{"gene":"HINT1","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":1997,"finding":"Crystal structures of HINT in complex with nucleotides demonstrated that HINT is a dimeric purine nucleotide-binding protein; the HIT motif forms part of the phosphate-binding loop, and the most conserved residues mediate nucleotide binding. These structures revealed that HINT shares the same fold and mode of nucleotide binding as galactose-1-phosphate uridylyltransferase (GalT) despite no overall sequence similarity.","method":"X-ray crystallography of HINT-nucleotide complexes","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct crystal structures with functional interpretation, foundational structural paper replicated across the field","pmids":["9164465"],"is_preprint":false},{"year":2002,"finding":"Rabbit HINT1 and yeast Hnt1 hydrolyze adenosine-5'-monophosphoramidate (AMP-NH2) as natural substrates in an active-site-dependent manner, with second-order rates exceeding 1,000,000 M−1 s−1. Loss of Hnt1 enzyme activity in yeast leads to failure to grow on galactose at elevated temperature and hypersensitivity to mutations in Kin28, Ccl1, and Tfb3 (the TFIIK kinase subcomplex of TFIIH), placing Hnt1 as a positive regulator of this transcription kinase pathway downstream of Cak1.","method":"In vitro enzymatic assay with AMP-NH2; yeast genetic epistasis with temperature-sensitive alleles and deletion strains; functional complementation with rabbit Hint","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinetic assay plus multi-allele genetic epistasis; rabbit Hint complementation confirms conservation","pmids":["11805111"],"is_preprint":false},{"year":2002,"finding":"HINT belongs to the adenosine 5'-monophosphoramide hydrolase branch of the HIT superfamily; a new enzyme mechanism was proposed for Hint and Fhit based on comparative structural and biochemical analysis of the three HIT branches (Hint, Fhit, GalT).","method":"Biochemical and structural comparative analysis; sequence/substrate specificity review with mechanistic inference","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — comprehensive review integrating structural and biochemical data from multiple sources, but primarily a review paper synthesizing existing experimental data","pmids":["12119013"],"is_preprint":false},{"year":2000,"finding":"HINT/PKCI-1 physically interacts with Cdk7; this interaction was identified by yeast two-hybrid and confirmed by co-immunoprecipitation. Overexpression of Cdk7 leads to partial relocalization of Hint to the nucleus. The interaction is independent of cyclin H binding or Cdk7 kinase activity. The orthologous interaction between yeast Kin28 and Hnt1 is conserved, and genetic combination of hnt1 deletion with a kin28 temperature-sensitive allele causes elongated cell morphology and reduced colony formation.","method":"Yeast two-hybrid, co-immunoprecipitation, subcellular localization by fluorescence microscopy, yeast genetic interaction (double mutant analysis)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus yeast genetic epistasis plus localization data, replicated across two organisms","pmids":["10958787"],"is_preprint":false},{"year":2004,"finding":"Biochemical and structural characterization of HINT enzymatic activity as an AMP-lysine (adenosine phosphoramidate) hydrolase. The 1.8-Å co-crystal structure with N-ethylsulfamoyl adenosine revealed a binding site for the alkyl group against Trp-123 across the dimer interface. Ser-107 donates a hydrogen bond to the leaving-group nitrogen; the S107A mutant displays markedly depressed catalytic activity, confirming a role for Ser-107 in acid-base catalysis. A novel substrate AMP-pNA and inhibitors (AdoOSO2NH2, AdoOSO2NHCH2CH3) were characterized kinetically.","method":"X-ray crystallography (1.8 Å co-crystal), active-site mutagenesis (S107A), in vitro kinetic assay with novel substrates and inhibitors","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro kinetic assays in a single rigorous study","pmids":["14982931"],"is_preprint":false},{"year":2005,"finding":"HINT1/PKCI-1 directly binds to Pontin and Reptin; pull-down and co-immunoprecipitation experiments mapped the HINT1 binding site to amino acids 214–295 of Pontin and 218–289 of Reptin, while Pontin and Reptin bind the N-terminus of HINT1. Through this interaction, HINT1 associates with the LEF-1/TCF–β-catenin transcription complex and acts as a negative regulator of TCF–β-catenin transcriptional activity, repressing cyclin D1 and axin2 expression. RNAi knockdown of HINT1 increases these target genes.","method":"Pull-down assay, co-immunoprecipitation, reporter gene assay (TCF-luciferase), RNAi knockdown with RT-PCR/qPCR","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with domain mapping, orthogonal reporter assays, and RNAi phenotype in multiple cell contexts","pmids":["16014379"],"is_preprint":false},{"year":2006,"finding":"HINT1 overexpression induces apoptosis in SW480 and MCF-7 cells via upregulation of p53 and proapoptotic Bax and downregulation of Bcl-2. HINT1 associates with the Bax promoter and is a component of the Tip60 histone acetyltransferase complex. A catalytically inactive mutant HINT1 (H112N) retains full pro-apoptotic activity, demonstrating that the apoptotic function is independent of AMP-NH2 hydrolase enzymatic activity.","method":"Transient transfection, caspase-3/PARP cleavage assay, cytochrome c release, DNA fragmentation ELISA, shRNA knockdown, chromatin immunoprecipitation (ChIP), co-immunoprecipitation with Tip60 complex, active-site mutant (H112N)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ChIP, Co-IP, mutant analysis, KD), single lab but rigorous experimental design","pmids":["16835243"],"is_preprint":false},{"year":2007,"finding":"HINT1 inhibits AP-1 transcriptional activity in colon cancer cells by forming an in vivo complex with POSH (plenty of SH3) and JNK2, thereby inhibiting phosphorylation of c-Jun. This pathway requires JNK2 but not JNK1, confirmed in JNK1-/- and JNK2-/- MEFs. Both wild-type HINT1 and the enzymatically inactive H112N mutant inhibit AP-1 activity equivalently, indicating the inhibition is independent of enzymatic function.","method":"Co-immunoprecipitation (HINT1-POSH-JNK complex), reporter gene assay (AP-1-luciferase), JNK1-/- and JNK2-/- MEF epistasis, phospho-c-Jun western blot, retroviral overexpression","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP of endogenous complex, genetic epistasis using KO MEFs, and enzymatic mutant confirming mechanism-independent activity","pmids":["17510397"],"is_preprint":false},{"year":2008,"finding":"HINT1 participates in ionizing radiation (IR)-induced DNA damage responses: it is recruited to IR-induced foci (IRIF) and co-associates with γ-H2AX and ATM. HINT1 deficiency does not prevent γ-H2AX foci formation but impairs their removal and impairs acetylation of γ-H2AX. HINT1 deficiency also impairs acetylation and activation of ATM and retards DNA repair; HINT1-deficient cells resist IR-induced apoptosis and exhibit chromosomal abnormalities.","method":"Immunofluorescence (IRIF recruitment), co-immunoprecipitation (HINT1-γ-H2AX-ATM), acetylation assays, DNA repair kinetics, apoptosis assays in Hint1-/- cells vs. WT","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including Co-IP, immunofluorescence, functional assays in KO cells","pmids":["18852295"],"is_preprint":false},{"year":2008,"finding":"In neurons, the C-terminus of the mu-opioid receptor (MOR) binds PKCI/HINT1, which in turn binds the regulator of G-protein signaling RGSZ1/Z2 proteins. Morphine administration recruits PKCγ (mostly) to the MOR via the HINT1/RGSZ complex. This recruitment requires zinc ions, HINT1, and RGSZ proteins, and specifically involves the cysteine-rich domains (CRDs) of PKCγ. NMDAR antagonist MK801 and NOS inhibition prevent PKCγ recruitment, implicating the NMDAR/nNOS cascade in providing zinc ions required for PKCγ to bind the HINT1/RGSZ complex at MOR.","method":"Co-immunoprecipitation from mouse brain; intracerebroventricular drug administration; siRNA knockdown of HINT1; zinc chelation (TPEN); phorbol ester and NO donor treatments","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo and ex vivo Co-IP experiments, pharmacological dissection with multiple reagents, and knockdown controls across multiple studies","pmids":["18652891"],"is_preprint":false},{"year":2009,"finding":"HINT1 co-immunoprecipitates with USF2 in hepatoma cell extracts and inhibits transcriptional activities of β-catenin/TCF4, USF2, and NF-κB in HepG2 cells. HINT1 overexpression inhibits nuclear translocation of p65 (NF-κB subunit) and reduces expression of cyclin D1 and TGFβ2.","method":"Co-immunoprecipitation, reporter gene assays (β-catenin/TCF4, USF2, NF-κB luciferase), nuclear fractionation, western blot","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and reporter assays in single lab, orthogonal methods but limited mechanistic depth","pmids":["19089909"],"is_preprint":false},{"year":2010,"finding":"HINT1 (Hint-1) phosphoramidase catalyzes the desulfuration of nucleoside 5'-O-phosphorothioates ((d)NMPS) to the corresponding nucleoside 5'-O-phosphates with release of hydrogen sulfide. Crystallographic analysis (1.08–1.37 Å) of three engineered cysteine mutants confirmed minimal structural perturbation. Substrate specificity order was determined: GMPS > AMPS > dGMPS ≥ CMPS > UMPS > dAMPS >> dCMPS > TMPS.","method":"In vitro enzymatic assay (HPLC-based), active-site mutagenesis, X-ray crystallography (1.08–1.37 Å resolution) of mutant structures","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus in vitro kinetic assays plus mutagenesis in a single rigorous study","pmids":["20940308"],"is_preprint":false},{"year":2011,"finding":"Morphine-generated nitric oxide (NO) causes release of endogenous zinc ions that recruit the Ras/Raf-1/ERK1/2 cassette to HINT1 at the MOR C-terminus; zinc bridges the Raf-1 cysteine-rich domain (CRD) with HINT1. Simultaneously, PKCγ is also recruited via NO/zinc to the MOR-HINT1 complex; Raf-1 and PKCγ CRDs bind simultaneously to HINT1, enabling PKCγ to enhance Raf-1 function and thereby MEK/ERK1/2 activation. A-Raf and B-Raf show little or no MOR association.","method":"Co-immunoprecipitation from mouse brain, pharmacological dissection (NOS inhibitors, zinc chelator TPEN), intracerebroventricular injection paradigms","journal":"Antioxidants & redox signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP from brain tissue with pharmacological controls; single lab, multiple experiments","pmids":["21235400"],"is_preprint":false},{"year":2012,"finding":"HINT1 co-immunoprecipitates with MITF and β-catenin in melanoma cell lines. HINT1 inhibits MITF and β-catenin transcriptional activity, reduces cyclin D1 and BCL2 expression (known MITF and β-catenin targets), and ChIP assays demonstrate HINT1 occupancy at MITF and β-catenin binding sites in BCL2 and cyclin D1 promoters. HINT1 co-immunoprecipitates with mSIN3a and HDAC1, suggesting recruitment of a repressor complex.","method":"Co-immunoprecipitation, ChIP assay, reporter gene assay, stable HINT1 overexpression with xenograft, rescue by BCL2/cyclin D1 overexpression","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP plus Co-IP of repressor complex plus functional rescue experiments, multiple orthogonal methods","pmids":["22647378"],"is_preprint":false},{"year":2012,"finding":"Human HINT1 has broad specificity for aminoacyl adenylate (aa-AMP) hydrolysis, recognizing only the common main-chain of the aminoacyl moiety and having no contact with the amino acid side chain. High-resolution crystal structures of HINT1 with three different aa-AMP analogues revealed that the α-amino group is anchored by a cation-π interaction with Trp123 at the C-terminus.","method":"X-ray crystallography (HINT1 with aa-AMP analogues), in vitro hydrolysis assays","journal":"The journal of physical chemistry B","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures with functional biochemical validation in a single study","pmids":["22329685"],"is_preprint":false},{"year":2012,"finding":"Loss-of-function mutations in HINT1 cause autosomal recessive axonal neuropathy with neuromyotonia, identified by combining linkage analysis and next-generation sequencing in 33 families with 8 distinct mutations.","method":"Linkage analysis, next-generation sequencing, cohort mutation screening","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — large multi-family genetic study with multiple mutations independently identified; replicated in subsequent studies","pmids":["22961002"],"is_preprint":false},{"year":2013,"finding":"HINT1 protein influences NMDAR activity in the presence of GPCRs: in CNR1+/+/HINT1-/- cortical neurons, NMDAR activation was enhanced and cannabinoid agonist WIN55,212-2 failed to protect against NMDA insult. Lentiviral re-expression of HINT1 normalized NMDAR activity and restored cannabinoid-mediated neuroprotection. In the absence of receptor activation, GPCRs collaborate with HINT1 to negatively control NMDAR activity.","method":"HINT1-/- and CNR1-/- mouse cortical neuron culture, lentiviral HINT1 rescue, NMDA excitotoxicity assay, electrophysiology","journal":"Molecular brain","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO neurons with viral rescue, multiple receptor and KO combinations, functional neuroprotection readout","pmids":["24093505"],"is_preprint":false},{"year":2015,"finding":"The teneurin-1 intracellular domain (ICD) binds HINT1 (identified by yeast two-hybrid and confirmed in human cells), disrupting HINT1-mediated repression of MITF transcriptional targets. Teneurin-1 ICD overexpression upregulates MITF target genes including GPNMB; promoter reporter assays showed the teneurin-1 ICD switches on MITF-dependent GPNMB transcription by binding and titrating HINT1 away from MITF.","method":"Yeast two-hybrid, co-immunoprecipitation in human cells, transcriptome analysis, RT-qPCR, promoter reporter assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP in human cells plus functional reporter assays, single lab","pmids":["25648896"],"is_preprint":false},{"year":2015,"finding":"The sigma 1 receptor (σ1R) engages HINT1 at the MOR C-terminus through a redox-regulated mechanism: activated MORs stimulate nitric oxide (NO) production; the RGSZ2 redox zinc switch converts this into free zinc ions that recruit PKCγ to HINT1 proteins, impairing HINT1-RGSZ2 association and enabling σ1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid signaling. σ1R antagonists remove σ1R from NR1, facilitate Ca2+-CaM entry, and prevent NR1-HINT1 interaction, blocking the negative feedback on opioid analgesia.","method":"Co-immunoprecipitation from mouse brain, intracerebroventricular pharmacology, σ1R-/- and HINT1-/- mice, NOS inhibition, in vitro binding assays","journal":"Antioxidants & redox signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple KO models, pharmacological dissection, and Co-IP with multiple orthogonal manipulations","pmids":["25557043"],"is_preprint":false},{"year":2015,"finding":"HINT1 and σ1R coordinate GPCR-NMDAR interactions: HINT1 binds GPCRs and NMDAR NR1 subunits in a calcium-independent manner, while σ1R binding to these proteins increases with calcium. σ1R agonists retain HINT1 at the GPCR and promote GPCR-NMDAR interaction; σ1R antagonists transfer HINT1 to NR1 subunits and disengage GPCRs from NMDARs. In σ1R-/- mice, HINT1 proteins mostly associate with NMDARs and GPCRs are functionally disconnected from NMDARs. In HINT1-/- mice, ischemia produces reduced NMDAR-mediated brain damage.","method":"Co-immunoprecipitation, in vivo pharmacology, σ1R-/- and HINT1-/- mice, ischemia model, electrophysiology","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 2 / Strong — two genetic KO models with biochemical Co-IP and in vivo functional paradigms","pmids":["26461475"],"is_preprint":false},{"year":2015,"finding":"The HINT1 enzymatic active site is critical for its regulatory role at the MOR-NMDAR interface; the HINT1 inhibitor guanosine-5'-tryptamine carbamate (TpGc) significantly enhances morphine antinociception, prevents tolerance development, and reduces MOR recruitment of NMDAR activity. Intracerebroventricular TpGc also attenuates NMDAR function and alleviates mechanical allodynia in chronic constriction injury mice.","method":"In vivo pharmacology with HINT1 active-site inhibitor, antinociception assays, mouse model of neuropathic pain, molecular interaction assays","journal":"Neuropharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition with in vivo behavioral readouts; enzymatic dependency inferred from active-site inhibitor; single lab","pmids":["25445489"],"is_preprint":false},{"year":2017,"finding":"HINT1 undergoes post-translational modifications in activated mast cells: K21 acetylation and Y109 phosphorylation. Mutational analysis confirmed that these modifications promote MITF transcriptional and oncogenic activity in melanoma cell lines, linked to the Ap4A-triggered dissociation of HINT1 from MITF. The LysRS-Ap4A-HINT1-MITF signaling pathway can be modulated through HINT1 PTMs to affect MITF-driven transcription.","method":"Mass spectrometry identification of PTMs in activated mast cells, site-directed mutagenesis (K21, Y109), reporter assays in melanoma cell lines","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PTMs identified by MS and confirmed by mutagenesis with functional reporter readout; single lab","pmids":["28394346"],"is_preprint":false},{"year":2019,"finding":"HINT1 exhibits zinc- and redox-regulated cysteine SUMO isopeptidase (sumoylase) activity, removing SUMO from signaling proteins. The catalytic triad Cys84-Asp87-His114 in the C-terminal region and a SUMO-interacting motif (residues 110-116 HIHLHVL) were identified. HINT1 sumoylase activity is blocked by zinc and released by nitric oxide or calcium-activated calmodulin (CaM). All 15 human HINT1 mutants causing ARAN-NM showed altered sumoylase activity.","method":"In vitro sumoylase assay, site-directed mutagenesis of catalytic triad, zinc/NO/CaM pharmacological manipulation, analysis of 15 disease-causing mutants","journal":"Antioxidants & redox signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzymatic assay with mutagenesis and pharmacological controls; novel activity claim from single lab","pmids":["31088288"],"is_preprint":false},{"year":2019,"finding":"Ap4A specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. Eight crystal structures revealed that Ap4A binding drives HINT1 polymerization; the polymerization interface overlaps with the HINT1-MITF interaction surface, suggesting a competitive mechanism to release MITF for transcriptional activation. The polymerization depends precisely on the length of the phosphodiester linkage of Ap4A.","method":"X-ray crystallography (8 structures), negative stain electron microscopy, biochemical polymerization assays, cellular experiments in RBL cells","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — eight crystal structures combined with EM and cellular biochemical assays in a single rigorous study","pmids":["31604935"],"is_preprint":false},{"year":2020,"finding":"HINT1 is acetylated by CBP at K21 and K30 and deacetylated by SIRT1. Deacetylation of HINT1 by SIRT1 increases the binding capacity of HINT1 for β-catenin or MITF, enhancing its tumor-suppressive function. The deacetylation-mimetic double mutant HINT1-2KR (K21R/K30R) significantly reduced cellular proliferation in colon cancer and melanoma cells in both cell-based and xenograft assays.","method":"Co-immunoprecipitation (HINT1 with CBP/SIRT1), acetylation assays, site-directed mutagenesis (K21R, K30R), in vitro binding assays, xenograft tumor assays","journal":"Experimental & molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — writer (CBP) and eraser (SIRT1) identified for specific sites, confirmed by mutagenesis and functional in vivo xenograft rescue","pmids":["32636443"],"is_preprint":false},{"year":2021,"finding":"HINT1 inhibits PKCβ1 membrane translocation and phosphorylation via direct interaction, attenuating the MEK/ERK/YY1 signaling pathway and thereby downregulating HOXA5 expression. HOXA5, in turn, affects hypertrophy through the TGF-β signaling pathway. In Hint1-deficient mice, cardiac hypertrophy was exacerbated after pressure overload; cardiac-specific HINT1 overexpression (via AAV9) alleviated it. The cardioprotective role of HINT1 was abolished by HOXA5 knockdown in vivo.","method":"Co-immunoprecipitation (HINT1-PKCβ1), cellular fractionation (PKCβ1 membrane translocation assay), Hint1 KO and AAV9-HINT1 OE mice with transverse aortic constriction, RNA sequencing, PCR array, AAV9-shHoxa5 in vivo","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP with fractionation, multiple genetic models (KO + OE), in vivo pathway rescue by target KD, orthogonal transcriptomics","pmids":["34098726"],"is_preprint":false},{"year":1996,"finding":"Human PKCI-1 (HINT1/PRKCNH1) localizes to cytoskeletal structures in the cytoplasm of human fibroblast cell lines by indirect immunofluorescence and is largely excluded from the nucleus, consistent with a role in mediating membrane-derived signals.","method":"Indirect immunofluorescence in human fibroblast cell line","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment replicated in later studies; functional link speculative at time","pmids":["8812426"],"is_preprint":false},{"year":2021,"finding":"HINT1 and σ1R control α2δ1 binding to NMDAR NR1 subunits: α2δ1 peptides require σ1Rs to interact with the NMDAR NR1 C1 segment. σ1R antagonists or low calcium dissociate σ1R-NR1 C1 dimers. HINT1 removes α2δ1 from the δ1-σ1R-NR1 trimer, facilitating σ1R dissociation from NMDARs. In σ1R-/- mice, sciatic nerve injury does not promote NMDAR-α2δ1 complex formation and allodynia does not develop; in HINT1-/- mice, α2δ1-σ1R-NMDAR complexes are increased and allodynia is exacerbated.","method":"Co-immunoprecipitation from PAG tissue, σ1R-/- and HINT1-/- mice with chronic constriction injury model, in vivo pharmacology","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two KO models with Co-IP and behavioral readouts; single lab building on prior series","pmids":["34827679"],"is_preprint":false},{"year":2019,"finding":"Spinal inhibition of HINT1 enzymatic activity by active-site inhibitors (TrpGc vs. acyl-sulfamate analogues) differentially affects MOR-NMDAR cross-talk: TrpGc blocked both the development of opioid tolerance and the inhibitory effect of opioids on NMDA activation, while acyl-sulfamate analogues could only block the latter. X-ray crystallographic and thermodynamic binding studies revealed key differences between bound HINT1-inhibitor surfaces that may account for their distinct pharmacological profiles.","method":"X-ray crystallography of HINT1-inhibitor complexes, isothermal titration calorimetry, in vivo spinal pharmacology, opioid tolerance behavioral assay","journal":"ACS chemical neuroscience","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structures plus in vivo pharmacology; single lab but two orthogonal methods","pmids":["31503445"],"is_preprint":false},{"year":2022,"finding":"HINT1 catalyzes the hydrolysis of deoxyguanosine-5'-O-selenophosphate (dGMPSe) with similar kcat and Km to the corresponding thiophosphate (dGMPS), releasing hydrogen selenide (H2Se). This H2Se release is toxic to HeLa cancer cells, demonstrating that HINT1 enzymatic activity toward selenophosphate nucleotides can generate cytotoxic H2Se intracellularly.","method":"HPLC-based and fluorescence-based enzymatic assay, MTT viability assay, fluorescence detection of H2Se in living cells, electroporation of substrate","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinetic assays plus cellular delivery and detection of product; single lab","pmids":["35054788"],"is_preprint":false}],"current_model":"HINT1 is a universally conserved, dimeric adenosine phosphoramidase/AMP-lysine hydrolase (HIT superfamily) whose enzymatic active site (centered on the HIT motif, Ser107, Trp123, and catalytic triad Cys84-Asp87-His114) hydrolyzes AMP-phosphoramidate, aminoacyl-AMP, and nucleoside phosphorothioate substrates; independently of enzymatic activity, HINT1 functions as a scaffold at the C-terminus of GPCRs (particularly the mu-opioid receptor) where it recruits RGSZ2, PKCγ, Raf-1, and σ1R in a redox/zinc-dependent manner to mediate GPCR-NMDAR cross-regulation; HINT1 also suppresses oncogenic transcription by binding and co-repressing β-catenin/TCF, MITF, AP-1/JNK2, NF-κB, and USF2 complexes—a function regulated by SIRT1-mediated deacetylation at K21/K30 and disrupted by Ap4A-induced HINT1 polymerization; HINT1 additionally inhibits PKCβ1 membrane translocation to suppress the MEK/ERK/YY1/HOXA5 cardiac hypertrophy pathway and exhibits zinc/CaM-regulated SUMO isopeptidase activity; loss-of-function mutations cause autosomal recessive axonal neuropathy with neuromyotonia (ARAN-NM)."},"narrative":{"mechanistic_narrative":"HINT1 is a universally conserved, dimeric purine nucleotide-binding enzyme of the HIT superfamily that doubles as a redox-regulated signaling scaffold and transcriptional co-repressor [PMID:9164465, PMID:11805111]. Its active site—built on the HIT phosphate-binding loop with Ser107 in acid-base catalysis and Trp123 anchoring substrate across the dimer interface—hydrolyzes adenosine 5'-monophosphoramidate, AMP-lysine, aminoacyl-adenylates, and nucleoside phosphorothioate/selenophosphate substrates, the latter releasing H2S or cytotoxic H2Se [PMID:11805111, PMID:14982931, PMID:20940308, PMID:22329685, PMID:35054788]. Independently of catalysis, HINT1 acts as a tumor suppressor by binding and co-repressing oncogenic transcription factors including β-catenin/TCF (via Pontin/Reptin), MITF, AP-1/JNK2, USF2, and NF-κB, recruiting mSIN3a/HDAC1 and Tip60 repressor/acetyltransferase complexes to target promoters such as cyclin D1, BCL2, and Bax; catalytically dead H112N mutants retain this activity [PMID:16014379, PMID:16835243, PMID:17510397, PMID:22647378]. This repressive function is tuned by post-translational control: SIRT1-mediated deacetylation at K21/K30 strengthens factor binding, while Ap4A-driven HINT1 polymerization competitively displaces MITF to relieve repression [PMID:31604935, PMID:32636443]. In neurons, HINT1 scaffolds the C-terminus of GPCRs—particularly the mu-opioid receptor—recruiting RGSZ proteins, PKCγ, Raf-1, and σ1R through a NO/zinc redox switch to govern GPCR-NMDAR cross-regulation, opioid analgesia, and neuropathic pain [PMID:18652891, PMID:21235400, PMID:25557043, PMID:26461475]. HINT1 also restrains cardiac hypertrophy by blocking PKCβ1 membrane translocation and the downstream MEK/ERK/YY1/HOXA5 axis [PMID:34098726]. Loss-of-function mutations in HINT1 cause autosomal recessive axonal neuropathy with neuromyotonia [PMID:22961002].","teleology":[{"year":1997,"claim":"Established the structural basis of HINT as a nucleotide-binding protein, defining the HIT motif's role and an unexpected fold relationship to GalT.","evidence":"X-ray crystallography of HINT-nucleotide complexes","pmids":["9164465"],"confidence":"High","gaps":["Did not identify the physiological substrate","Catalytic mechanism not resolved from binding alone"]},{"year":2002,"claim":"Identified the natural enzymatic activity (AMP-phosphoramidate hydrolysis) and placed HINT in a transcription-kinase regulatory pathway via yeast genetics.","evidence":"In vitro kinetics with AMP-NH2 plus yeast epistasis with TFIIK/Cak1 alleles and rabbit Hint complementation","pmids":["11805111"],"confidence":"High","gaps":["Direct enzymatic substrate in transcription pathway not pinpointed","Mechanism linking hydrolase activity to kinase regulation unresolved"]},{"year":2000,"claim":"Linked HINT physically to the cell-cycle/transcription kinase Cdk7, showing a kinase-activity-independent interaction conserved to yeast Kin28.","evidence":"Yeast two-hybrid, reciprocal Co-IP, localization, and yeast double-mutant analysis","pmids":["10958787"],"confidence":"High","gaps":["Functional consequence of Cdk7 binding for HINT unclear","Mechanism of Cdk7-driven nuclear relocalization unknown"]},{"year":2004,"claim":"Defined the catalytic machinery for AMP-lysine/phosphoramidate hydrolysis, assigning Ser107 to acid-base catalysis and Trp123 to substrate binding.","evidence":"1.8-Å co-crystal structure, S107A mutagenesis, and kinetics with novel substrates/inhibitors","pmids":["14982931"],"confidence":"High","gaps":["In vivo substrate of the AMP-lysine activity not established","Physiological context of catalysis unaddressed"]},{"year":2005,"claim":"Revealed HINT1's enzyme-independent role as a transcriptional repressor of β-catenin/TCF signaling through Pontin/Reptin binding.","evidence":"Pull-down/Co-IP with domain mapping, TCF-luciferase reporters, and RNAi with target qPCR","pmids":["16014379"],"confidence":"High","gaps":["Whether repression requires the catalytic site untested here","Recruitment of corepressor machinery not yet defined"]},{"year":2006,"claim":"Demonstrated catalysis-independent pro-apoptotic/tumor-suppressor function through Bax promoter occupancy and Tip60 complex association.","evidence":"ChIP, Co-IP with Tip60, apoptosis assays, shRNA, and H112N catalytic mutant in cancer cells","pmids":["16835243"],"confidence":"High","gaps":["How HINT1 is recruited to the Bax promoter unknown","Relationship between p53 induction and direct chromatin binding unclear"]},{"year":2007,"claim":"Extended HINT1 repression to AP-1 by showing it forms a POSH-JNK2 complex blocking c-Jun phosphorylation, JNK2-specifically.","evidence":"Endogenous Co-IP, AP-1 reporters, JNK1/JNK2 KO MEF epistasis, and H112N mutant","pmids":["17510397"],"confidence":"High","gaps":["Structural basis of HINT1-POSH-JNK2 assembly unknown","Why JNK2 but not JNK1 is targeted unexplained"]},{"year":2008,"claim":"Placed HINT1 in the DNA damage response, showing it is needed for γ-H2AX/ATM acetylation and efficient repair.","evidence":"IRIF immunofluorescence, Co-IP with γ-H2AX/ATM, acetylation and repair kinetics in Hint1-/- cells","pmids":["18852295"],"confidence":"High","gaps":["Direct enzymatic contribution to acetylation events unclear","Mechanism of HINT1 recruitment to damage foci unknown"]},{"year":2008,"claim":"Defined HINT1 as a scaffold at the mu-opioid receptor C-terminus that recruits PKCγ via RGSZ proteins in a zinc/NMDAR-dependent manner.","evidence":"Brain Co-IP, ICV pharmacology, HINT1 siRNA, zinc chelation, and NOS/NMDAR inhibitors","pmids":["18652891"],"confidence":"High","gaps":["Direct structural map of the MOR-HINT1-RGSZ interface absent","Source/handling of recruited zinc not fully resolved"]},{"year":2009,"claim":"Generalized HINT1 transcriptional repression to USF2 and NF-κB, including blockade of p65 nuclear translocation.","evidence":"Co-IP, multiple reporter assays, and nuclear fractionation in hepatoma cells","pmids":["19089909"],"confidence":"Medium","gaps":["Single-lab Co-IP/reporter without domain mapping","Mechanism of p65 nuclear-translocation block undefined"]},{"year":2010,"claim":"Expanded the substrate repertoire to nucleoside phosphorothioates, showing HINT1 catalyzes desulfuration with H2S release.","evidence":"HPLC enzymatic assays, mutagenesis, and high-resolution crystallography of mutants","pmids":["20940308"],"confidence":"High","gaps":["Physiological relevance of phosphorothioate desulfuration unknown","In vivo H2S generation not demonstrated"]},{"year":2011,"claim":"Showed NO-released zinc recruits the Ras/Raf-1/ERK cassette to HINT1 at MOR alongside PKCγ, integrating two CRD-bearing effectors.","evidence":"Brain Co-IP with NOS inhibitors and zinc chelation, ICV paradigms","pmids":["21235400"],"confidence":"Medium","gaps":["Single-lab pharmacological dissection","Stoichiometry of simultaneous Raf-1/PKCγ binding unresolved"]},{"year":2012,"claim":"Identified HINT1 as a melanoma tumor suppressor co-repressing MITF and β-catenin via mSIN3a/HDAC1 recruitment at BCL2 and cyclin D1 promoters.","evidence":"Co-IP, ChIP, reporters, xenograft, and rescue by target overexpression","pmids":["22647378"],"confidence":"High","gaps":["Determinants of promoter selectivity unclear","Whether the same complex operates on all repressed factors untested"]},{"year":2012,"claim":"Defined the structural basis for broad aminoacyl-adenylate hydrolysis, showing Trp123 cation-π anchoring of the α-amino group.","evidence":"Crystal structures with aa-AMP analogues and in vitro hydrolysis assays","pmids":["22329685"],"confidence":"High","gaps":["Cellular substrate among aminoacyl-adenylates not identified","Link to translation/aaRS biology not tested"]},{"year":2012,"claim":"Established HINT1 as the causative gene for autosomal recessive axonal neuropathy with neuromyotonia.","evidence":"Linkage analysis and NGS across 33 families identifying 8 mutations","pmids":["22961002"],"confidence":"High","gaps":["Which HINT1 molecular function underlies neuropathy not resolved by genetics","Cell-type-specific disease mechanism unknown"]},{"year":2013,"claim":"Showed HINT1 functionally constrains NMDAR activity in concert with GPCRs in neurons, with viral rescue restoring control.","evidence":"HINT1-/- and CNR1-/- cortical neurons, lentiviral rescue, excitotoxicity and electrophysiology","pmids":["24093505"],"confidence":"High","gaps":["Molecular bridge between GPCR-HINT1 and NMDAR not fully mapped","Generalizability across GPCR types untested here"]},{"year":2015,"claim":"Defined a redox/zinc switch by which σ1R is recruited to MOR-HINT1 complexes to restrain opioid signaling and NMDAR coupling.","evidence":"Brain Co-IP, ICV pharmacology, σ1R-/- and HINT1-/- mice, NOS inhibition","pmids":["25557043","26461475"],"confidence":"High","gaps":["Atomic-level interface of σ1R-HINT1-NR1 unknown","Quantitative thresholds of the zinc switch undefined"]},{"year":2015,"claim":"Identified teneurin-1 ICD as a HINT1-binding partner that titrates HINT1 away from MITF to derepress targets like GPNMB.","evidence":"Yeast two-hybrid, Co-IP in human cells, transcriptomics, and promoter reporters","pmids":["25648896"],"confidence":"Medium","gaps":["Single-lab interaction without structural mapping","Physiological contexts of teneurin-HINT1 competition unclear"]},{"year":2015,"claim":"Linked HINT1 enzymatic active site to opioid pharmacology, showing an active-site inhibitor enhances analgesia and prevents tolerance.","evidence":"In vivo pharmacology with HINT1 inhibitor TpGc, antinociception and neuropathic pain assays","pmids":["25445489"],"confidence":"Medium","gaps":["Enzymatic dependency inferred from inhibitor, not genetic active-site mutant in vivo","Single lab"]},{"year":2017,"claim":"Connected HINT1 PTMs (K21 acetylation, Y109 phosphorylation) and Ap4A signaling to switching MITF oncogenic activity on.","evidence":"Mass spectrometry of PTMs in mast cells, mutagenesis, and melanoma reporter assays","pmids":["28394346"],"confidence":"Medium","gaps":["Writers/erasers for Y109 not identified","In vivo relevance of PTM switch untested"]},{"year":2019,"claim":"Proposed a SUMO isopeptidase activity for HINT1 gated by zinc/NO/CaM and altered in all tested ARAN-NM mutants.","evidence":"In vitro sumoylase assay, catalytic-triad mutagenesis, pharmacological gating, and disease-mutant panel","pmids":["31088288"],"confidence":"Medium","gaps":["Novel activity from a single lab awaits independent confirmation","In vivo SUMO substrates not identified"]},{"year":2019,"claim":"Demonstrated structurally that Ap4A drives HINT1 polymerization through an interface overlapping the MITF-binding surface, providing a competitive derepression mechanism.","evidence":"Eight crystal structures, negative-stain EM, polymerization assays, and RBL cell experiments","pmids":["31604935"],"confidence":"High","gaps":["Cellular triggers of Ap4A accumulation for polymerization not delineated","Reversibility kinetics in vivo unclear"]},{"year":2019,"claim":"Showed distinct HINT1 active-site inhibitors yield different effects on opioid tolerance versus NMDAR cross-talk, tied to differential binding surfaces.","evidence":"Crystallography of HINT1-inhibitor complexes, ITC, and spinal in vivo pharmacology","pmids":["31503445"],"confidence":"Medium","gaps":["Structural basis for divergent behavioral outcomes incompletely defined","Single lab"]},{"year":2020,"claim":"Identified CBP/SIRT1 as writer/eraser at K21/K30 controlling HINT1's affinity for β-catenin/MITF and its tumor-suppressive output.","evidence":"Co-IP, acetylation assays, K21R/K30R mutagenesis, binding assays, and xenografts","pmids":["32636443"],"confidence":"High","gaps":["How acetylation alters the binding surface structurally unknown","Upstream signals regulating SIRT1/CBP on HINT1 undefined"]},{"year":2021,"claim":"Defined a cardioprotective HINT1 axis that blocks PKCβ1 membrane translocation to suppress MEK/ERK/YY1/HOXA5-driven hypertrophy.","evidence":"Co-IP, fractionation, Hint1 KO and AAV9-HINT1 mice with TAC, RNA-seq, and AAV9-shHoxa5 rescue","pmids":["34098726"],"confidence":"High","gaps":["Direct HINT1-PKCβ1 binding interface not mapped","Whether enzymatic activity contributes untested"]},{"year":2021,"claim":"Showed HINT1/σ1R control α2δ1 incorporation into NMDAR complexes, linking the scaffold to neuropathic allodynia.","evidence":"PAG tissue Co-IP, σ1R-/- and HINT1-/- mice with constriction injury, and in vivo pharmacology","pmids":["34827679"],"confidence":"Medium","gaps":["Single-lab series extension","Structural arrangement of the α2δ1-σ1R-NR1-HINT1 assembly unknown"]},{"year":2022,"claim":"Extended HINT1 catalysis to selenophosphate nucleotides, generating cytotoxic H2Se in cancer cells.","evidence":"HPLC/fluorescence enzymatic assays, viability assays, and intracellular H2Se detection","pmids":["35054788"],"confidence":"Medium","gaps":["Endogenous selenophosphate substrate existence unknown","Therapeutic exploitation untested in vivo"]},{"year":null,"claim":"How HINT1's conserved enzymatic activities mechanistically connect to its scaffolding and transcriptional-repressor roles—and which function underlies ARAN-NM—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No endogenous physiological substrate has been definitively assigned to the catalytic activity","The molecular basis by which loss-of-function mutations cause peripheral neuropathy is unestablished","Whether the proposed SUMO isopeptidase activity is independently reproducible is open"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,4,11,14,29]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[11,29]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[5,6,13]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,6,7,13,10]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[9,18,19,25]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[22]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[26]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,6,13]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[9,18]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[26]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,6,7,13,10]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,12,18,19,25]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[16,19,27]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[6]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[15]}],"complexes":["Tip60 histone acetyltransferase complex","mSIN3a/HDAC1 corepressor complex","MOR-HINT1-RGSZ scaffold"],"partners":["RGSZ2","PKCG","RAF1","SIGMAR1","CTNNB1","MITF","RUVBL1","CDK7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P49773","full_name":"Adenosine 5'-monophosphoramidase HINT1","aliases":["Desumoylating isopeptidase HINT1","Histidine triad nucleotide-binding protein 1","Protein kinase C inhibitor 1","Protein kinase C-interacting protein 1","PKCI-1"],"length_aa":126,"mass_kda":13.8,"function":"Exhibits adenosine 5'-monophosphoramidase activity, hydrolyzing purine nucleotide phosphoramidates with a single phosphate group such as adenosine 5'monophosphoramidate (AMP-NH2) to yield AMP and NH2 (PubMed:15703176, PubMed:16835243, PubMed:17217311, PubMed:17337452, PubMed:22329685, PubMed:23614568, PubMed:28691797, PubMed:29787766, PubMed:31990367). Hydrolyzes adenosine 5'monophosphomorpholidate (AMP-morpholidate) and guanosine 5'monophosphomorpholidate (GMP-morpholidate) (PubMed:15703176, PubMed:16835243). Hydrolyzes lysyl-AMP (AMP-N-epsilon-(N-alpha-acetyl lysine methyl ester)) generated by lysine tRNA ligase, as well as Met-AMP, His-AMP and Asp-AMP, lysyl-GMP (GMP-N-epsilon-(N-alpha-acetyl lysine methyl ester)) and AMP-N-alanine methyl ester (PubMed:15703176, PubMed:17337452, PubMed:22329685). Hydrolyzes 3-indolepropionic acyl-adenylate, tryptamine adenosine phosphoramidate monoester and other fluorogenic purine nucleoside tryptamine phosphoramidates in vitro (PubMed:17217311, PubMed:17337452, PubMed:23614568, PubMed:28691797, PubMed:29787766, PubMed:31990367). Can also convert adenosine 5'-O-phosphorothioate and guanosine 5'-O-phosphorothioate to the corresponding nucleoside 5'-O-phosphates with concomitant release of hydrogen sulfide (PubMed:30772266). In addition, functions as scaffolding protein that modulates transcriptional activation by the LEF1/TCF1-CTNNB1 complex and by the complex formed with MITF and CTNNB1 (PubMed:16014379, PubMed:22647378). Modulates p53/TP53 levels and p53/TP53-mediated apoptosis (PubMed:16835243). Modulates proteasomal degradation of target proteins by the SCF (SKP2-CUL1-F-box protein) E3 ubiquitin-protein ligase complex (PubMed:19112177). Also exhibits SUMO-specific isopeptidase activity, deconjugating SUMO1 from RGS17 (PubMed:31088288). Deconjugates SUMO1 from RANGAP1 (By similarity)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/P49773/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HINT1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SAR1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/HINT1","total_profiled":1310},"omim":[{"mim_id":"609998","title":"HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN 3; HINT3","url":"https://www.omim.org/entry/609998"},{"mim_id":"609997","title":"HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN 2; HINT2","url":"https://www.omim.org/entry/609997"},{"mim_id":"601314","title":"HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN 1; HINT1","url":"https://www.omim.org/entry/601314"},{"mim_id":"137200","title":"NEUROMYOTONIA AND AXONAL NEUROPATHY, AUTOSOMAL RECESSIVE; NMAN","url":"https://www.omim.org/entry/137200"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HINT1"},"hgnc":{"alias_symbol":["PKCI-1"],"prev_symbol":["PRKCNH1","HINT"]},"alphafold":{"accession":"P49773","domains":[{"cath_id":"3.30.428.10","chopping":"1-126","consensus_level":"medium","plddt":96.3135,"start":1,"end":126}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P49773","model_url":"https://alphafold.ebi.ac.uk/files/AF-P49773-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P49773-F1-predicted_aligned_error_v6.png","plddt_mean":96.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HINT1","jax_strain_url":"https://www.jax.org/strain/search?query=HINT1"},"sequence":{"accession":"P49773","fasta_url":"https://rest.uniprot.org/uniprotkb/P49773.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P49773/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P49773"}},"corpus_meta":[{"pmid":"22846459","id":"PMC_22846459","title":"HINT: High-quality 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These structures revealed that HINT shares the same fold and mode of nucleotide binding as galactose-1-phosphate uridylyltransferase (GalT) despite no overall sequence similarity.\",\n      \"method\": \"X-ray crystallography of HINT-nucleotide complexes\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct crystal structures with functional interpretation, foundational structural paper replicated across the field\",\n      \"pmids\": [\"9164465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Rabbit HINT1 and yeast Hnt1 hydrolyze adenosine-5'-monophosphoramidate (AMP-NH2) as natural substrates in an active-site-dependent manner, with second-order rates exceeding 1,000,000 M−1 s−1. Loss of Hnt1 enzyme activity in yeast leads to failure to grow on galactose at elevated temperature and hypersensitivity to mutations in Kin28, Ccl1, and Tfb3 (the TFIIK kinase subcomplex of TFIIH), placing Hnt1 as a positive regulator of this transcription kinase pathway downstream of Cak1.\",\n      \"method\": \"In vitro enzymatic assay with AMP-NH2; yeast genetic epistasis with temperature-sensitive alleles and deletion strains; functional complementation with rabbit Hint\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinetic assay plus multi-allele genetic epistasis; rabbit Hint complementation confirms conservation\",\n      \"pmids\": [\"11805111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"HINT belongs to the adenosine 5'-monophosphoramide hydrolase branch of the HIT superfamily; a new enzyme mechanism was proposed for Hint and Fhit based on comparative structural and biochemical analysis of the three HIT branches (Hint, Fhit, GalT).\",\n      \"method\": \"Biochemical and structural comparative analysis; sequence/substrate specificity review with mechanistic inference\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — comprehensive review integrating structural and biochemical data from multiple sources, but primarily a review paper synthesizing existing experimental data\",\n      \"pmids\": [\"12119013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"HINT/PKCI-1 physically interacts with Cdk7; this interaction was identified by yeast two-hybrid and confirmed by co-immunoprecipitation. Overexpression of Cdk7 leads to partial relocalization of Hint to the nucleus. The interaction is independent of cyclin H binding or Cdk7 kinase activity. The orthologous interaction between yeast Kin28 and Hnt1 is conserved, and genetic combination of hnt1 deletion with a kin28 temperature-sensitive allele causes elongated cell morphology and reduced colony formation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, subcellular localization by fluorescence microscopy, yeast genetic interaction (double mutant analysis)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus yeast genetic epistasis plus localization data, replicated across two organisms\",\n      \"pmids\": [\"10958787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Biochemical and structural characterization of HINT enzymatic activity as an AMP-lysine (adenosine phosphoramidate) hydrolase. The 1.8-Å co-crystal structure with N-ethylsulfamoyl adenosine revealed a binding site for the alkyl group against Trp-123 across the dimer interface. Ser-107 donates a hydrogen bond to the leaving-group nitrogen; the S107A mutant displays markedly depressed catalytic activity, confirming a role for Ser-107 in acid-base catalysis. A novel substrate AMP-pNA and inhibitors (AdoOSO2NH2, AdoOSO2NHCH2CH3) were characterized kinetically.\",\n      \"method\": \"X-ray crystallography (1.8 Å co-crystal), active-site mutagenesis (S107A), in vitro kinetic assay with novel substrates and inhibitors\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro kinetic assays in a single rigorous study\",\n      \"pmids\": [\"14982931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"HINT1/PKCI-1 directly binds to Pontin and Reptin; pull-down and co-immunoprecipitation experiments mapped the HINT1 binding site to amino acids 214–295 of Pontin and 218–289 of Reptin, while Pontin and Reptin bind the N-terminus of HINT1. Through this interaction, HINT1 associates with the LEF-1/TCF–β-catenin transcription complex and acts as a negative regulator of TCF–β-catenin transcriptional activity, repressing cyclin D1 and axin2 expression. RNAi knockdown of HINT1 increases these target genes.\",\n      \"method\": \"Pull-down assay, co-immunoprecipitation, reporter gene assay (TCF-luciferase), RNAi knockdown with RT-PCR/qPCR\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with domain mapping, orthogonal reporter assays, and RNAi phenotype in multiple cell contexts\",\n      \"pmids\": [\"16014379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"HINT1 overexpression induces apoptosis in SW480 and MCF-7 cells via upregulation of p53 and proapoptotic Bax and downregulation of Bcl-2. HINT1 associates with the Bax promoter and is a component of the Tip60 histone acetyltransferase complex. A catalytically inactive mutant HINT1 (H112N) retains full pro-apoptotic activity, demonstrating that the apoptotic function is independent of AMP-NH2 hydrolase enzymatic activity.\",\n      \"method\": \"Transient transfection, caspase-3/PARP cleavage assay, cytochrome c release, DNA fragmentation ELISA, shRNA knockdown, chromatin immunoprecipitation (ChIP), co-immunoprecipitation with Tip60 complex, active-site mutant (H112N)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ChIP, Co-IP, mutant analysis, KD), single lab but rigorous experimental design\",\n      \"pmids\": [\"16835243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HINT1 inhibits AP-1 transcriptional activity in colon cancer cells by forming an in vivo complex with POSH (plenty of SH3) and JNK2, thereby inhibiting phosphorylation of c-Jun. This pathway requires JNK2 but not JNK1, confirmed in JNK1-/- and JNK2-/- MEFs. Both wild-type HINT1 and the enzymatically inactive H112N mutant inhibit AP-1 activity equivalently, indicating the inhibition is independent of enzymatic function.\",\n      \"method\": \"Co-immunoprecipitation (HINT1-POSH-JNK complex), reporter gene assay (AP-1-luciferase), JNK1-/- and JNK2-/- MEF epistasis, phospho-c-Jun western blot, retroviral overexpression\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP of endogenous complex, genetic epistasis using KO MEFs, and enzymatic mutant confirming mechanism-independent activity\",\n      \"pmids\": [\"17510397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HINT1 participates in ionizing radiation (IR)-induced DNA damage responses: it is recruited to IR-induced foci (IRIF) and co-associates with γ-H2AX and ATM. HINT1 deficiency does not prevent γ-H2AX foci formation but impairs their removal and impairs acetylation of γ-H2AX. HINT1 deficiency also impairs acetylation and activation of ATM and retards DNA repair; HINT1-deficient cells resist IR-induced apoptosis and exhibit chromosomal abnormalities.\",\n      \"method\": \"Immunofluorescence (IRIF recruitment), co-immunoprecipitation (HINT1-γ-H2AX-ATM), acetylation assays, DNA repair kinetics, apoptosis assays in Hint1-/- cells vs. WT\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including Co-IP, immunofluorescence, functional assays in KO cells\",\n      \"pmids\": [\"18852295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In neurons, the C-terminus of the mu-opioid receptor (MOR) binds PKCI/HINT1, which in turn binds the regulator of G-protein signaling RGSZ1/Z2 proteins. Morphine administration recruits PKCγ (mostly) to the MOR via the HINT1/RGSZ complex. This recruitment requires zinc ions, HINT1, and RGSZ proteins, and specifically involves the cysteine-rich domains (CRDs) of PKCγ. NMDAR antagonist MK801 and NOS inhibition prevent PKCγ recruitment, implicating the NMDAR/nNOS cascade in providing zinc ions required for PKCγ to bind the HINT1/RGSZ complex at MOR.\",\n      \"method\": \"Co-immunoprecipitation from mouse brain; intracerebroventricular drug administration; siRNA knockdown of HINT1; zinc chelation (TPEN); phorbol ester and NO donor treatments\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo and ex vivo Co-IP experiments, pharmacological dissection with multiple reagents, and knockdown controls across multiple studies\",\n      \"pmids\": [\"18652891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"HINT1 co-immunoprecipitates with USF2 in hepatoma cell extracts and inhibits transcriptional activities of β-catenin/TCF4, USF2, and NF-κB in HepG2 cells. HINT1 overexpression inhibits nuclear translocation of p65 (NF-κB subunit) and reduces expression of cyclin D1 and TGFβ2.\",\n      \"method\": \"Co-immunoprecipitation, reporter gene assays (β-catenin/TCF4, USF2, NF-κB luciferase), nuclear fractionation, western blot\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and reporter assays in single lab, orthogonal methods but limited mechanistic depth\",\n      \"pmids\": [\"19089909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HINT1 (Hint-1) phosphoramidase catalyzes the desulfuration of nucleoside 5'-O-phosphorothioates ((d)NMPS) to the corresponding nucleoside 5'-O-phosphates with release of hydrogen sulfide. Crystallographic analysis (1.08–1.37 Å) of three engineered cysteine mutants confirmed minimal structural perturbation. Substrate specificity order was determined: GMPS > AMPS > dGMPS ≥ CMPS > UMPS > dAMPS >> dCMPS > TMPS.\",\n      \"method\": \"In vitro enzymatic assay (HPLC-based), active-site mutagenesis, X-ray crystallography (1.08–1.37 Å resolution) of mutant structures\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus in vitro kinetic assays plus mutagenesis in a single rigorous study\",\n      \"pmids\": [\"20940308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Morphine-generated nitric oxide (NO) causes release of endogenous zinc ions that recruit the Ras/Raf-1/ERK1/2 cassette to HINT1 at the MOR C-terminus; zinc bridges the Raf-1 cysteine-rich domain (CRD) with HINT1. Simultaneously, PKCγ is also recruited via NO/zinc to the MOR-HINT1 complex; Raf-1 and PKCγ CRDs bind simultaneously to HINT1, enabling PKCγ to enhance Raf-1 function and thereby MEK/ERK1/2 activation. A-Raf and B-Raf show little or no MOR association.\",\n      \"method\": \"Co-immunoprecipitation from mouse brain, pharmacological dissection (NOS inhibitors, zinc chelator TPEN), intracerebroventricular injection paradigms\",\n      \"journal\": \"Antioxidants & redox signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP from brain tissue with pharmacological controls; single lab, multiple experiments\",\n      \"pmids\": [\"21235400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"HINT1 co-immunoprecipitates with MITF and β-catenin in melanoma cell lines. HINT1 inhibits MITF and β-catenin transcriptional activity, reduces cyclin D1 and BCL2 expression (known MITF and β-catenin targets), and ChIP assays demonstrate HINT1 occupancy at MITF and β-catenin binding sites in BCL2 and cyclin D1 promoters. HINT1 co-immunoprecipitates with mSIN3a and HDAC1, suggesting recruitment of a repressor complex.\",\n      \"method\": \"Co-immunoprecipitation, ChIP assay, reporter gene assay, stable HINT1 overexpression with xenograft, rescue by BCL2/cyclin D1 overexpression\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP plus Co-IP of repressor complex plus functional rescue experiments, multiple orthogonal methods\",\n      \"pmids\": [\"22647378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human HINT1 has broad specificity for aminoacyl adenylate (aa-AMP) hydrolysis, recognizing only the common main-chain of the aminoacyl moiety and having no contact with the amino acid side chain. High-resolution crystal structures of HINT1 with three different aa-AMP analogues revealed that the α-amino group is anchored by a cation-π interaction with Trp123 at the C-terminus.\",\n      \"method\": \"X-ray crystallography (HINT1 with aa-AMP analogues), in vitro hydrolysis assays\",\n      \"journal\": \"The journal of physical chemistry B\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures with functional biochemical validation in a single study\",\n      \"pmids\": [\"22329685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Loss-of-function mutations in HINT1 cause autosomal recessive axonal neuropathy with neuromyotonia, identified by combining linkage analysis and next-generation sequencing in 33 families with 8 distinct mutations.\",\n      \"method\": \"Linkage analysis, next-generation sequencing, cohort mutation screening\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — large multi-family genetic study with multiple mutations independently identified; replicated in subsequent studies\",\n      \"pmids\": [\"22961002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HINT1 protein influences NMDAR activity in the presence of GPCRs: in CNR1+/+/HINT1-/- cortical neurons, NMDAR activation was enhanced and cannabinoid agonist WIN55,212-2 failed to protect against NMDA insult. Lentiviral re-expression of HINT1 normalized NMDAR activity and restored cannabinoid-mediated neuroprotection. In the absence of receptor activation, GPCRs collaborate with HINT1 to negatively control NMDAR activity.\",\n      \"method\": \"HINT1-/- and CNR1-/- mouse cortical neuron culture, lentiviral HINT1 rescue, NMDA excitotoxicity assay, electrophysiology\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO neurons with viral rescue, multiple receptor and KO combinations, functional neuroprotection readout\",\n      \"pmids\": [\"24093505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The teneurin-1 intracellular domain (ICD) binds HINT1 (identified by yeast two-hybrid and confirmed in human cells), disrupting HINT1-mediated repression of MITF transcriptional targets. Teneurin-1 ICD overexpression upregulates MITF target genes including GPNMB; promoter reporter assays showed the teneurin-1 ICD switches on MITF-dependent GPNMB transcription by binding and titrating HINT1 away from MITF.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation in human cells, transcriptome analysis, RT-qPCR, promoter reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP in human cells plus functional reporter assays, single lab\",\n      \"pmids\": [\"25648896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The sigma 1 receptor (σ1R) engages HINT1 at the MOR C-terminus through a redox-regulated mechanism: activated MORs stimulate nitric oxide (NO) production; the RGSZ2 redox zinc switch converts this into free zinc ions that recruit PKCγ to HINT1 proteins, impairing HINT1-RGSZ2 association and enabling σ1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid signaling. σ1R antagonists remove σ1R from NR1, facilitate Ca2+-CaM entry, and prevent NR1-HINT1 interaction, blocking the negative feedback on opioid analgesia.\",\n      \"method\": \"Co-immunoprecipitation from mouse brain, intracerebroventricular pharmacology, σ1R-/- and HINT1-/- mice, NOS inhibition, in vitro binding assays\",\n      \"journal\": \"Antioxidants & redox signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple KO models, pharmacological dissection, and Co-IP with multiple orthogonal manipulations\",\n      \"pmids\": [\"25557043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HINT1 and σ1R coordinate GPCR-NMDAR interactions: HINT1 binds GPCRs and NMDAR NR1 subunits in a calcium-independent manner, while σ1R binding to these proteins increases with calcium. σ1R agonists retain HINT1 at the GPCR and promote GPCR-NMDAR interaction; σ1R antagonists transfer HINT1 to NR1 subunits and disengage GPCRs from NMDARs. In σ1R-/- mice, HINT1 proteins mostly associate with NMDARs and GPCRs are functionally disconnected from NMDARs. In HINT1-/- mice, ischemia produces reduced NMDAR-mediated brain damage.\",\n      \"method\": \"Co-immunoprecipitation, in vivo pharmacology, σ1R-/- and HINT1-/- mice, ischemia model, electrophysiology\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two genetic KO models with biochemical Co-IP and in vivo functional paradigms\",\n      \"pmids\": [\"26461475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The HINT1 enzymatic active site is critical for its regulatory role at the MOR-NMDAR interface; the HINT1 inhibitor guanosine-5'-tryptamine carbamate (TpGc) significantly enhances morphine antinociception, prevents tolerance development, and reduces MOR recruitment of NMDAR activity. Intracerebroventricular TpGc also attenuates NMDAR function and alleviates mechanical allodynia in chronic constriction injury mice.\",\n      \"method\": \"In vivo pharmacology with HINT1 active-site inhibitor, antinociception assays, mouse model of neuropathic pain, molecular interaction assays\",\n      \"journal\": \"Neuropharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition with in vivo behavioral readouts; enzymatic dependency inferred from active-site inhibitor; single lab\",\n      \"pmids\": [\"25445489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HINT1 undergoes post-translational modifications in activated mast cells: K21 acetylation and Y109 phosphorylation. Mutational analysis confirmed that these modifications promote MITF transcriptional and oncogenic activity in melanoma cell lines, linked to the Ap4A-triggered dissociation of HINT1 from MITF. The LysRS-Ap4A-HINT1-MITF signaling pathway can be modulated through HINT1 PTMs to affect MITF-driven transcription.\",\n      \"method\": \"Mass spectrometry identification of PTMs in activated mast cells, site-directed mutagenesis (K21, Y109), reporter assays in melanoma cell lines\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PTMs identified by MS and confirmed by mutagenesis with functional reporter readout; single lab\",\n      \"pmids\": [\"28394346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HINT1 exhibits zinc- and redox-regulated cysteine SUMO isopeptidase (sumoylase) activity, removing SUMO from signaling proteins. The catalytic triad Cys84-Asp87-His114 in the C-terminal region and a SUMO-interacting motif (residues 110-116 HIHLHVL) were identified. HINT1 sumoylase activity is blocked by zinc and released by nitric oxide or calcium-activated calmodulin (CaM). All 15 human HINT1 mutants causing ARAN-NM showed altered sumoylase activity.\",\n      \"method\": \"In vitro sumoylase assay, site-directed mutagenesis of catalytic triad, zinc/NO/CaM pharmacological manipulation, analysis of 15 disease-causing mutants\",\n      \"journal\": \"Antioxidants & redox signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzymatic assay with mutagenesis and pharmacological controls; novel activity claim from single lab\",\n      \"pmids\": [\"31088288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Ap4A specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. Eight crystal structures revealed that Ap4A binding drives HINT1 polymerization; the polymerization interface overlaps with the HINT1-MITF interaction surface, suggesting a competitive mechanism to release MITF for transcriptional activation. The polymerization depends precisely on the length of the phosphodiester linkage of Ap4A.\",\n      \"method\": \"X-ray crystallography (8 structures), negative stain electron microscopy, biochemical polymerization assays, cellular experiments in RBL cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — eight crystal structures combined with EM and cellular biochemical assays in a single rigorous study\",\n      \"pmids\": [\"31604935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HINT1 is acetylated by CBP at K21 and K30 and deacetylated by SIRT1. Deacetylation of HINT1 by SIRT1 increases the binding capacity of HINT1 for β-catenin or MITF, enhancing its tumor-suppressive function. The deacetylation-mimetic double mutant HINT1-2KR (K21R/K30R) significantly reduced cellular proliferation in colon cancer and melanoma cells in both cell-based and xenograft assays.\",\n      \"method\": \"Co-immunoprecipitation (HINT1 with CBP/SIRT1), acetylation assays, site-directed mutagenesis (K21R, K30R), in vitro binding assays, xenograft tumor assays\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — writer (CBP) and eraser (SIRT1) identified for specific sites, confirmed by mutagenesis and functional in vivo xenograft rescue\",\n      \"pmids\": [\"32636443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HINT1 inhibits PKCβ1 membrane translocation and phosphorylation via direct interaction, attenuating the MEK/ERK/YY1 signaling pathway and thereby downregulating HOXA5 expression. HOXA5, in turn, affects hypertrophy through the TGF-β signaling pathway. In Hint1-deficient mice, cardiac hypertrophy was exacerbated after pressure overload; cardiac-specific HINT1 overexpression (via AAV9) alleviated it. The cardioprotective role of HINT1 was abolished by HOXA5 knockdown in vivo.\",\n      \"method\": \"Co-immunoprecipitation (HINT1-PKCβ1), cellular fractionation (PKCβ1 membrane translocation assay), Hint1 KO and AAV9-HINT1 OE mice with transverse aortic constriction, RNA sequencing, PCR array, AAV9-shHoxa5 in vivo\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP with fractionation, multiple genetic models (KO + OE), in vivo pathway rescue by target KD, orthogonal transcriptomics\",\n      \"pmids\": [\"34098726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Human PKCI-1 (HINT1/PRKCNH1) localizes to cytoskeletal structures in the cytoplasm of human fibroblast cell lines by indirect immunofluorescence and is largely excluded from the nucleus, consistent with a role in mediating membrane-derived signals.\",\n      \"method\": \"Indirect immunofluorescence in human fibroblast cell line\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment replicated in later studies; functional link speculative at time\",\n      \"pmids\": [\"8812426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HINT1 and σ1R control α2δ1 binding to NMDAR NR1 subunits: α2δ1 peptides require σ1Rs to interact with the NMDAR NR1 C1 segment. σ1R antagonists or low calcium dissociate σ1R-NR1 C1 dimers. HINT1 removes α2δ1 from the δ1-σ1R-NR1 trimer, facilitating σ1R dissociation from NMDARs. In σ1R-/- mice, sciatic nerve injury does not promote NMDAR-α2δ1 complex formation and allodynia does not develop; in HINT1-/- mice, α2δ1-σ1R-NMDAR complexes are increased and allodynia is exacerbated.\",\n      \"method\": \"Co-immunoprecipitation from PAG tissue, σ1R-/- and HINT1-/- mice with chronic constriction injury model, in vivo pharmacology\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two KO models with Co-IP and behavioral readouts; single lab building on prior series\",\n      \"pmids\": [\"34827679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Spinal inhibition of HINT1 enzymatic activity by active-site inhibitors (TrpGc vs. acyl-sulfamate analogues) differentially affects MOR-NMDAR cross-talk: TrpGc blocked both the development of opioid tolerance and the inhibitory effect of opioids on NMDA activation, while acyl-sulfamate analogues could only block the latter. X-ray crystallographic and thermodynamic binding studies revealed key differences between bound HINT1-inhibitor surfaces that may account for their distinct pharmacological profiles.\",\n      \"method\": \"X-ray crystallography of HINT1-inhibitor complexes, isothermal titration calorimetry, in vivo spinal pharmacology, opioid tolerance behavioral assay\",\n      \"journal\": \"ACS chemical neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures plus in vivo pharmacology; single lab but two orthogonal methods\",\n      \"pmids\": [\"31503445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HINT1 catalyzes the hydrolysis of deoxyguanosine-5'-O-selenophosphate (dGMPSe) with similar kcat and Km to the corresponding thiophosphate (dGMPS), releasing hydrogen selenide (H2Se). This H2Se release is toxic to HeLa cancer cells, demonstrating that HINT1 enzymatic activity toward selenophosphate nucleotides can generate cytotoxic H2Se intracellularly.\",\n      \"method\": \"HPLC-based and fluorescence-based enzymatic assay, MTT viability assay, fluorescence detection of H2Se in living cells, electroporation of substrate\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinetic assays plus cellular delivery and detection of product; single lab\",\n      \"pmids\": [\"35054788\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HINT1 is a universally conserved, dimeric adenosine phosphoramidase/AMP-lysine hydrolase (HIT superfamily) whose enzymatic active site (centered on the HIT motif, Ser107, Trp123, and catalytic triad Cys84-Asp87-His114) hydrolyzes AMP-phosphoramidate, aminoacyl-AMP, and nucleoside phosphorothioate substrates; independently of enzymatic activity, HINT1 functions as a scaffold at the C-terminus of GPCRs (particularly the mu-opioid receptor) where it recruits RGSZ2, PKCγ, Raf-1, and σ1R in a redox/zinc-dependent manner to mediate GPCR-NMDAR cross-regulation; HINT1 also suppresses oncogenic transcription by binding and co-repressing β-catenin/TCF, MITF, AP-1/JNK2, NF-κB, and USF2 complexes—a function regulated by SIRT1-mediated deacetylation at K21/K30 and disrupted by Ap4A-induced HINT1 polymerization; HINT1 additionally inhibits PKCβ1 membrane translocation to suppress the MEK/ERK/YY1/HOXA5 cardiac hypertrophy pathway and exhibits zinc/CaM-regulated SUMO isopeptidase activity; loss-of-function mutations cause autosomal recessive axonal neuropathy with neuromyotonia (ARAN-NM).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HINT1 is a universally conserved, dimeric purine nucleotide-binding enzyme of the HIT superfamily that doubles as a redox-regulated signaling scaffold and transcriptional co-repressor [#0, #1]. Its active site\\u2014built on the HIT phosphate-binding loop with Ser107 in acid-base catalysis and Trp123 anchoring substrate across the dimer interface\\u2014hydrolyzes adenosine 5'-monophosphoramidate, AMP-lysine, aminoacyl-adenylates, and nucleoside phosphorothioate/selenophosphate substrates, the latter releasing H2S or cytotoxic H2Se [#1, #4, #11, #14, #29]. Independently of catalysis, HINT1 acts as a tumor suppressor by binding and co-repressing oncogenic transcription factors including \\u03b2-catenin/TCF (via Pontin/Reptin), MITF, AP-1/JNK2, USF2, and NF-\\u03baB, recruiting mSIN3a/HDAC1 and Tip60 repressor/acetyltransferase complexes to target promoters such as cyclin D1, BCL2, and Bax; catalytically dead H112N mutants retain this activity [#5, #6, #7, #13]. This repressive function is tuned by post-translational control: SIRT1-mediated deacetylation at K21/K30 strengthens factor binding, while Ap4A-driven HINT1 polymerization competitively displaces MITF to relieve repression [#23, #24]. In neurons, HINT1 scaffolds the C-terminus of GPCRs\\u2014particularly the mu-opioid receptor\\u2014recruiting RGSZ proteins, PKC\\u03b3, Raf-1, and \\u03c31R through a NO/zinc redox switch to govern GPCR-NMDAR cross-regulation, opioid analgesia, and neuropathic pain [#9, #12, #18, #19]. HINT1 also restrains cardiac hypertrophy by blocking PKC\\u03b21 membrane translocation and the downstream MEK/ERK/YY1/HOXA5 axis [#25]. Loss-of-function mutations in HINT1 cause autosomal recessive axonal neuropathy with neuromyotonia [#15].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established the structural basis of HINT as a nucleotide-binding protein, defining the HIT motif's role and an unexpected fold relationship to GalT.\",\n      \"evidence\": \"X-ray crystallography of HINT-nucleotide complexes\",\n      \"pmids\": [\"9164465\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the physiological substrate\", \"Catalytic mechanism not resolved from binding alone\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identified the natural enzymatic activity (AMP-phosphoramidate hydrolysis) and placed HINT in a transcription-kinase regulatory pathway via yeast genetics.\",\n      \"evidence\": \"In vitro kinetics with AMP-NH2 plus yeast epistasis with TFIIK/Cak1 alleles and rabbit Hint complementation\",\n      \"pmids\": [\"11805111\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic substrate in transcription pathway not pinpointed\", \"Mechanism linking hydrolase activity to kinase regulation unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Linked HINT physically to the cell-cycle/transcription kinase Cdk7, showing a kinase-activity-independent interaction conserved to yeast Kin28.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, localization, and yeast double-mutant analysis\",\n      \"pmids\": [\"10958787\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Cdk7 binding for HINT unclear\", \"Mechanism of Cdk7-driven nuclear relocalization unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the catalytic machinery for AMP-lysine/phosphoramidate hydrolysis, assigning Ser107 to acid-base catalysis and Trp123 to substrate binding.\",\n      \"evidence\": \"1.8-\\u00c5 co-crystal structure, S107A mutagenesis, and kinetics with novel substrates/inhibitors\",\n      \"pmids\": [\"14982931\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo substrate of the AMP-lysine activity not established\", \"Physiological context of catalysis unaddressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealed HINT1's enzyme-independent role as a transcriptional repressor of \\u03b2-catenin/TCF signaling through Pontin/Reptin binding.\",\n      \"evidence\": \"Pull-down/Co-IP with domain mapping, TCF-luciferase reporters, and RNAi with target qPCR\",\n      \"pmids\": [\"16014379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether repression requires the catalytic site untested here\", \"Recruitment of corepressor machinery not yet defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated catalysis-independent pro-apoptotic/tumor-suppressor function through Bax promoter occupancy and Tip60 complex association.\",\n      \"evidence\": \"ChIP, Co-IP with Tip60, apoptosis assays, shRNA, and H112N catalytic mutant in cancer cells\",\n      \"pmids\": [\"16835243\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How HINT1 is recruited to the Bax promoter unknown\", \"Relationship between p53 induction and direct chromatin binding unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Extended HINT1 repression to AP-1 by showing it forms a POSH-JNK2 complex blocking c-Jun phosphorylation, JNK2-specifically.\",\n      \"evidence\": \"Endogenous Co-IP, AP-1 reporters, JNK1/JNK2 KO MEF epistasis, and H112N mutant\",\n      \"pmids\": [\"17510397\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of HINT1-POSH-JNK2 assembly unknown\", \"Why JNK2 but not JNK1 is targeted unexplained\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Placed HINT1 in the DNA damage response, showing it is needed for \\u03b3-H2AX/ATM acetylation and efficient repair.\",\n      \"evidence\": \"IRIF immunofluorescence, Co-IP with \\u03b3-H2AX/ATM, acetylation and repair kinetics in Hint1-/- cells\",\n      \"pmids\": [\"18852295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic contribution to acetylation events unclear\", \"Mechanism of HINT1 recruitment to damage foci unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined HINT1 as a scaffold at the mu-opioid receptor C-terminus that recruits PKC\\u03b3 via RGSZ proteins in a zinc/NMDAR-dependent manner.\",\n      \"evidence\": \"Brain Co-IP, ICV pharmacology, HINT1 siRNA, zinc chelation, and NOS/NMDAR inhibitors\",\n      \"pmids\": [\"18652891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural map of the MOR-HINT1-RGSZ interface absent\", \"Source/handling of recruited zinc not fully resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Generalized HINT1 transcriptional repression to USF2 and NF-\\u03baB, including blockade of p65 nuclear translocation.\",\n      \"evidence\": \"Co-IP, multiple reporter assays, and nuclear fractionation in hepatoma cells\",\n      \"pmids\": [\"19089909\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Co-IP/reporter without domain mapping\", \"Mechanism of p65 nuclear-translocation block undefined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Expanded the substrate repertoire to nucleoside phosphorothioates, showing HINT1 catalyzes desulfuration with H2S release.\",\n      \"evidence\": \"HPLC enzymatic assays, mutagenesis, and high-resolution crystallography of mutants\",\n      \"pmids\": [\"20940308\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of phosphorothioate desulfuration unknown\", \"In vivo H2S generation not demonstrated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed NO-released zinc recruits the Ras/Raf-1/ERK cassette to HINT1 at MOR alongside PKC\\u03b3, integrating two CRD-bearing effectors.\",\n      \"evidence\": \"Brain Co-IP with NOS inhibitors and zinc chelation, ICV paradigms\",\n      \"pmids\": [\"21235400\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab pharmacological dissection\", \"Stoichiometry of simultaneous Raf-1/PKC\\u03b3 binding unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified HINT1 as a melanoma tumor suppressor co-repressing MITF and \\u03b2-catenin via mSIN3a/HDAC1 recruitment at BCL2 and cyclin D1 promoters.\",\n      \"evidence\": \"Co-IP, ChIP, reporters, xenograft, and rescue by target overexpression\",\n      \"pmids\": [\"22647378\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of promoter selectivity unclear\", \"Whether the same complex operates on all repressed factors untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the structural basis for broad aminoacyl-adenylate hydrolysis, showing Trp123 cation-\\u03c0 anchoring of the \\u03b1-amino group.\",\n      \"evidence\": \"Crystal structures with aa-AMP analogues and in vitro hydrolysis assays\",\n      \"pmids\": [\"22329685\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular substrate among aminoacyl-adenylates not identified\", \"Link to translation/aaRS biology not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Established HINT1 as the causative gene for autosomal recessive axonal neuropathy with neuromyotonia.\",\n      \"evidence\": \"Linkage analysis and NGS across 33 families identifying 8 mutations\",\n      \"pmids\": [\"22961002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which HINT1 molecular function underlies neuropathy not resolved by genetics\", \"Cell-type-specific disease mechanism unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showed HINT1 functionally constrains NMDAR activity in concert with GPCRs in neurons, with viral rescue restoring control.\",\n      \"evidence\": \"HINT1-/- and CNR1-/- cortical neurons, lentiviral rescue, excitotoxicity and electrophysiology\",\n      \"pmids\": [\"24093505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular bridge between GPCR-HINT1 and NMDAR not fully mapped\", \"Generalizability across GPCR types untested here\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined a redox/zinc switch by which \\u03c31R is recruited to MOR-HINT1 complexes to restrain opioid signaling and NMDAR coupling.\",\n      \"evidence\": \"Brain Co-IP, ICV pharmacology, \\u03c31R-/- and HINT1-/- mice, NOS inhibition\",\n      \"pmids\": [\"25557043\", \"26461475\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-level interface of \\u03c31R-HINT1-NR1 unknown\", \"Quantitative thresholds of the zinc switch undefined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified teneurin-1 ICD as a HINT1-binding partner that titrates HINT1 away from MITF to derepress targets like GPNMB.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP in human cells, transcriptomics, and promoter reporters\",\n      \"pmids\": [\"25648896\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab interaction without structural mapping\", \"Physiological contexts of teneurin-HINT1 competition unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked HINT1 enzymatic active site to opioid pharmacology, showing an active-site inhibitor enhances analgesia and prevents tolerance.\",\n      \"evidence\": \"In vivo pharmacology with HINT1 inhibitor TpGc, antinociception and neuropathic pain assays\",\n      \"pmids\": [\"25445489\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Enzymatic dependency inferred from inhibitor, not genetic active-site mutant in vivo\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected HINT1 PTMs (K21 acetylation, Y109 phosphorylation) and Ap4A signaling to switching MITF oncogenic activity on.\",\n      \"evidence\": \"Mass spectrometry of PTMs in mast cells, mutagenesis, and melanoma reporter assays\",\n      \"pmids\": [\"28394346\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Writers/erasers for Y109 not identified\", \"In vivo relevance of PTM switch untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Proposed a SUMO isopeptidase activity for HINT1 gated by zinc/NO/CaM and altered in all tested ARAN-NM mutants.\",\n      \"evidence\": \"In vitro sumoylase assay, catalytic-triad mutagenesis, pharmacological gating, and disease-mutant panel\",\n      \"pmids\": [\"31088288\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Novel activity from a single lab awaits independent confirmation\", \"In vivo SUMO substrates not identified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated structurally that Ap4A drives HINT1 polymerization through an interface overlapping the MITF-binding surface, providing a competitive derepression mechanism.\",\n      \"evidence\": \"Eight crystal structures, negative-stain EM, polymerization assays, and RBL cell experiments\",\n      \"pmids\": [\"31604935\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular triggers of Ap4A accumulation for polymerization not delineated\", \"Reversibility kinetics in vivo unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed distinct HINT1 active-site inhibitors yield different effects on opioid tolerance versus NMDAR cross-talk, tied to differential binding surfaces.\",\n      \"evidence\": \"Crystallography of HINT1-inhibitor complexes, ITC, and spinal in vivo pharmacology\",\n      \"pmids\": [\"31503445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for divergent behavioral outcomes incompletely defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified CBP/SIRT1 as writer/eraser at K21/K30 controlling HINT1's affinity for \\u03b2-catenin/MITF and its tumor-suppressive output.\",\n      \"evidence\": \"Co-IP, acetylation assays, K21R/K30R mutagenesis, binding assays, and xenografts\",\n      \"pmids\": [\"32636443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How acetylation alters the binding surface structurally unknown\", \"Upstream signals regulating SIRT1/CBP on HINT1 undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a cardioprotective HINT1 axis that blocks PKC\\u03b21 membrane translocation to suppress MEK/ERK/YY1/HOXA5-driven hypertrophy.\",\n      \"evidence\": \"Co-IP, fractionation, Hint1 KO and AAV9-HINT1 mice with TAC, RNA-seq, and AAV9-shHoxa5 rescue\",\n      \"pmids\": [\"34098726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct HINT1-PKC\\u03b21 binding interface not mapped\", \"Whether enzymatic activity contributes untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed HINT1/\\u03c31R control \\u03b12\\u03b41 incorporation into NMDAR complexes, linking the scaffold to neuropathic allodynia.\",\n      \"evidence\": \"PAG tissue Co-IP, \\u03c31R-/- and HINT1-/- mice with constriction injury, and in vivo pharmacology\",\n      \"pmids\": [\"34827679\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab series extension\", \"Structural arrangement of the \\u03b12\\u03b41-\\u03c31R-NR1-HINT1 assembly unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended HINT1 catalysis to selenophosphate nucleotides, generating cytotoxic H2Se in cancer cells.\",\n      \"evidence\": \"HPLC/fluorescence enzymatic assays, viability assays, and intracellular H2Se detection\",\n      \"pmids\": [\"35054788\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous selenophosphate substrate existence unknown\", \"Therapeutic exploitation untested in vivo\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HINT1's conserved enzymatic activities mechanistically connect to its scaffolding and transcriptional-repressor roles\\u2014and which function underlies ARAN-NM\\u2014remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No endogenous physiological substrate has been definitively assigned to the catalytic activity\", \"The molecular basis by which loss-of-function mutations cause peripheral neuropathy is unestablished\", \"Whether the proposed SUMO isopeptidase activity is independently reproducible is open\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 4, 11, 14, 29]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [11, 29]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [5, 6, 13]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 6, 7, 13, 10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [9, 18, 19, 25]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [26]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 6, 13]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 18]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [26]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 6, 7, 13, 10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 12, 18, 19, 25]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [16, 19, 27]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"complexes\": [\"Tip60 histone acetyltransferase complex\", \"mSIN3a/HDAC1 corepressor complex\", \"MOR-HINT1-RGSZ scaffold\"],\n    \"partners\": [\"RGSZ2\", \"PKCG\", \"RAF1\", \"SIGMAR1\", \"CTNNB1\", \"MITF\", \"RUVBL1\", \"CDK7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}