xaa-Pro dipeptides from cytosolic substrates (including the antigenic peptide RU1(34-42), the kinase Syk, adenylate kinase AK2, and BRCA2) to control their stability via the N-degron/proteasomal pathway, and simultaneously acts as a non-catalytic scaffold that sequesters the C-terminal fragments of the NLRP1 and CARD8 inflammasome sensors in inhibitory ternary complexes (DPP9:full-length sensor:sensor-CT), thereby suppressing inflammasome activation; DPP9 also engages non-catalytically with KEAP1, BRISC, CYLD-SPATA2, and Filamin A to regulate redox sensing, ubiquitin signaling, and B-cell receptor signaling, and its enzymatic activity is allosterically stimulated by SUMO1 binding."},"narrative":{"mechanistic_narrative":"DPP9 is an intracellular serine dipeptidyl peptidase of the DPP IV gene family that removes N-terminal Xaa-Pro dipeptides from cytosolic proteins and peptides, coupling N-terminal processing to protein stability and immune regulation [PMID:12459266, PMID:19667070]. As a rate-limiting cytoplasmic prolyl-peptidase, it degrades proline-containing antigenic peptides such as RU1(34-42), so that its loss enhances antigen presentation [PMID:19667070]. By generating neo-N-termini that feed substrates into the N-degron/proteasomal pathway, DPP9 controls turnover of the tyrosine kinase Syk—recruited via Filamin A and primed for Cbl-mediated ubiquitination—and of adenylate kinase AK2, modulating B-cell receptor signaling and preventing cytosolic accumulation of active AK2 [PMID:27614019, PMID:32815200]. The most extensively defined role of DPP9 is as an endogenous brake on the NLRP1 and CARD8 inflammasomes: it forms inhibitory ternary complexes (DPP9:full-length sensor:sensor C-terminal fragment) in which the N-terminus of the sensor CT inserts into the DPP9 active site, with both scaffolding and catalytic activity enforcing autoinhibition; small-molecule DPP8/9 inhibitors or viral antagonists disrupt these complexes to trigger pro-caspase-1-dependent, gasdermin-D-mediated pyroptosis [PMID:27820798, PMID:30291141, PMID:31525884, PMID:33731932, PMID:33731929, PMID:40608794]. DPP9 enzymatic activity is allosterically stimulated by SUMO1 binding to an extended arm flanking the substrate entry site [PMID:23152501]. Beyond its protease functions, DPP9 acts non-catalytically: it competes with NRF2 for KEAP1 binding to drive an antioxidant response, and it scaffolds the BRISC and CYLD-SPATA2 ubiquitin-signaling complexes [PMID:37713596, PMID:39615677, PMID:41826729, PMID:41636814]. A de novo DPP9 mutation (p.Arg252Pro) that destabilizes the protein and abolishes inflammasome restraint causes severe infancy-onset autoinflammation with HLH-like features [PMID:37544411].","teleology":[{"year":2002,"claim":"Establishing that DPP9 is a cytosolic member of the DPP IV serine protease family defined its catalytic machinery and subcellular compartment, framing it as an intracellular peptidase rather than a cell-surface enzyme.","evidence":"In silico identification, in vitro translation, SDS-PAGE, and subcellular fractionation","pmids":["12459266"],"confidence":"Medium","gaps":["No natural substrate identified at this stage","Physiological function unaddressed"]},{"year":2009,"claim":"Identifying RU1(34-42) as the first natural substrate showed DPP9 is rate-limiting for cytoplasmic proline-peptide degradation and links its activity to antigen presentation.","evidence":"In vitro peptidase assay with cell extracts and siRNA knockdown with antigen presentation readout","pmids":["19667070"],"confidence":"High","gaps":["Broader substrate repertoire unknown","No structural basis for substrate selectivity"]},{"year":2012,"claim":"Discovery of SUMO1 binding to an extended arm flanking the substrate entry site revealed an allosteric mechanism that stimulates DPP9 catalytic activity.","evidence":"Pulldown/co-IP, mutagenesis of the binding arm, enzymatic activity assays, and SUMO1 silencing","pmids":["23152501"],"confidence":"High","gaps":["Physiological contexts where SUMO1-dependent regulation matters not defined","SUMO2 selectivity basis only partially explained"]},{"year":2016,"claim":"Defining Syk as an N-end rule substrate processed by DPP9 (recruited via Filamin A and primed for Cbl ubiquitination) connected DPP9 N-terminal processing to protein turnover and B-cell signaling.","evidence":"Co-IP of DPP9-Filamin A-Syk complex, N-terminal sequencing, pulse-chase/mutagenesis, ubiquitination assays, and siRNA knockdown","pmids":["27614019"],"confidence":"High","gaps":["Generality of the Filamin A-mediated recruitment to other substrates unknown"]},{"year":2016,"claim":"Showing that DPP8/9 inhibition triggers ASC-independent pro-caspase-1 activation and gasdermin-D-mediated pyroptosis placed DPP9 as a suppressor of an inflammatory death pathway.","evidence":"Val-boroPro inhibition, caspase-1 KO mice, caspase-1 activity assays, and gasdermin D cleavage assays","pmids":["27820798"],"confidence":"High","gaps":["The upstream sensor restrained by DPP9 was not yet identified at this stage"]},{"year":2018,"claim":"Identifying NLRP1 and CARD8 as DPP9-bound inflammasome sensors, and dissecting whether catalytic or scaffolding activity restrains each, established DPP9 as a direct endogenous inflammasome brake.","evidence":"Proteomics, co-IP, CRISPR deletion, catalytic-mutant rescue, reconstituted inflammasome and pyroptosis assays, and patient mutation analysis","pmids":["30291141","29967349","31525884"],"confidence":"High","gaps":["Structural basis of the inhibitory complex not yet resolved","Differential requirement of binding vs. catalysis for NLRP1 vs. CARD8 mechanistically distinct"]},{"year":2017,"claim":"A catalytically inactive S729A knock-in mouse demonstrated that DPP9 enzymatic activity is required in vivo for survival of migratory tongue muscle progenitors, giving the protease an essential developmental role.","evidence":"Catalytic knock-in mouse, histology, apoptosis quantification, and phenotypic rescue by manual feeding","pmids":["28887018"],"confidence":"High","gaps":["The relevant in vivo substrate driving progenitor apoptosis is not identified"]},{"year":2020,"claim":"Identifying AK2 as a substrate whose N-terminal processing triggers proteasomal degradation, plus >100 candidate mitochondrial-derived substrates, broadened DPP9's role in cytosolic proteostasis.","evidence":"Biochemical fractionation, DPP8/9 and proteasome inhibition, AK2 validation, and MS-based proteomics","pmids":["32815200"],"confidence":"High","gaps":["Functional consequences for most candidate substrates untested","Physiological role of clearing cytosolic AK2 not fully defined"]},{"year":2021,"claim":"Cryo-EM structures of the NLRP1-DPP9 ternary complex resolved how DPP9 binds full-length NLRP1 and inserts the CT N-terminus into its active site, explaining how Val-boroPro disrupts autoinhibition to activate the inflammasome.","evidence":"Cryo-EM/crystal structures, biochemical reconstitution, structure-guided mutagenesis, and functional inflammasome assays (human and rat)","pmids":["33731932","33731929"],"confidence":"High","gaps":["Structural detail of the DPP9-CARD8 complex less defined","How sensor CT degradation kinetics couple to oligomerization quantitatively"]},{"year":2018,"claim":"Linking DPP9 to CaMKII-phospholamban and PKC signaling in cardiomyocytes extended its functional reach to cardiac signaling, distinguishing it from DPP8.","evidence":"DPP9-specific siRNA, pharmacological inhibition, phosphorylation and PKC activity assays, Ca2+ transient and action potential recordings","pmids":["30487758"],"confidence":"Medium","gaps":["Direct molecular substrate/partner mediating cardiac effects not identified","Single-lab finding"]},{"year":2023,"claim":"Demonstrating that DPP9 competes with NRF2 for KEAP1 binding via an ESGE motif in an enzyme-independent manner revealed a non-catalytic role in redox sensing and ferroptosis/drug resistance.","evidence":"Affinity purification, competition co-IP, ESGE motif mutagenesis, NRF2 stability and ROS assays, ferroptosis and sorafenib-resistance assays in ccRCC","pmids":["37713596"],"confidence":"Medium","gaps":["In vivo relevance of the DPP9-KEAP1-NRF2 axis untested","Single lab"]},{"year":2024,"claim":"Showing KEAP1 traps DPP9 in an inactive non-dimeric conformation, which in turn blocks KEAP1 from degrading NRF2, defined a reciprocal endogenous inhibition mechanism coupling DPP9 to redox state.","evidence":"Co-IP, biochemical characterization of DPP9 conformational states, and NRF2 stability/competition assays","pmids":["39615677"],"confidence":"Medium","gaps":["Physiological trigger driving the conformational switch not identified","Single lab"]},{"year":2023,"claim":"Reporting BRCA2 as a DPP9 substrate whose N-terminal processing accelerates proteasomal turnover positioned DPP9 upstream of the N-degron pathway for a tumor suppressor.","evidence":"Enzymatic assays and substrate identification described in a methods review chapter","pmids":["37230592"],"confidence":"Low","gaps":["Reported in a methods review rather than detailed in an original research study","Cellular and physiological consequences not independently characterized"]},{"year":2026,"claim":"Proximity-labeling interactome mapping identified DPP9 as a non-catalytic scaffold disrupting BRISC and CYLD-SPATA2 ubiquitin-signaling complexes, linking it to IFNAR1 stability, PD-L1 transcription, and antitumor immunity.","evidence":"TurboID proximity labeling, NanoBRET in living cells, IFNAR1 ubiquitination, PD-L1, and T cell cytotoxicity assays with DPP9 inhibition","pmids":["41826729","41636814"],"confidence":"Medium","gaps":["Structural basis of BRISC/CYLD-SPATA2 disruption unknown","Single-lab findings awaiting independent replication"]},{"year":2023,"claim":"A destabilizing de novo DPP9 mutation causing constitutive NLRP1/CARD8 activation and severe autoinflammation established DPP9 loss-of-function as a Mendelian autoinflammatory disease mechanism.","evidence":"Patient genetics, transfection in HEK293T and patient iPSCs, inflammasome activity assays, and protein stability assessment","pmids":["37544411"],"confidence":"Medium","gaps":["Genotype-phenotype spectrum across patients not defined","Single index case"]},{"year":null,"claim":"The full physiological substrate repertoire of DPP9, the mechanisms switching it between catalytic and non-catalytic scaffolding roles, and how its redox-sensing, ubiquitin-signaling, and inflammasome functions are coordinated in vivo remain open.","evidence":"","pmids":[],"confidence":"Low","gaps":["Most candidate substrates lack functional validation","Conformational/redox triggers governing DPP9 activity state unresolved","Integration of DPP9's diverse functions at the organismal level unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,4,9]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[6,10,13,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,8,13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1,9]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,6,8,10,11]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,5,16]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,9,15]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[13,18]}],"complexes":["NLRP1-DPP9 inflammasome ternary complex","CARD8-DPP9 complex","BRISC","CYLD-SPATA2"],"partners":["NLRP1","CARD8","SUMO1","FLNA","SYK","KEAP1","BRCC3","ABRAXAS2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86TI2","full_name":"Dipeptidyl peptidase 9","aliases":["Dipeptidyl peptidase IV-related protein 2","DPRP-2","Dipeptidyl peptidase IX","DPP IX","Dipeptidyl peptidase-like protein 9","DPLP9"],"length_aa":863,"mass_kda":98.3,"function":"Dipeptidyl peptidase that cleaves off N-terminal dipeptides from proteins having a Pro or Ala residue at position 2 (PubMed:12662155, PubMed:16475979, PubMed:19667070, PubMed:29382749, PubMed:30291141, PubMed:33731929, PubMed:36112693). Acts as a key inhibitor of caspase-1-dependent monocyte and macrophage pyroptosis in resting cells by preventing activation of NLRP1 and CARD8 (PubMed:27820798, PubMed:29967349, PubMed:30291141, PubMed:31525884, PubMed:32796818, PubMed:36112693, PubMed:36357533). Sequesters the cleaved C-terminal part of NLRP1 and CARD8, which respectively constitute the active part of the NLRP1 and CARD8 inflammasomes, in a ternary complex, thereby preventing their oligomerization and activation (PubMed:33731929, PubMed:33731932, PubMed:34019797). The dipeptidyl peptidase activity is required to suppress NLRP1 and CARD8; however, neither NLRP1 nor CARD8 are bona fide substrates of DPP9, suggesting the existence of substrate(s) required for NLRP1 and CARD8 inhibition (PubMed:33731929)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q86TI2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DPP9","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DPP9","total_profiled":1310},"omim":[{"mim_id":"620331","title":"HATIPOGLU IMMUNODEFICIENCY SYNDROME; HATIS","url":"https://www.omim.org/entry/620331"},{"mim_id":"609051","title":"CASPASE RECRUITMENT DOMAIN-CONTAINING PROTEIN 8; CARD8","url":"https://www.omim.org/entry/609051"},{"mim_id":"608258","title":"DIPEPTIDYL PEPTIDASE IX; DPP9","url":"https://www.omim.org/entry/608258"},{"mim_id":"606819","title":"DIPEPTIDYL PEPTIDASE VIII; DPP8","url":"https://www.omim.org/entry/606819"},{"mim_id":"606636","title":"NLR FAMILY, PYRIN DOMAIN-CONTAINING 1; NLRP1","url":"https://www.omim.org/entry/606636"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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It lacks transmembrane domains and a signal sequence, consistent with its cytosolic localization, and migrates at ~98 kDa on SDS-PAGE.\",\n      \"method\": \"In silico identification, in vitro translation, SDS-PAGE, subcellular fractionation\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (in vitro translation, fractionation) in a single characterization study; foundational identification paper\",\n      \"pmids\": [\"12459266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DPP9 is rate-limiting for degradation of proline-containing peptides in the cytoplasm. Its first identified natural substrate is the RU1(34-42) antigenic peptide (VPYGSFKHV); DPP9 degrades this peptide in vitro, and DPP9 knockdown in intact cells increases antigen presentation of RU1(34-42).\",\n      \"method\": \"In vitro peptidase assay with cell extracts; siRNA knockdown with antigen presentation readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro biochemical assay plus orthogonal cell-based functional readout (antigen presentation), demonstrating substrate identity and physiological relevance\",\n      \"pmids\": [\"19667070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DPP9 binds SUMO1 (but not SUMO2) via a novel SUMO-interacting motif in an extended arm structure flanking the substrate entry site, independent of the canonical SIM. SUMO1 binding stimulates DPP9 enzymatic activity; mutations in the SUMO1-binding arm reduce DPP9 activity, and silencing SUMO1 decreases cytosolic prolyl-peptidase activity.\",\n      \"method\": \"Pulldown/co-IP, mutagenesis, enzymatic activity assays, siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays, mutagenesis of binding arm, enzymatic activity measurements, and cellular silencing providing multiple orthogonal lines of evidence in one study\",\n      \"pmids\": [\"23152501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DPP8 and DPP9 inhibition (by Val-boroPro/Talabostat) triggers pyroptosis in monocytes and macrophages by activating pro-caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 cleaves gasdermin D to induce pyroptosis but does not efficiently process itself or IL-1β. Caspase-1 knockout mice do not show immune stimulation after Val-boroPro treatment.\",\n      \"method\": \"Small-molecule inhibition, genetic knockout (caspase-1 KO mice), caspase-1 activity assays, gasdermin D cleavage assay\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological inhibition corroborated by genetic KO, multiple downstream effector assays, mechanistic pathway dissection\",\n      \"pmids\": [\"27820798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DPP9 interacts with Filamin A, which recruits DPP9 to the tyrosine kinase Syk. DPP9 cleaves Syk to expose a neo-N-terminus with serine at position 1 (N-end rule substrate), which strongly influences Syk stability. DPP9 processing is a prerequisite for Syk ubiquitination by the E3 ligase Cbl, and DPP9 inhibition stabilizes Syk and modulates B-cell signaling.\",\n      \"method\": \"Co-IP (DPP9-Filamin A-Syk complex), N-terminal sequencing, pulse-chase/mutagenesis, siRNA knockdown, ubiquitination assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — complex identified by co-IP, substrate cleavage site defined biochemically, N-end rule mechanism validated by mutagenesis and pulse-chase, functional consequences confirmed by siRNA and signaling readouts\",\n      \"pmids\": [\"27614019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. DPP8/9 inhibitors induce pyroptosis in the majority of human AML cell lines and primary AML samples but not in cells from many other lineages.\",\n      \"method\": \"Genetic knockdown/knockout, reconstituted inflammasome assays, primary patient samples, mouse xenograft models\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function (CARD8 KO/KD) combined with primary human samples and in vivo mouse models; mechanism pathway placed\",\n      \"pmids\": [\"29967349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DPP9 directly interacts with human NLRP1 and CARD8 via their FIIND (Function-to-Find) autoproteolytic domains and acts as an endogenous inhibitor of the NLRP1 inflammasome. Both DPP9's scaffolding/binding function and its catalytic activity act synergistically to maintain NLRP1 in its inactive state. A patient-derived germline missense mutation in NLRP1's FIIND domain abrogates DPP9 binding and causes inflammasome hyperactivation.\",\n      \"method\": \"Proteomics screen, co-IP, CRISPR/Cas9 deletion, small-molecule inhibitors, ASC speck formation assay, IL-1β secretion, pyroptosis assays, patient mutation functional analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — interaction confirmed by proteomics and co-IP, genetic deletion by CRISPR, pharmacological inhibition, patient mutation validation; multiple orthogonal methods across multiple labs\",\n      \"pmids\": [\"30291141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DPP9 enzyme activity is required for survival of migratory tongue muscle progenitors in vivo. Catalytically inactive DPP9 knock-in mice (S729A) die within 12–18 h of birth due to microglossia caused by increased apoptosis of occipital somite-derived migratory muscle progenitors forming distal tongue intrinsic muscles.\",\n      \"method\": \"Catalytic knock-in mouse (S729A), histology, apoptosis quantification, phenotypic rescue by manual feeding\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — defined catalytic-inactive knock-in with specific cell-type apoptosis phenotype and mechanistic rescue experiment; single lab but rigorous in vivo genetics\",\n      \"pmids\": [\"28887018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DPP9's enzymatic catalytic activity, but not its binding to CARD8, restrains the CARD8 inflammasome. Wild-type but not catalytically inactive DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. The DPP9-CARD8 interaction (unlike the DPP9-NLRP1 interaction) is not disrupted by DPP9 inhibitors or CARD8 mutations blocking autoproteolysis.\",\n      \"method\": \"Activity-based probes, reconstituted inflammasome assays, mass spectrometry-based proteomics, DPP9 knockout cell lines, catalytic mutant rescue\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted inflammasome assay, catalytic mutant rescue, activity-based probes; multiple orthogonal methods definitively separating catalytic vs. scaffolding functions for CARD8\",\n      \"pmids\": [\"31525884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DPP8/9 mediate N-terminal processing of adenylate kinase 2 (AK2) in the cytoplasm. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Over 100 mitochondrial proteins with putative DPP8/9 recognition sites were identified, and DPP8/9 influence cellular levels of a subset of these.\",\n      \"method\": \"Biochemical fractionation, DPP8/9 inhibition, proteasome inhibition, AK2 substrate validation, MS-based proteomics\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — biochemical substrate identification with functional consequences (proteasomal degradation), proteomics of additional substrates, multiple inhibitor and genetic approaches\",\n      \"pmids\": [\"32815200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structures of human NLRP1-DPP9 complex (alone and with Val-boroPro) reveal a ternary complex comprising DPP9, full-length NLRP1, and the NLRP1 C-terminal fragment (CT). The N-terminus of NLRP1 CT inserts into the DPP9 active site; Val-boroPro disrupts this interaction. Binding of NLRP1 CT to DPP9 requires full-length NLRP1. VbP weakens the NLRP1-DPP9 interaction and accelerates degradation of the N-terminal fragment to induce inflammasome activation.\",\n      \"method\": \"Cryo-EM structure determination, biochemical binding assays, functional inflammasome assays, ectopic expression rescue experiments\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures plus orthogonal functional assays in a single rigorous study; replicated by parallel paper (PMID 33731929)\",\n      \"pmids\": [\"33731932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Structural and biochemical analysis of rat NLRP1-DPP9 shows a 2:1 complex with one autoinhibited full-length NLRP1 and one active UPA-CARD fragment. The ZU5 domain is required for both NLRP1 autoinhibition and 2:1 complex assembly. Complex formation prevents UPA-mediated higher-order oligomerization and strengthens ZU5-mediated autoinhibition. Both NLRP1 binding and DPP9 enzymatic activity are required to suppress NLRP1 in human cells.\",\n      \"method\": \"Cryo-EM/crystal structure, biochemical reconstitution, structure-guided mutagenesis, functional inflammasome assays in human cells\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural determination plus reconstitution, mutagenesis, and cell-based functional validation; replicated by parallel paper (PMID 33731932)\",\n      \"pmids\": [\"33731929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Saxagliptin (but not sitagliptin) inhibits DPP9 in cardiomyocytes and impairs CaMKII phosphorylation of phospholamban and PKC activity. DPP9 knockdown (but not DPP8 knockdown) and pharmacological DPP8/9 inhibition recapitulate these effects, implicating DPP9 specifically in CaMKII-PLB and PKC signaling in cardiomyocytes.\",\n      \"method\": \"siRNA knockdown of DPP9 vs DPP8, pharmacological inhibition (TC-E-5007, saxagliptin, sitagliptin), CaMKII/PLB phosphorylation assays, PKC activity assays, Ca2+ transient measurements, action potential duration recording in cardiomyocytes\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gene-specific knockdown with pharmacological corroboration and multiple downstream readouts, single lab but two orthogonal approaches\",\n      \"pmids\": [\"30487758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DPP9 binds KEAP1 via a conserved ESGE motif and disrupts KEAP1-NRF2 binding by competing with NRF2 for KEAP1 binding in an enzyme-independent (non-catalytic) manner. DPP9 overexpression stabilizes NRF2, drives NRF2-dependent transcription, decreases cellular ROS, suppresses ferroptosis, and induces sorafenib resistance in ccRCC cells, largely dependent on the NRF2 target SLC7A11.\",\n      \"method\": \"Protein affinity purification, co-IP competition assays, ESGE motif mutagenesis (enzyme-independent binding), NRF2 stability assays, ROS measurement, ferroptosis assays, drug resistance assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity purification plus competition co-IP and motif mutagenesis showing non-catalytic mechanism; single lab with multiple orthogonal cellular readouts\",\n      \"pmids\": [\"37713596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The NLRP1 variant M1184V stabilizes the FIIND domain in a monomeric conformation, promotes DPP9 binding (confirmed by surface plasmon resonance and co-IP), and enhances formation of the autoinhibited NLRP1-DPP9 complex, leading to reduced inflammasome activation.\",\n      \"method\": \"Size-exclusion chromatography, molecular dynamics simulation, surface plasmon resonance, immunoprecipitation, inflammasome activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SPR binding measurement plus co-IP and functional assays confirm enhanced DPP9 interaction; single lab, multiple methods\",\n      \"pmids\": [\"36309085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In addition to Syk and AK2, BRCA2 (tumor suppressor) was identified as a DPP9 substrate; N-terminal processing of BRCA2 by DPP9 triggers its rapid proteasomal turnover, placing DPP9 as an upstream component of the N-degron pathway.\",\n      \"method\": \"Enzymatic assays, substrate identification (described in review/methods context referencing experimental data)\",\n      \"journal\": \"Methods in enzymology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — substrate mention in a methods review chapter with reference to experimental work; not independently detailed in an original research abstract\",\n      \"pmids\": [\"37230592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A de novo DPP9 mutation (p.Arg252Pro) destabilizes the DPP9 protein and causes it to fail to restrain the NLRP1 and CARD8 inflammasomes, resulting in constitutive inflammasome activation and severe infancy-onset autoinflammation with HLH-like features.\",\n      \"method\": \"Patient genetic analysis, transfection in HEK293T cells and patient iPSCs, functional inflammasome assays (caspase-1 activation, IL-1β/IL-18 secretion), protein stability assessment\",\n      \"journal\": \"The Journal of allergy and clinical immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — disease variant functionally validated in heterologous cells and patient-derived iPSCs with inflammasome assays; single lab, two orthogonal cell models\",\n      \"pmids\": [\"37544411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FAP (Fibroblast Activation Protein) interacts intracellularly with DPP9 (identified by IP-MS), and FAP overexpression leads to reduction in DPP9 expression, promoting EMT in oral squamous cell carcinoma. DPP9 overexpression reverses FAP-induced proliferation, migration, invasion, and EMT, acting in a non-enzymatic manner.\",\n      \"method\": \"IP-mass spectrometry, DPP9 knockdown/overexpression, cell proliferation, migration and invasion assays, EMT marker analysis, in vivo xenograft\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP/MS interaction identification; functional assays are phenotypic with limited mechanistic dissection of DPP9's molecular role\",\n      \"pmids\": [\"32273729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KEAP1 binds DPP9 in an inactive (non-native) conformation and stabilizes this non-dimeric fold. Reciprocally, this inactive form of DPP9 inhibits KEAP1 from binding and degrading NRF2, thereby inducing an antioxidant response. This reveals an endogenous DPP9 inhibition mechanism and links DPP9, like thioredoxin-1, to the intracellular redox potential.\",\n      \"method\": \"Co-IP, biochemical characterization of DPP9 conformational states, NRF2 stability assays, binding competition assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal inhibition demonstrated by co-IP and functional NRF2 stability assays; single lab, multiple orthogonal readouts but conformational trigger not yet identified\",\n      \"pmids\": [\"39615677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SFTSV non-structural protein NSs activates the NLRP1 and CARD8 inflammasomes by disrupting the DPP9-mediated inhibitory ternary complex: NSs binds NLRP1 and CARD8 via their FIIND domains (competing with DPP8/9 for binding) and promotes degradation of DPP8 and DPP9, releasing activated C-terminal fragments.\",\n      \"method\": \"Viral infection of primary keratinocytes and macrophages, co-IP (NSs-NLRP1/CARD8 interaction), DPP9 protein level measurement, CARD8 deletion functional assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of NSs-NLRP1/CARD8 interaction, genetic deletion validation, and functional inflammasome readouts; single lab with multiple approaches\",\n      \"pmids\": [\"40608794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"DPP9 interacts with the BRISC deubiquitinase complex (BRCC36/BRCC3 and ABRO1/ABRAXAS2) and CYLD-SPATA2 complex. DPP9 disrupts BRISC-SHMT2 complex formation, reducing BRISC-mediated deubiquitination and stabilization of IFNAR1, thereby suppressing JAK/STAT-driven PD-L1 transcription in ccRCC. DPP9 inhibition restores T cell cytotoxicity and enhances checkpoint blockade.\",\n      \"method\": \"TurboID proximity labeling, NanoBRET in living cells, pharmacological DPP9 inhibition (1G244), IFNAR1 ubiquitination assays, PD-L1 expression assays, T cell cytotoxicity assays, co-IP\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling plus NanoBRET competition assays and functional ubiquitination/signaling readouts; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"41826729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"DPP9 interactome mapped by TurboID proximity labeling reveals DPP8, the E3 ligase CBL, the deubiquitinase complex CYLD-SPATA2, and BRISC complex components (BRCC36/BRCC3, ABRO1/ABRAXAS2) as DPP9 interactors. NanoBRET assays show DPP9 disrupts BRCC36-ABRO1 binding and CYLD-SPATA2 interaction, revealing non-catalytic scaffolding functions of DPP9 in ubiquitin signaling.\",\n      \"method\": \"TurboID proximity labeling in DPP9-KO HEK293 cells, NanoBRET in living cells, validation co-IPs\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling plus NanoBRET in living cells for interaction disruption; single lab, two orthogonal methods with inducible physiological expression controls\",\n      \"pmids\": [\"41636814\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DPP9 is an intracellular serine dipeptidyl peptidase that removes N-terminal Xaa-Pro dipeptides from cytosolic substrates (including the antigenic peptide RU1(34-42), the kinase Syk, adenylate kinase AK2, and BRCA2) to control their stability via the N-degron/proteasomal pathway, and simultaneously acts as a non-catalytic scaffold that sequesters the C-terminal fragments of the NLRP1 and CARD8 inflammasome sensors in inhibitory ternary complexes (DPP9:full-length sensor:sensor-CT), thereby suppressing inflammasome activation; DPP9 also engages non-catalytically with KEAP1, BRISC, CYLD-SPATA2, and Filamin A to regulate redox sensing, ubiquitin signaling, and B-cell receptor signaling, and its enzymatic activity is allosterically stimulated by SUMO1 binding.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DPP9 is an intracellular serine dipeptidyl peptidase of the DPP IV gene family that removes N-terminal Xaa-Pro dipeptides from cytosolic proteins and peptides, coupling N-terminal processing to protein stability and immune regulation [#0, #1]. As a rate-limiting cytoplasmic prolyl-peptidase, it degrades proline-containing antigenic peptides such as RU1(34-42), so that its loss enhances antigen presentation [#1]. By generating neo-N-termini that feed substrates into the N-degron/proteasomal pathway, DPP9 controls turnover of the tyrosine kinase Syk—recruited via Filamin A and primed for Cbl-mediated ubiquitination—and of adenylate kinase AK2, modulating B-cell receptor signaling and preventing cytosolic accumulation of active AK2 [#4, #9]. The most extensively defined role of DPP9 is as an endogenous brake on the NLRP1 and CARD8 inflammasomes: it forms inhibitory ternary complexes (DPP9:full-length sensor:sensor C-terminal fragment) in which the N-terminus of the sensor CT inserts into the DPP9 active site, with both scaffolding and catalytic activity enforcing autoinhibition; small-molecule DPP8/9 inhibitors or viral antagonists disrupt these complexes to trigger pro-caspase-1-dependent, gasdermin-D-mediated pyroptosis [#3, #6, #8, #10, #11, #19]. DPP9 enzymatic activity is allosterically stimulated by SUMO1 binding to an extended arm flanking the substrate entry site [#2]. Beyond its protease functions, DPP9 acts non-catalytically: it competes with NRF2 for KEAP1 binding to drive an antioxidant response, and it scaffolds the BRISC and CYLD-SPATA2 ubiquitin-signaling complexes [#13, #18, #20, #21]. A de novo DPP9 mutation (p.Arg252Pro) that destabilizes the protein and abolishes inflammasome restraint causes severe infancy-onset autoinflammation with HLH-like features [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that DPP9 is a cytosolic member of the DPP IV serine protease family defined its catalytic machinery and subcellular compartment, framing it as an intracellular peptidase rather than a cell-surface enzyme.\",\n      \"evidence\": \"In silico identification, in vitro translation, SDS-PAGE, and subcellular fractionation\",\n      \"pmids\": [\"12459266\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No natural substrate identified at this stage\", \"Physiological function unaddressed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying RU1(34-42) as the first natural substrate showed DPP9 is rate-limiting for cytoplasmic proline-peptide degradation and links its activity to antigen presentation.\",\n      \"evidence\": \"In vitro peptidase assay with cell extracts and siRNA knockdown with antigen presentation readout\",\n      \"pmids\": [\"19667070\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Broader substrate repertoire unknown\", \"No structural basis for substrate selectivity\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery of SUMO1 binding to an extended arm flanking the substrate entry site revealed an allosteric mechanism that stimulates DPP9 catalytic activity.\",\n      \"evidence\": \"Pulldown/co-IP, mutagenesis of the binding arm, enzymatic activity assays, and SUMO1 silencing\",\n      \"pmids\": [\"23152501\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where SUMO1-dependent regulation matters not defined\", \"SUMO2 selectivity basis only partially explained\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defining Syk as an N-end rule substrate processed by DPP9 (recruited via Filamin A and primed for Cbl ubiquitination) connected DPP9 N-terminal processing to protein turnover and B-cell signaling.\",\n      \"evidence\": \"Co-IP of DPP9-Filamin A-Syk complex, N-terminal sequencing, pulse-chase/mutagenesis, ubiquitination assays, and siRNA knockdown\",\n      \"pmids\": [\"27614019\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of the Filamin A-mediated recruitment to other substrates unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showing that DPP8/9 inhibition triggers ASC-independent pro-caspase-1 activation and gasdermin-D-mediated pyroptosis placed DPP9 as a suppressor of an inflammatory death pathway.\",\n      \"evidence\": \"Val-boroPro inhibition, caspase-1 KO mice, caspase-1 activity assays, and gasdermin D cleavage assays\",\n      \"pmids\": [\"27820798\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The upstream sensor restrained by DPP9 was not yet identified at this stage\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identifying NLRP1 and CARD8 as DPP9-bound inflammasome sensors, and dissecting whether catalytic or scaffolding activity restrains each, established DPP9 as a direct endogenous inflammasome brake.\",\n      \"evidence\": \"Proteomics, co-IP, CRISPR deletion, catalytic-mutant rescue, reconstituted inflammasome and pyroptosis assays, and patient mutation analysis\",\n      \"pmids\": [\"30291141\", \"29967349\", \"31525884\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the inhibitory complex not yet resolved\", \"Differential requirement of binding vs. catalysis for NLRP1 vs. CARD8 mechanistically distinct\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A catalytically inactive S729A knock-in mouse demonstrated that DPP9 enzymatic activity is required in vivo for survival of migratory tongue muscle progenitors, giving the protease an essential developmental role.\",\n      \"evidence\": \"Catalytic knock-in mouse, histology, apoptosis quantification, and phenotypic rescue by manual feeding\",\n      \"pmids\": [\"28887018\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The relevant in vivo substrate driving progenitor apoptosis is not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying AK2 as a substrate whose N-terminal processing triggers proteasomal degradation, plus >100 candidate mitochondrial-derived substrates, broadened DPP9's role in cytosolic proteostasis.\",\n      \"evidence\": \"Biochemical fractionation, DPP8/9 and proteasome inhibition, AK2 validation, and MS-based proteomics\",\n      \"pmids\": [\"32815200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences for most candidate substrates untested\", \"Physiological role of clearing cytosolic AK2 not fully defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Cryo-EM structures of the NLRP1-DPP9 ternary complex resolved how DPP9 binds full-length NLRP1 and inserts the CT N-terminus into its active site, explaining how Val-boroPro disrupts autoinhibition to activate the inflammasome.\",\n      \"evidence\": \"Cryo-EM/crystal structures, biochemical reconstitution, structure-guided mutagenesis, and functional inflammasome assays (human and rat)\",\n      \"pmids\": [\"33731932\", \"33731929\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of the DPP9-CARD8 complex less defined\", \"How sensor CT degradation kinetics couple to oligomerization quantitatively\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linking DPP9 to CaMKII-phospholamban and PKC signaling in cardiomyocytes extended its functional reach to cardiac signaling, distinguishing it from DPP8.\",\n      \"evidence\": \"DPP9-specific siRNA, pharmacological inhibition, phosphorylation and PKC activity assays, Ca2+ transient and action potential recordings\",\n      \"pmids\": [\"30487758\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular substrate/partner mediating cardiac effects not identified\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrating that DPP9 competes with NRF2 for KEAP1 binding via an ESGE motif in an enzyme-independent manner revealed a non-catalytic role in redox sensing and ferroptosis/drug resistance.\",\n      \"evidence\": \"Affinity purification, competition co-IP, ESGE motif mutagenesis, NRF2 stability and ROS assays, ferroptosis and sorafenib-resistance assays in ccRCC\",\n      \"pmids\": [\"37713596\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of the DPP9-KEAP1-NRF2 axis untested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showing KEAP1 traps DPP9 in an inactive non-dimeric conformation, which in turn blocks KEAP1 from degrading NRF2, defined a reciprocal endogenous inhibition mechanism coupling DPP9 to redox state.\",\n      \"evidence\": \"Co-IP, biochemical characterization of DPP9 conformational states, and NRF2 stability/competition assays\",\n      \"pmids\": [\"39615677\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological trigger driving the conformational switch not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Reporting BRCA2 as a DPP9 substrate whose N-terminal processing accelerates proteasomal turnover positioned DPP9 upstream of the N-degron pathway for a tumor suppressor.\",\n      \"evidence\": \"Enzymatic assays and substrate identification described in a methods review chapter\",\n      \"pmids\": [\"37230592\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Reported in a methods review rather than detailed in an original research study\", \"Cellular and physiological consequences not independently characterized\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Proximity-labeling interactome mapping identified DPP9 as a non-catalytic scaffold disrupting BRISC and CYLD-SPATA2 ubiquitin-signaling complexes, linking it to IFNAR1 stability, PD-L1 transcription, and antitumor immunity.\",\n      \"evidence\": \"TurboID proximity labeling, NanoBRET in living cells, IFNAR1 ubiquitination, PD-L1, and T cell cytotoxicity assays with DPP9 inhibition\",\n      \"pmids\": [\"41826729\", \"41636814\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of BRISC/CYLD-SPATA2 disruption unknown\", \"Single-lab findings awaiting independent replication\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A destabilizing de novo DPP9 mutation causing constitutive NLRP1/CARD8 activation and severe autoinflammation established DPP9 loss-of-function as a Mendelian autoinflammatory disease mechanism.\",\n      \"evidence\": \"Patient genetics, transfection in HEK293T and patient iPSCs, inflammasome activity assays, and protein stability assessment\",\n      \"pmids\": [\"37544411\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genotype-phenotype spectrum across patients not defined\", \"Single index case\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full physiological substrate repertoire of DPP9, the mechanisms switching it between catalytic and non-catalytic scaffolding roles, and how its redox-sensing, ubiquitin-signaling, and inflammasome functions are coordinated in vivo remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Most candidate substrates lack functional validation\", \"Conformational/redox triggers governing DPP9 activity state unresolved\", \"Integration of DPP9's diverse functions at the organismal level unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 4, 9]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [6, 10, 13, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 8, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 6, 8, 10, 11]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 5, 16]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 9, 15]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [13, 18]}\n    ],\n    \"complexes\": [\n      \"NLRP1-DPP9 inflammasome ternary complex\",\n      \"CARD8-DPP9 complex\",\n      \"BRISC\",\n      \"CYLD-SPATA2\"\n    ],\n    \"partners\": [\n      \"NLRP1\",\n      \"CARD8\",\n      \"SUMO1\",\n      \"FLNA\",\n      \"SYK\",\n      \"KEAP1\",\n      \"BRCC3\",\n      \"ABRAXAS2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}