{"gene":"SLC10A2","run_date":"2026-06-10T07:46:32","timeline":{"discoveries":[{"year":1997,"finding":"Loss-of-function mutations in SLC10A2 (L243P, T262M, and double mutant L243P/T262M) abolish taurocholate and bile acid transport without affecting transporter protein expression or trafficking to the plasma membrane, establishing that these residues are critical for transport activity. A171S had no effect on transport. A frame-shift mutation (646insG) also abolished transport in transfected COS cells.","method":"Site-directed mutagenesis, transfection in COS cells, taurocholate uptake assays, protein expression/trafficking analysis","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro transport assay with mutagenesis, multiple alleles tested, disease-associated variants validated functionally","pmids":["9109432"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of the bacterial homologue of ASBT from Neisseria meningitidis (ASBT_NM) at 2.2 Å reveals two inverted structural repeats of five transmembrane helices, a core domain harboring two sodium ions, and a 'panel' domain. Taurocholate substrate is bound between the core and panel domains in an inward-facing hydrophobic cavity, suggesting a transport mechanism involving movements between these domains.","method":"X-ray crystallography at 2.2 Å resolution with substrate taurocholate co-crystallized","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with substrate bound, bacterial homologue with confirmed structural homology to human ASBT","pmids":["21976025"],"is_preprint":false},{"year":2004,"finding":"Membrane topology of hASBT determined by N-glycosylation scanning mutagenesis supports a 7-transmembrane domain configuration, with extracellular loops 1 and 3 containing glycosylation-accessible reporter sites. A 3D model was built and site-directed mutagenesis of the predicted bile acid binding domain (including P290S pathological mutation) validated the model.","method":"N-glycosylation scanning mutagenesis, 3D homology modeling, site-directed mutagenesis, cholic acid docking","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (glycosylation scanning + mutagenesis + modeling), single lab","pmids":["15350125"],"is_preprint":false},{"year":2006,"finding":"Dual label epitope insertion scanning mutagenesis confirmed a 7-transmembrane topology for hASBT, with extracellular loops 1 (residues 99-130), 2 (180-191), and 3 (253-287) and cytosolic localization of loops in the 7TM model. N-terminus is exofacial and C-terminus is cytosolic. This refutes the previously proposed 9TM model.","method":"Dual label epitope (HA and FLAG) insertion mutagenesis, cell surface biotinylation, confocal microscopy in permeabilized and non-permeabilized COS-1 and MDCK cells","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic epitope insertion at multiple positions with two orthogonal tags, confocal imaging with and without permeabilization, activity measurements","pmids":["16411770"],"is_preprint":false},{"year":2006,"finding":"Cysteine-scanning mutagenesis of TM7 of hASBT showed that TM7 lines the substrate translocation pathway. Residues in the extracellular half of TM7 are accessible to membrane-impermeant MTSES and MTSET, while the entire TM7 length is accessible to MTSEA. Substrate co-application significantly amended MTSEA sensitivity, consistent with conformational changes during transport. Loss-of-function mutants T289C, Y293C, Q297C, and A301C are MTS-inaccessible, suggesting a structural role.","method":"Cysteine-scanning mutagenesis, SCAM (substituted cysteine accessibility method) with MTSEA/MTSET/MTSES in COS-1 cells, taurocholate uptake assay","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic SCAM across 22 consecutive residues with substrate protection experiments and multiple MTS reagents","pmids":["16899538"],"is_preprint":false},{"year":2005,"finding":"Cysteine scanning mutagenesis identified C51A/T, C105A/T, C144A, and C255A/T as loss-of-function mutations in hASBT, and C74A/T abolishes cell surface expression suggesting a role in protein folding/trafficking. C270A remains functional and insensitive to polar MTS reagents but shows decreased K_T and J_max with cholyl- and chenodeoxycholyl-MTS conjugates, implicating C270 in a putative substrate binding domain.","method":"Ala/Thr scanning mutagenesis of all Cys residues, MTS bile acid conjugate affinity inactivators, taurocholate transport assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis of all cysteines combined with novel bile acid-MTS affinity tools and kinetic analysis","pmids":["15952798"],"is_preprint":false},{"year":2008,"finding":"Conserved residue Asp-122 in extracellular loop 1 of ASBT functions as a Na+ sensor (binding one of two co-transported Na+ ions), and Asp-124 interacts with 7α-OH groups of bile acids. Neutralization of charge at Asp-120 and Asp-122 abolishes transport, while D124A yields a functionally active transporter with low affinity for common bile acids except deoxycholic acid (which lacks a 7α-OH group).","method":"Cysteine-scanning mutagenesis of EL1 residues V99-S126, thiol modification, taurocholate uptake assay, in silico modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis with mechanistic substrate specificity analysis (deoxycholic acid lacking 7α-OH) across multiple residues","pmids":["18508772"],"is_preprint":false},{"year":2008,"finding":"Cysteine-scanning mutagenesis of TM4 (21 amino acids) of hASBT showed that primarily the cytosolic half is solvent-accessible via MTSES and MTSET modification, implicating it in substrate translocation. TM4 mutants were not sensitive to equilibrative sodium conditions, ruling out a direct role for TM4 in sodium translocation.","method":"Cysteine-scanning mutagenesis of TM4, MTS reagent accessibility (MTSES/MTSET), cell surface expression assays, taurocholate uptake","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic SCAM of full TM domain with sodium sensitivity controls, in COS-1 cells","pmids":["18311924"],"is_preprint":false},{"year":2009,"finding":"Cysteine-scanning mutagenesis of TM3 (V127-T149) showed that the cytosolic half forms portions of the substrate exit route. Residues S128C and L145C-T149C are solvent-accessible and show significant loss of taurocholate affinity. P142 is structurally and functionally important during the transport cycle (P142C mutation severely decreases activity).","method":"Cysteine-scanning mutagenesis of TM3, SCAM with MTS reagents, kinetic analysis (K_T and J_max) for taurocholate","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic SCAM of full TM3, substrate kinetic analysis, multiple MTS reagents","pmids":["19653651"],"is_preprint":false},{"year":2011,"finding":"Cysteine-scanning mutagenesis of TM1 (I29-G50) showed that TM1 plays a pivotal role in hASBT function and stability. G50 is involved in protein folding (G50C/A expression rescued by MG132 and cyclosporine A but not FK506). Residues L30, L38, T39, and M46 participate in sodium co-transport, while L34, T36, and L38 show temperature-dependent substrate protection from MTSET, indicating conformational changes in TM1 during transport.","method":"Cysteine-scanning mutagenesis, SCAM with MTSET, proteasome inhibitor rescue (MG132), cyclosporine A treatment, kinetic analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic SCAM of TM1 with substrate/sodium kinetics and folding rescue experiments","pmids":["21646357"],"is_preprint":false},{"year":2013,"finding":"Cysteine-scanning mutagenesis of TM2 (systematic approach) showed that TM2 is inaccessible to membrane-impermeant MTSET, indicating it does not directly form the bile acid translocation pathway. Kinetic studies identified residues Q75, F76, M79, G83, L86, F90, and D91 as sodium-sensitive, and computational analysis suggests D91 coordinates sodium during transport. TM2 residues likely form the sodium binding and translocation pathway together with TM3 residues.","method":"Cysteine-scanning mutagenesis of TM2, MTSET accessibility, sodium dependence kinetics, computational analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic SCAM plus kinetics for sodium sensitivity, computational model, single rigorous study","pmids":["24045943"],"is_preprint":false},{"year":2013,"finding":"Cysteine-scanning mutagenesis of TM5 showed that residues G197 and G201 form a GxxxG motif important for helix-helix interactions with TM4 (interacting with G179 and P175), and G212 interacts with G237 in a GxxxG domain in TM6. TM5 residues are critical for ASBT function but are not directly involved in substrate translocation pathway.","method":"Cysteine-scanning mutagenesis of TM5, alanine conservative mutations, cell surface expression, taurocholate uptake, 3D modeling","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — systematic TM5 mutagenesis with conservative mutations, but no direct SCAM accessibility experiments for TM5","pmids":["23815591"],"is_preprint":false},{"year":2002,"finding":"The human ASBT gene promoter contains three functional HNF1α recognition sites essential for expression, and a DR1 motif ~1.6 kb upstream that binds PPARα/RXR heterodimer. PPARα ligands (WY14643, ciprofibrate) transactivate ASBT promoter constructs in Caco2 cells; targeted mutagenesis of the DR1 motif abolished PPARα responsiveness. Ciprofibrate treatment of SK-ChA cholangiocytes increased ASBT mRNA.","method":"Promoter-reporter (luciferase) assays, targeted mutagenesis of DR1 motif, EMSA, PPARα ligand treatment, mRNA analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis of specific promoter element, EMSA binding confirmation, both in vitro and cell-based experiments","pmids":["12055195"],"is_preprint":false},{"year":2004,"finding":"Human ASBT is transactivated by the glucocorticoid receptor (GR). Two glucocorticoid response elements (IR3 elements, inverted hexanucleotide repeats) in the ASBT promoter confer inducibility by GR and dexamethasone. These elements bind GR in EMSA. In vivo, budesonide treatment increased ASBT protein expression in healthy volunteers.","method":"Luciferase reporter assays with GR co-expression, EMSA, western blot of human ileal biopsies, in vivo budesonide treatment","journal":"Gut","confidence":"High","confidence_rationale":"Tier 1 / Moderate — promoter mutagenesis, EMSA, and in vivo human data confirming regulation","pmids":["14684580"],"is_preprint":false},{"year":2005,"finding":"FXR-activating bile acids repress rabbit ASBT expression via the cascade FXR→SHP→FTF. A cis-acting FTF binding element in the rabbit ASBT promoter (-1166/-1158) mediates this regulation. FTF stimulates ASBT promoter 4-fold; increasing SHP protein inhibits FTF-dependent transactivation. Only FXR-activating ligands (DCA, CDCA, CA) repress ASBT; non-FXR ligands (UDCA, UCA) do not.","method":"In vivo rabbit feeding experiments, 5'-flanking ASBT promoter cloning, luciferase reporter assays in Caco-2 cells, SHP overexpression, FTF site deletion mutagenesis","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vivo plus in vitro assays with promoter deletion/mutagenesis, mechanistic cascade validated","pmids":["15591588"],"is_preprint":false},{"year":2011,"finding":"CDX1 and CDX2 transcription factors transcriptionally activate the human ASBT promoter. Six CDX binding sites within the ASBT promoter were verified by EMSA in vitro and chromatin immunoprecipitation in living cells. siRNA knockdown of CDX1/2 reduced ASBT mRNA expression in intestinal cells.","method":"siRNA knockdown, luciferase reporter assays, EMSA, chromatin immunoprecipitation (ChIP), real-time PCR in human biopsies","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — siRNA knockdown, EMSA, and ChIP in living cells; multiple orthogonal methods","pmids":["22016432"],"is_preprint":false},{"year":2009,"finding":"PKCζ (an atypical PKC isoform) mediates posttranscriptional regulation of ileal ASBT function and membrane expression. PMA (PKC activator) inhibits ASBT-mediated taurocholate uptake in Caco-2 cells; inhibition is blocked by bisindolylmaleimide I (PKC inhibitor) and myristoylated PKCζ pseudosubstrate peptide but not by Ca2+ chelation or PI3K inhibition. PMA decreases transporter V_max and reduces ASBT plasma membrane content, suggesting vesicular recycling.","method":"Taurocholate uptake assay in Caco-2 cells, PKC inhibitors (bisindolylmaleimide I, PKCζ pseudosubstrate), kinetic analysis (V_max), plasma membrane fractionation/immunoblot","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological dissection of PKC isoform, kinetic analysis, membrane expression measurements; single lab","pmids":["19571234"],"is_preprint":false},{"year":2007,"finding":"ASBT associates with lipid raft microdomains in plasma membrane. Depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) disrupts lipid raft association of ASBT and significantly reduces ASBT activity (decreased V_max) without altering plasma membrane expression. Cholesterol repletion with MβCD-cholesterol complexes restores activity.","method":"Optiprep density gradient flotation of lipid rafts, MβCD cholesterol depletion, taurocholate uptake kinetics, plasma membrane expression by biotinylation in HEK-293 cells stably transfected with ASBT, Caco-2 cells, and human ileal brush-border membrane vesicles","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell systems, flotation gradient, kinetic analysis, cholesterol rescue experiment; single lab","pmids":["18063707"],"is_preprint":false},{"year":2010,"finding":"EGCG (epigallocatechin-3-gallate) inhibits ASBT activity by displacing ASBT from lipid raft fractions of the plasma membrane into detergent-soluble fractions, reducing V_max without changing total ASBT content on plasma membrane. Inhibition of PKC, PI3K, and MAPK pathways did not block EGCG effect.","method":"Taurocholate uptake assay, kinetic analysis, Optiprep density gradient fractionation, plasma membrane biotinylation, pathway inhibitor studies in HEK-293/ASBT cells and Caco-2","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell systems, kinetic and membrane fractionation data, pharmacological dissection of pathways; single lab","pmids":["20056894"],"is_preprint":false},{"year":2012,"finding":"Enteropathogenic E. coli (EPEC) inhibits ileal ASBT function via its type-III secretion system (TTSS) and bundle-forming pili. Mutations in escN, espA, espB, espD (TTSS components) and BFP (pili) gene ablate EPEC inhibitory effects on ASBT. EPEC inhibition is associated with decreased V_max and reduced ASBT plasma membrane levels, and is blocked by protein tyrosine phosphatase inhibitors.","method":"Taurocholate uptake in Caco-2 and HEK-293/ASBT cells infected with EPEC and TTSS/BFP mutant strains, kinetic analysis, plasma membrane expression, tyrosine phosphatase inhibitors","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple bacterial mutants plus pharmacological inhibitors with functional and expression readouts; single lab","pmids":["22403793"],"is_preprint":false},{"year":2014,"finding":"The lipid flippase heterodimer ATP8B1-CDC50A is essential for apical membrane insertion and surface expression of SLC10A2/ASBT in intestinal Caco-2 cells. ATP8B1 depletion strongly impairs SLC10A2-mediated bile salt uptake and reduces apical membrane localization of SLC10A2, caused by impaired apical membrane insertion (not reduced synthesis). Endogenous ATP8B1 exists in a functional heterodimer with CDC50A in Caco-2 cells.","method":"siRNA depletion of ATP8B1 in Caco-2 cells, bile salt uptake assay, apical membrane biotinylation, co-immunoprecipitation of ATP8B1-CDC50A, fecal bile salt analysis from PFIC1 patients","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 2 / Strong — siRNA KD with functional readout, membrane localization by biotinylation, reciprocal Co-IP of heterodimer, human patient data correlation","pmids":["25239307"],"is_preprint":false},{"year":2014,"finding":"Resveratrol promotes ASBT protein degradation via the ubiquitin-proteasome pathway in a SIRT1-independent manner. Proteasome inhibitors MG132 and lactacystin, and ubiquitin inhibitor LDN57444, reverse RSV-mediated ASBT inhibition. Immunoprecipitation revealed high levels of ubiquitinated ASBT after RSV treatment. The effect is specific to ASBT; NTCP, OSTα, and ABCG1 are unaffected. Phosphorylation at the corresponding residues of rat Ser335/Thr339 does not contribute to RSV-mediated degradation.","method":"Proteasome inhibitor rescue assays, ubiquitin inhibitor, co-immunoprecipitation for ubiquitinated ASBT, site-directed mutagenesis of phosphorylation sites, transporter expression/function assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological rescue with multiple inhibitors, immunoprecipitation of ubiquitinated ASBT, mutagenesis of phosphorylation sites; single lab","pmids":["24498857"],"is_preprint":false},{"year":2015,"finding":"N-glycosylation of ASBT is essential for function and protein stability but not for targeting to the plasma membrane. Fully mature N-acetylglucosamine-rich ASBT (41 kDa band) and core mannose-rich glycoprotein (35 kDa band) are present. The N10Q glycosylation-deficient mutant reaches the plasma membrane but has reduced half-life and increased trypsin susceptibility. High glucose increases mature glycosylated ASBT along with increased ASBT function.","method":"Tunicamycin treatment, PNGase F and Endo H glycosidase treatment, N10Q mutant expression, pulse-chase/half-life analysis, protease susceptibility assay, taurocholate uptake","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — glycosidase mapping, glycosylation site mutation, protein stability analysis; single lab","pmids":["25855079"],"is_preprint":false},{"year":2017,"finding":"hASBT forms functional non-covalent homodimers and higher-order oligomers, independent of endogenous cysteine residues. Chemical cross-linking and co-immunoprecipitation of differentially tagged (HA, FLAG) wild-type and cysteine-less hASBT confirmed dimerization. Cysteine-less hASBT showed dominant-negative effect when co-expressed with wild-type hASBT, validating functional heterodimerization/oligomerization.","method":"Site-directed mutagenesis (all 13 cysteines mutated stepwise), chemical cross-linking, co-immunoprecipitation of HA-/FLAG-tagged species, dominant-negative functional assay","journal":"Biochimica et biophysica acta. Biomembranes","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP with differentially tagged constructs, cross-linking, dominant-negative functional validation; multiple orthogonal approaches in one study","pmids":["29198943"],"is_preprint":false},{"year":2019,"finding":"Tyrosine phosphorylation mediated by Src family kinases (SFKs) regulates surface expression, function, and stability of hASBT. SFK inhibition (PP2) reduces hASBT function and surface expression, rescued by proteasome inhibitor MG132, indicating dephosphorylation leads to proteasome-dependent degradation. Five intracellular tyrosine residues (Y148, Y216, Y308, Y311, Y337) individually alter ASBT function without changing total cellular expression; sequential mutation of all five renders ASBT nonfunctional with diminished expression.","method":"SFK inhibitor PP2, PTP inhibitor orthovanadate, proteasome inhibitor MG132, site-directed mutagenesis of five intracellular Tyr residues, taurocholate transport assays, surface expression measurements","journal":"Molecular pharmaceutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and mutagenesis approaches with functional and expression readouts; single lab","pmids":["31194565"],"is_preprint":false},{"year":2024,"finding":"PKC regulates hASBT activity by phosphorylation at Ser335, established as the predominant phosphosite. Parallel reaction monitoring targeted mass spectrometry identified phosphorylation at Thr330, Ser334, and Ser335, with Ser335 being dominant. A proportional relationship between Ser335 phosphorylation level and ASBT bile acid uptake activity was demonstrated.","method":"Parallel reaction monitoring (PRM) targeted mass spectrometry, kinase inhibitor/activator treatments, phosphorylation site-specific analysis, bile acid uptake activity assay","journal":"ACS omega","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — targeted mass spectrometry for site-specific phosphorylation occupancy with functional correlation; single study","pmids":["39310206"],"is_preprint":false},{"year":2005,"finding":"ASBT-mediated bile salt (taurocholate) absorption in the distal ileum triggers CFTR activation and consequent Cl- secretion. TC-evoked Cl- secretion was abrogated in Cftr-null mice, blocked by a selective ASBT inhibitor, and was restricted to the distal ileum. The mechanism involves CFTR channel gating modulation rather than changes in driving force, and is independent of cAMP/cGMP signaling.","method":"Short circuit current measurements across ileal tissue, Cftr-null mice, cGMP-dependent kinase II-null mice, ASBT inhibitor, neurotransmitter/prostaglandin suppression, nystatin permeabilization, H89 (PKA inhibitor)","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout, pharmacological inhibition, electrophysiology in intact tissue; multiple orthogonal approaches","pmids":["16037545"],"is_preprint":false},{"year":2015,"finding":"Gut microbiota suppresses intestinal Asbt expression via the transcription factor Gata4. In germ-free or antibiotic-treated mice, Asbt expression increased in the ileum and extended more proximally in the small intestine. Genetic inactivation of either Asbt or Gata4 prevented most metabolic effects of antibiotic treatment on bile acid homeostasis, establishing Gata4 as a mediator of microbiota-dependent Asbt suppression.","method":"Germ-free and antibiotic-treated mouse models, Asbt-KO mice, intestinal-specific Gata4-iKO mice, gene expression analysis, metabolic profiling","journal":"Journal of hepatology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis using Asbt-KO and Gata4-iKO mice with metabolic phenotyping; multiple mouse models","pmids":["26022694"],"is_preprint":false},{"year":2009,"finding":"Mutational analysis of conserved uncharged polar residues and proline in mouse Slc10a2 (Thr130-Pro142 region) revealed that T130A causes loss of cell surface localization, P142V abolishes almost all transport activity, and T134A affects taurocholic acid affinity. This region contains residues involved in substrate interaction, function, and cellular localization.","method":"Site-directed mutagenesis of conserved residues in mouse Slc10a2, taurocholic acid uptake assay, cell surface localization assessment","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro mutagenesis with transport and localization readouts, single lab, limited number of residues","pmids":["19584562"],"is_preprint":false},{"year":2013,"finding":"Mutational analysis of conserved proline and uncharged polar residues in mouse Slc10a2 (Pro107-Ser128 region) revealed that P107N/L impairs cell surface localization, S126A completely impairs cellular expression, S112A abolishes transport activity while maintaining surface expression, and T110A/S128A mutations enhance membrane expression. Tyr117 mutations show reduced activity proportional to side chain van der Waals volume.","method":"Site-directed mutagenesis, taurocholate uptake assay, cell surface localization assessment in heterologous expression system","journal":"BMC physiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis with transport and localization readouts; extends prior work on conserved region","pmids":["23374508"],"is_preprint":false},{"year":2011,"finding":"SLC10A2 coding region variants 292G>A and 431G>A show partially impaired taurocholate transport in vitro, and a novel variant 790A>G shows near complete loss of taurocholate transport, without affecting protein expression or cell surface trafficking.","method":"PCR-based temperature gradient capillary electrophoresis (TGCE) for SNP identification, heterologous expression, taurocholate transport assay, western blot, immunofluorescence confocal microscopy","journal":"Journal of gastroenterology and hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic SNP functional characterization with transport and trafficking readouts in heterologous system; single lab","pmids":["21649730"],"is_preprint":false},{"year":2021,"finding":"Systematic comparative transport experiments with NTCP, ASBT, and SOAT using stably transfected HEK293 cells identified taurolithocholic acid (TLC) as the first common substrate of all three SLC10 carriers (K_m values: NTCP 18.4 μM, ASBT 5.9 μM, SOAT 19.3 μM). Lithocholic acid was the only bile acid not transported by any. Troglitazone, BSP, and erythrosine B were pan-SLC10 inhibitors, while cyclosporine A, irbesartan, and others only inhibited NTCP and SOAT but not ASBT.","method":"Stably transfected HEK293 cells (NTCP-, ASBT-, SOAT-), transport assays with >20 substrates, inhibition experiments","journal":"Frontiers in molecular biosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic substrate and inhibitor profiling in stable cell lines; single lab but comprehensive substrate panel","pmids":["34079822"],"is_preprint":false},{"year":2021,"finding":"The bacterial ASBT homologue ASBT_NM is a monomer (not a dimer or trimer as expected for elevator-type transporters). Na+ ions shift the conformational equilibrium toward the inward-facing state, facilitating cytoplasmic uptake of substrate. Site-directed alkylation monitored by in-gel fluorescence (SDAF) and DEER EPR spectroscopy validated the conformational states.","method":"Site-directed alkylation with in-gel fluorescence (SDAF), crosslinking experiments, DEER (double electron-electron resonance) EPR spectroscopy","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — two orthogonal biophysical methods (SDAF and DEER) on bacterial homologue; sodium-dependent conformational change demonstrated","pmids":["33359100"],"is_preprint":false},{"year":2010,"finding":"Ileal SLC10A2 mRNA and protein levels are significantly increased by antibiotic-mediated reduction of enterobacteria in mice (FXR-independent), with increased bile acid concentrations in portal blood. Re-administration of taurodeoxycholic acid or cholic acid (enterobacteria-biotransformed bile acids) to antibiotic-treated mice decreased ileal SLC10A2 expression, suggesting that enterobacteria-mediated bile acid biotransformation modulates SLC10A2 expression.","method":"Antibiotic treatment of C57BL/6N and FXR-null mice, ileal SLC10A2 mRNA/protein measurement, portal blood bile acid measurement, bile acid re-administration experiments","journal":"The Journal of pharmacology and experimental therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FXR-null mouse epistasis, bile acid rescue experiment, protein and mRNA analysis; single lab","pmids":["20884752"],"is_preprint":false},{"year":2010,"finding":"ASBT expression and promoter activity are significantly decreased by insulin in Caco-2 cells. In streptozotocin-induced diabetic rats, ASBT mRNA and protein are significantly elevated, and ileal Na+-dependent taurocholate uptake is increased. Insulin treatment of diabetic rats reversed increased ASBT protein expression to control levels.","method":"STZ-induced diabetes in rats, Western blot, real-time qRT-PCR, isolated intestinal epithelial cell taurocholate uptake, Caco-2 promoter activity assays with insulin treatment","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo model plus in vitro mechanistic studies; promoter and expression analysis; single lab","pmids":["20651004"],"is_preprint":false},{"year":2022,"finding":"KDM6B histone demethylase directly transcriptionally activates SLC10A2; KDM6B loss increases H3K27me3 repression at the SLC10A2 promoter, activating the ERK/AP-1 pathway and CXCL/CXCR2-dependent MDSC recruitment in colorectal cancer.","method":"Intestinal epithelial-specific KDM6B deletion mouse model, ChIP for H3K27me3 at SLC10A2 promoter, ERK/AP-1 pathway analysis, MDSC recruitment assays","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for histone modification at SLC10A2 promoter, genetic KO with downstream signaling analysis; single study","pmids":["41387297"],"is_preprint":false},{"year":2022,"finding":"Transcription factor KLF9 promotes intestinal Asbt expression in the terminal ileum to enhance bile acid absorption. Klf9 knockout mice show increased bile acids in gallbladder and feces with decreased serum BA levels; intestine-specific Klf9 deletion recapitulates this phenotype. Klf9 transgenic mice show the opposite. Biochemical and molecular assays confirmed KLF9 directly promotes Asbt expression.","method":"Systemic Klf9-/- mice, intestine-specific Klf9vil-/- mice, intestinal Klf9 transgenic mice, biochemical/molecular/functional assays for Asbt expression and bile acid homeostasis","journal":"Acta pharmacologica Sinica","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple independent genetic mouse models (KO, tissue-specific KO, transgenic overexpression) with consistent phenotype","pmids":["35105957"],"is_preprint":false},{"year":2023,"finding":"Apple-derived extracellular vesicles (APEVs) downregulate ASBT/SLC10A2 expression indirectly by reducing RARα/NR1B1 protein expression. APEVs decrease binding of RARα to the SLC10A2 promoter. MicroRNAs in APEVs reduce NR1B1 mRNA stability by targeting its 3'UTR, and apple microRNA mimics suppress NR1B1 mRNA expression.","method":"Proteomics, RARα binding to SLC10A2 promoter assay, NR1B1-3'UTR reporter assay, apple microRNA mimic transfection, ASBT expression and uptake activity","journal":"Drug metabolism and pharmacokinetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — mechanistic cascade proposed with promoter binding and miRNA mimic data but indirect mechanism; single lab","pmids":["37517353"],"is_preprint":false}],"current_model":"SLC10A2 (ASBT) is an intestinal apical sodium-dependent bile acid cotransporter with a 7-transmembrane topology (N-terminus extracellular, C-terminus cytosolic) that functions as a non-covalent homodimer/oligomer; it uses two sodium ions (with Na+-sensing residues in EL1 and TM2/TM3) to drive uphill transport of bile acids via an elevator-like mechanism involving a core domain and a panel domain (established by bacterial homologue crystal structure), with specific transmembrane helices (TM1, TM3, TM4, TM7) lining the substrate translocation pathway; its activity and surface expression are acutely regulated by PKCζ-dependent vesicular recycling, Src family kinase-mediated tyrosine phosphorylation, PKC-mediated Ser335 phosphorylation, ubiquitin-proteasome degradation (promoted by resveratrol), N-glycosylation (required for stability/protease protection), and lipid raft association; its gene expression is transcriptionally controlled by HNF1α, PPARα, glucocorticoid receptor (via IR3 elements), CDX1/CDX2 homeobox factors, KLF9, FXR-SHP-FTF cascade (bile acid feedback), KDM6B epigenetic activation, and microbiota-dependent suppression via GATA4; loss-of-function mutations cause primary bile acid malabsorption, and ASBT-mediated bile acid uptake also activates CFTR-dependent Cl- secretion in the ileum."},"narrative":{"mechanistic_narrative":"SLC10A2 (ASBT) is the apical sodium-dependent bile acid cotransporter that drives reclamation of bile acids from the intestinal lumen, and loss-of-function mutations in it cause primary bile acid malabsorption [PMID:9109432]. The transporter adopts a 7-transmembrane topology with an exofacial N-terminus and cytosolic C-terminus [PMID:16411770], and homology to a high-resolution bacterial structure organizes the protein into a sodium-bearing core domain and a panel domain between which substrate binds in an inward-facing cavity [PMID:21976025]; sodium binding shifts the conformational equilibrium toward this inward-facing state to favor cytoplasmic substrate release [PMID:33359100]. Systematic cysteine-scanning of individual helices has mapped function onto specific helices: TM1, TM3, TM4, and TM7 line the substrate translocation pathway and undergo substrate-dependent conformational changes [PMID:16899538, PMID:18311924, PMID:19653651, PMID:21646357], while TM2 and TM3 residues form the sodium binding/translocation route [PMID:24045943], with EL1 residues Asp-122 sensing Na+ and Asp-124 recognizing the 7α-OH of bile acids [PMID:18508772]. ASBT functions as a non-covalent homodimer/oligomer [PMID:29198943] and transports a defined panel of bile acids, including taurolithocholic acid shared across the SLC10 family [PMID:34079822]. Surface expression and activity are tuned post-translationally by N-glycosylation required for stability [PMID:25855079], lipid raft association [PMID:18063707], PKC-mediated Ser335 phosphorylation [PMID:39310206], Src family kinase tyrosine phosphorylation that protects against proteasomal degradation [PMID:31194565], ubiquitin-proteasome turnover [PMID:24498857], and apical insertion dependent on the ATP8B1-CDC50A flippase heterodimer [PMID:25239307]. Gene expression is governed by a transcriptional network including HNF1α and PPARα [PMID:12055195], the glucocorticoid receptor [PMID:14684580], CDX1/CDX2 [PMID:22016432], KLF9 [PMID:35105957], the FXR→SHP→FTF bile acid feedback cascade [PMID:15591588], KDM6B-mediated chromatin activation [PMID:41387297], and microbiota-dependent suppression through GATA4 [PMID:26022694]. Functionally, ASBT-mediated bile acid uptake in the distal ileum activates CFTR-dependent Cl- secretion [PMID:16037545].","teleology":[{"year":1997,"claim":"Established that SLC10A2 dysfunction directly causes a human disease by showing that specific missense mutations abolish bile acid transport without disrupting protein expression or membrane trafficking.","evidence":"Site-directed mutagenesis and taurocholate uptake assays in transfected COS cells with disease-associated alleles","pmids":["9109432"],"confidence":"High","gaps":["Did not resolve the structural basis of the affected residues","Did not define the transport mechanism or sodium stoichiometry"]},{"year":2004,"claim":"Resolved the membrane architecture of the transporter, supporting a 7-transmembrane topology and an initial 3D model of the bile acid binding domain.","evidence":"N-glycosylation scanning mutagenesis, homology modeling, and cholic acid docking","pmids":["15350125"],"confidence":"Medium","gaps":["Model not experimentally confirmed by structure","Topology of cytosolic loops not fully resolved"]},{"year":2006,"claim":"Definitively settled the topology debate by confirming a 7TM model and refuting the alternative 9TM configuration, and identified TM7 as part of the substrate translocation pathway.","evidence":"Dual epitope-insertion mutagenesis with confocal imaging, plus cysteine-scanning SCAM of TM7 with substrate protection in COS-1 cells","pmids":["16411770","16899538"],"confidence":"High","gaps":["Did not localize sodium binding sites","Functional roles of other helices unaddressed"]},{"year":2008,"claim":"Dissected which helices and residues form the substrate path versus the sodium path, identifying Asp-122 as a Na+ sensor and Asp-124 as a 7α-OH bile acid recognition residue.","evidence":"Cysteine-scanning/SCAM of EL1 and TM4 with substrate-specificity and sodium-sensitivity kinetics","pmids":["18508772","18311924"],"confidence":"High","gaps":["Did not provide a holistic structural model integrating all helices","Number and exact coordination of both Na+ ions not fully defined"]},{"year":2009,"claim":"Extended the translocation-pathway map to TM3 and identified residues forming the substrate exit route, and demonstrated PKCζ-dependent post-translational control of surface expression.","evidence":"SCAM of TM3 with kinetic analysis; PKC isoform-selective pharmacology and membrane fractionation in Caco-2 cells","pmids":["19653651","19571234"],"confidence":"High","gaps":["Direct phosphorylation target of PKCζ not defined here","Vesicular recycling machinery not identified"]},{"year":2011,"claim":"Provided the structural framework for the elevator mechanism via a substrate-bound bacterial homologue crystal structure, while helix-level mutagenesis assigned TM1 to both sodium co-transport and folding.","evidence":"X-ray crystallography of ASBT_NM with taurocholate; SCAM of TM1 with sodium/substrate kinetics and proteasome-inhibitor folding rescue","pmids":["21976025","21646357"],"confidence":"High","gaps":["Structure is of a bacterial monomer, not human ASBT","Oligomeric state of human transporter unresolved at this point"]},{"year":2013,"claim":"Completed the helix-by-helix functional map by assigning TM2 to the sodium pathway and TM5 to helix-helix packing rather than direct translocation.","evidence":"SCAM/mutagenesis of TM2 and TM5 with sodium kinetics, conservative substitutions, and 3D modeling","pmids":["24045943","23815591"],"confidence":"Medium","gaps":["TM5 not tested by direct SCAM accessibility","Inter-helix dynamics during transport not directly observed"]},{"year":2017,"claim":"Resolved the oligomeric organization of the human transporter, demonstrating functional non-covalent homodimers/oligomers independent of cysteine residues.","evidence":"Cross-linking, reciprocal co-IP of differentially tagged constructs, and dominant-negative functional assays","pmids":["29198943"],"confidence":"High","gaps":["Dimer interface residues not mapped","Apparent conflict with monomeric bacterial homologue not reconciled"]},{"year":2021,"claim":"Defined the sodium-driven conformational mechanism of the homologue and benchmarked human ASBT substrate/inhibitor selectivity within the SLC10 family.","evidence":"SDAF and DEER EPR on ASBT_NM; comparative substrate/inhibitor profiling of NTCP, ASBT, SOAT in stable HEK293 cells","pmids":["33359100","34079822"],"confidence":"High","gaps":["Conformational dynamics not measured on human ASBT directly","Structural basis of inhibitor selectivity unresolved"]},{"year":2024,"claim":"Identified Ser335 as the predominant PKC phosphosite controlling ASBT activity, providing site-level resolution of post-translational regulation.","evidence":"Parallel reaction monitoring targeted mass spectrometry with kinase modulation and uptake assays","pmids":["39310206"],"confidence":"Medium","gaps":["Responsible PKC isoform at Ser335 not definitively assigned","Single study, mechanism linking phosphorylation to activity unresolved"]},{"year":null,"claim":"How the diverse post-translational inputs (glycosylation, raft association, tyrosine/serine phosphorylation, ubiquitination, flippase-dependent insertion) are coordinated in vivo, and a high-resolution structure of human ASBT, remain to be established.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No human ASBT structure in the corpus","Integration of competing regulatory signals not modeled","Dimer interface and physiological role of oligomerization undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,1,6,31]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[6,10,31]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[6,31]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,17,20,22]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,1,26]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[14,27,31]}],"complexes":[],"partners":["ATP8B1","CDC50A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q12908","full_name":"Ileal sodium/bile acid cotransporter","aliases":["Apical sodium-dependent bile acid transporter","ASBT","Ileal Na(+)/bile acid cotransporter","Ileal sodium-dependent bile acid transporter","IBAT","ISBT","Na(+)-dependent ileal bile acid transporter","Sodium/taurocholate cotransporting polypeptide, ileal","Solute carrier family 10 member 2"],"length_aa":348,"mass_kda":37.7,"function":"Plays a critical role in the sodium-dependent reabsorption of bile acids from the lumen of the small intestine (PubMed:7592981, PubMed:9458785, PubMed:9856990). Transports various bile acids, unconjugated or conjugated, such as cholate and taurocholate (PubMed:7592981, PubMed:9458785, PubMed:9856990). Also responsible for bile acid transport in the renal proximal tubules, a salvage mechanism that helps conserve bile acids (Probable). Works collaboratively with the Na(+)-taurocholate cotransporting polypeptide (NTCP), the organic solute transporter (OST), and the bile salt export pump (BSEP), to ensure efficacious biological recycling of bile acids during enterohepatic circulation (PubMed:33222321)","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q12908/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SLC10A2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SLC10A2","total_profiled":1310},"omim":[{"mim_id":"617382","title":"START DOMAIN-CONTAINING PROTEIN 10; STARD10","url":"https://www.omim.org/entry/617382"},{"mim_id":"617049","title":"CHOLESTASIS, PROGRESSIVE FAMILIAL INTRAHEPATIC, 5; PFIC5","url":"https://www.omim.org/entry/617049"},{"mim_id":"613291","title":"BILE ACID MALABSORPTION, PRIMARY, 1; PBAM1","url":"https://www.omim.org/entry/613291"},{"mim_id":"611899","title":"MICRO RNA 203A; MIR203A","url":"https://www.omim.org/entry/611899"},{"mim_id":"609972","title":"ACYL-CoA THIOESTERASE 2; ACOT2","url":"https://www.omim.org/entry/609972"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"intestine","ntpm":89.4}],"url":"https://www.proteinatlas.org/search/SLC10A2"},"hgnc":{"alias_symbol":["NTCP2","IBAT"],"prev_symbol":["ASBT","ISBT"]},"alphafold":{"accession":"Q12908","domains":[{"cath_id":"1.20.1530.20","chopping":"37-311","consensus_level":"high","plddt":92.0757,"start":37,"end":311}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12908","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q12908-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q12908-F1-predicted_aligned_error_v6.png","plddt_mean":82.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SLC10A2","jax_strain_url":"https://www.jax.org/strain/search?query=SLC10A2"},"sequence":{"accession":"Q12908","fasta_url":"https://rest.uniprot.org/uniprotkb/Q12908.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q12908/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12908"}},"corpus_meta":[{"pmid":"9109432","id":"PMC_9109432","title":"Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2).","date":"1997","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/9109432","citation_count":282,"is_preprint":false},{"pmid":"21976025","id":"PMC_21976025","title":"Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT.","date":"2011","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/21976025","citation_count":225,"is_preprint":false},{"pmid":"14684580","id":"PMC_14684580","title":"Human ileal bile acid transporter gene ASBT (SLC10A2) is transactivated by the glucocorticoid receptor.","date":"2004","source":"Gut","url":"https://pubmed.ncbi.nlm.nih.gov/14684580","citation_count":167,"is_preprint":false},{"pmid":"16749855","id":"PMC_16749855","title":"Apical sodium dependent bile acid transporter (ASBT, SLC10A2): a potential prodrug target.","date":"2006","source":"Molecular pharmaceutics","url":"https://pubmed.ncbi.nlm.nih.gov/16749855","citation_count":133,"is_preprint":false},{"pmid":"26022694","id":"PMC_26022694","title":"Gut microbiota inhibit Asbt-dependent intestinal bile acid reabsorption via Gata4.","date":"2015","source":"Journal of hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/26022694","citation_count":102,"is_preprint":false},{"pmid":"33578447","id":"PMC_33578447","title":"ABO blood group and COVID-19: a review on behalf of the ISBT COVID-19 Working Group.","date":"2021","source":"Vox sanguinis","url":"https://pubmed.ncbi.nlm.nih.gov/33578447","citation_count":100,"is_preprint":false},{"pmid":"12055195","id":"PMC_12055195","title":"Human apical sodium-dependent bile salt transporter gene (SLC10A2) is regulated by the peroxisome proliferator-activated receptor alpha.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12055195","citation_count":88,"is_preprint":false},{"pmid":"8607148","id":"PMC_8607148","title":"Practical guidelines for process validation and process control of white cell-reduced blood components: report of the Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion (ISBT).","date":"1996","source":"Transfusion","url":"https://pubmed.ncbi.nlm.nih.gov/8607148","citation_count":88,"is_preprint":false},{"pmid":"21341987","id":"PMC_21341987","title":"Genetic polymorphisms in Na+-taurocholate co-transporting polypeptide (NTCP) and ileal apical sodium-dependent bile acid transporter (ASBT) and ethnic comparisons of functional variants of NTCP among Asian populations.","date":"2011","source":"Xenobiotica; 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SLC10A2 (L243P, T262M, and double mutant L243P/T262M) abolish taurocholate and bile acid transport without affecting transporter protein expression or trafficking to the plasma membrane, establishing that these residues are critical for transport activity. A171S had no effect on transport. A frame-shift mutation (646insG) also abolished transport in transfected COS cells.\",\n      \"method\": \"Site-directed mutagenesis, transfection in COS cells, taurocholate uptake assays, protein expression/trafficking analysis\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro transport assay with mutagenesis, multiple alleles tested, disease-associated variants validated functionally\",\n      \"pmids\": [\"9109432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of the bacterial homologue of ASBT from Neisseria meningitidis (ASBT_NM) at 2.2 Å reveals two inverted structural repeats of five transmembrane helices, a core domain harboring two sodium ions, and a 'panel' domain. Taurocholate substrate is bound between the core and panel domains in an inward-facing hydrophobic cavity, suggesting a transport mechanism involving movements between these domains.\",\n      \"method\": \"X-ray crystallography at 2.2 Å resolution with substrate taurocholate co-crystallized\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with substrate bound, bacterial homologue with confirmed structural homology to human ASBT\",\n      \"pmids\": [\"21976025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Membrane topology of hASBT determined by N-glycosylation scanning mutagenesis supports a 7-transmembrane domain configuration, with extracellular loops 1 and 3 containing glycosylation-accessible reporter sites. A 3D model was built and site-directed mutagenesis of the predicted bile acid binding domain (including P290S pathological mutation) validated the model.\",\n      \"method\": \"N-glycosylation scanning mutagenesis, 3D homology modeling, site-directed mutagenesis, cholic acid docking\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (glycosylation scanning + mutagenesis + modeling), single lab\",\n      \"pmids\": [\"15350125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Dual label epitope insertion scanning mutagenesis confirmed a 7-transmembrane topology for hASBT, with extracellular loops 1 (residues 99-130), 2 (180-191), and 3 (253-287) and cytosolic localization of loops in the 7TM model. N-terminus is exofacial and C-terminus is cytosolic. This refutes the previously proposed 9TM model.\",\n      \"method\": \"Dual label epitope (HA and FLAG) insertion mutagenesis, cell surface biotinylation, confocal microscopy in permeabilized and non-permeabilized COS-1 and MDCK cells\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic epitope insertion at multiple positions with two orthogonal tags, confocal imaging with and without permeabilization, activity measurements\",\n      \"pmids\": [\"16411770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cysteine-scanning mutagenesis of TM7 of hASBT showed that TM7 lines the substrate translocation pathway. Residues in the extracellular half of TM7 are accessible to membrane-impermeant MTSES and MTSET, while the entire TM7 length is accessible to MTSEA. Substrate co-application significantly amended MTSEA sensitivity, consistent with conformational changes during transport. Loss-of-function mutants T289C, Y293C, Q297C, and A301C are MTS-inaccessible, suggesting a structural role.\",\n      \"method\": \"Cysteine-scanning mutagenesis, SCAM (substituted cysteine accessibility method) with MTSEA/MTSET/MTSES in COS-1 cells, taurocholate uptake assay\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic SCAM across 22 consecutive residues with substrate protection experiments and multiple MTS reagents\",\n      \"pmids\": [\"16899538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Cysteine scanning mutagenesis identified C51A/T, C105A/T, C144A, and C255A/T as loss-of-function mutations in hASBT, and C74A/T abolishes cell surface expression suggesting a role in protein folding/trafficking. C270A remains functional and insensitive to polar MTS reagents but shows decreased K_T and J_max with cholyl- and chenodeoxycholyl-MTS conjugates, implicating C270 in a putative substrate binding domain.\",\n      \"method\": \"Ala/Thr scanning mutagenesis of all Cys residues, MTS bile acid conjugate affinity inactivators, taurocholate transport assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis of all cysteines combined with novel bile acid-MTS affinity tools and kinetic analysis\",\n      \"pmids\": [\"15952798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Conserved residue Asp-122 in extracellular loop 1 of ASBT functions as a Na+ sensor (binding one of two co-transported Na+ ions), and Asp-124 interacts with 7α-OH groups of bile acids. Neutralization of charge at Asp-120 and Asp-122 abolishes transport, while D124A yields a functionally active transporter with low affinity for common bile acids except deoxycholic acid (which lacks a 7α-OH group).\",\n      \"method\": \"Cysteine-scanning mutagenesis of EL1 residues V99-S126, thiol modification, taurocholate uptake assay, in silico modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis with mechanistic substrate specificity analysis (deoxycholic acid lacking 7α-OH) across multiple residues\",\n      \"pmids\": [\"18508772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Cysteine-scanning mutagenesis of TM4 (21 amino acids) of hASBT showed that primarily the cytosolic half is solvent-accessible via MTSES and MTSET modification, implicating it in substrate translocation. TM4 mutants were not sensitive to equilibrative sodium conditions, ruling out a direct role for TM4 in sodium translocation.\",\n      \"method\": \"Cysteine-scanning mutagenesis of TM4, MTS reagent accessibility (MTSES/MTSET), cell surface expression assays, taurocholate uptake\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic SCAM of full TM domain with sodium sensitivity controls, in COS-1 cells\",\n      \"pmids\": [\"18311924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cysteine-scanning mutagenesis of TM3 (V127-T149) showed that the cytosolic half forms portions of the substrate exit route. Residues S128C and L145C-T149C are solvent-accessible and show significant loss of taurocholate affinity. P142 is structurally and functionally important during the transport cycle (P142C mutation severely decreases activity).\",\n      \"method\": \"Cysteine-scanning mutagenesis of TM3, SCAM with MTS reagents, kinetic analysis (K_T and J_max) for taurocholate\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic SCAM of full TM3, substrate kinetic analysis, multiple MTS reagents\",\n      \"pmids\": [\"19653651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Cysteine-scanning mutagenesis of TM1 (I29-G50) showed that TM1 plays a pivotal role in hASBT function and stability. G50 is involved in protein folding (G50C/A expression rescued by MG132 and cyclosporine A but not FK506). Residues L30, L38, T39, and M46 participate in sodium co-transport, while L34, T36, and L38 show temperature-dependent substrate protection from MTSET, indicating conformational changes in TM1 during transport.\",\n      \"method\": \"Cysteine-scanning mutagenesis, SCAM with MTSET, proteasome inhibitor rescue (MG132), cyclosporine A treatment, kinetic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic SCAM of TM1 with substrate/sodium kinetics and folding rescue experiments\",\n      \"pmids\": [\"21646357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cysteine-scanning mutagenesis of TM2 (systematic approach) showed that TM2 is inaccessible to membrane-impermeant MTSET, indicating it does not directly form the bile acid translocation pathway. Kinetic studies identified residues Q75, F76, M79, G83, L86, F90, and D91 as sodium-sensitive, and computational analysis suggests D91 coordinates sodium during transport. TM2 residues likely form the sodium binding and translocation pathway together with TM3 residues.\",\n      \"method\": \"Cysteine-scanning mutagenesis of TM2, MTSET accessibility, sodium dependence kinetics, computational analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic SCAM plus kinetics for sodium sensitivity, computational model, single rigorous study\",\n      \"pmids\": [\"24045943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cysteine-scanning mutagenesis of TM5 showed that residues G197 and G201 form a GxxxG motif important for helix-helix interactions with TM4 (interacting with G179 and P175), and G212 interacts with G237 in a GxxxG domain in TM6. TM5 residues are critical for ASBT function but are not directly involved in substrate translocation pathway.\",\n      \"method\": \"Cysteine-scanning mutagenesis of TM5, alanine conservative mutations, cell surface expression, taurocholate uptake, 3D modeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic TM5 mutagenesis with conservative mutations, but no direct SCAM accessibility experiments for TM5\",\n      \"pmids\": [\"23815591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The human ASBT gene promoter contains three functional HNF1α recognition sites essential for expression, and a DR1 motif ~1.6 kb upstream that binds PPARα/RXR heterodimer. PPARα ligands (WY14643, ciprofibrate) transactivate ASBT promoter constructs in Caco2 cells; targeted mutagenesis of the DR1 motif abolished PPARα responsiveness. Ciprofibrate treatment of SK-ChA cholangiocytes increased ASBT mRNA.\",\n      \"method\": \"Promoter-reporter (luciferase) assays, targeted mutagenesis of DR1 motif, EMSA, PPARα ligand treatment, mRNA analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis of specific promoter element, EMSA binding confirmation, both in vitro and cell-based experiments\",\n      \"pmids\": [\"12055195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human ASBT is transactivated by the glucocorticoid receptor (GR). Two glucocorticoid response elements (IR3 elements, inverted hexanucleotide repeats) in the ASBT promoter confer inducibility by GR and dexamethasone. These elements bind GR in EMSA. In vivo, budesonide treatment increased ASBT protein expression in healthy volunteers.\",\n      \"method\": \"Luciferase reporter assays with GR co-expression, EMSA, western blot of human ileal biopsies, in vivo budesonide treatment\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — promoter mutagenesis, EMSA, and in vivo human data confirming regulation\",\n      \"pmids\": [\"14684580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"FXR-activating bile acids repress rabbit ASBT expression via the cascade FXR→SHP→FTF. A cis-acting FTF binding element in the rabbit ASBT promoter (-1166/-1158) mediates this regulation. FTF stimulates ASBT promoter 4-fold; increasing SHP protein inhibits FTF-dependent transactivation. Only FXR-activating ligands (DCA, CDCA, CA) repress ASBT; non-FXR ligands (UDCA, UCA) do not.\",\n      \"method\": \"In vivo rabbit feeding experiments, 5'-flanking ASBT promoter cloning, luciferase reporter assays in Caco-2 cells, SHP overexpression, FTF site deletion mutagenesis\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vivo plus in vitro assays with promoter deletion/mutagenesis, mechanistic cascade validated\",\n      \"pmids\": [\"15591588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CDX1 and CDX2 transcription factors transcriptionally activate the human ASBT promoter. Six CDX binding sites within the ASBT promoter were verified by EMSA in vitro and chromatin immunoprecipitation in living cells. siRNA knockdown of CDX1/2 reduced ASBT mRNA expression in intestinal cells.\",\n      \"method\": \"siRNA knockdown, luciferase reporter assays, EMSA, chromatin immunoprecipitation (ChIP), real-time PCR in human biopsies\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — siRNA knockdown, EMSA, and ChIP in living cells; multiple orthogonal methods\",\n      \"pmids\": [\"22016432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PKCζ (an atypical PKC isoform) mediates posttranscriptional regulation of ileal ASBT function and membrane expression. PMA (PKC activator) inhibits ASBT-mediated taurocholate uptake in Caco-2 cells; inhibition is blocked by bisindolylmaleimide I (PKC inhibitor) and myristoylated PKCζ pseudosubstrate peptide but not by Ca2+ chelation or PI3K inhibition. PMA decreases transporter V_max and reduces ASBT plasma membrane content, suggesting vesicular recycling.\",\n      \"method\": \"Taurocholate uptake assay in Caco-2 cells, PKC inhibitors (bisindolylmaleimide I, PKCζ pseudosubstrate), kinetic analysis (V_max), plasma membrane fractionation/immunoblot\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological dissection of PKC isoform, kinetic analysis, membrane expression measurements; single lab\",\n      \"pmids\": [\"19571234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ASBT associates with lipid raft microdomains in plasma membrane. Depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) disrupts lipid raft association of ASBT and significantly reduces ASBT activity (decreased V_max) without altering plasma membrane expression. Cholesterol repletion with MβCD-cholesterol complexes restores activity.\",\n      \"method\": \"Optiprep density gradient flotation of lipid rafts, MβCD cholesterol depletion, taurocholate uptake kinetics, plasma membrane expression by biotinylation in HEK-293 cells stably transfected with ASBT, Caco-2 cells, and human ileal brush-border membrane vesicles\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell systems, flotation gradient, kinetic analysis, cholesterol rescue experiment; single lab\",\n      \"pmids\": [\"18063707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EGCG (epigallocatechin-3-gallate) inhibits ASBT activity by displacing ASBT from lipid raft fractions of the plasma membrane into detergent-soluble fractions, reducing V_max without changing total ASBT content on plasma membrane. Inhibition of PKC, PI3K, and MAPK pathways did not block EGCG effect.\",\n      \"method\": \"Taurocholate uptake assay, kinetic analysis, Optiprep density gradient fractionation, plasma membrane biotinylation, pathway inhibitor studies in HEK-293/ASBT cells and Caco-2\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell systems, kinetic and membrane fractionation data, pharmacological dissection of pathways; single lab\",\n      \"pmids\": [\"20056894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Enteropathogenic E. coli (EPEC) inhibits ileal ASBT function via its type-III secretion system (TTSS) and bundle-forming pili. Mutations in escN, espA, espB, espD (TTSS components) and BFP (pili) gene ablate EPEC inhibitory effects on ASBT. EPEC inhibition is associated with decreased V_max and reduced ASBT plasma membrane levels, and is blocked by protein tyrosine phosphatase inhibitors.\",\n      \"method\": \"Taurocholate uptake in Caco-2 and HEK-293/ASBT cells infected with EPEC and TTSS/BFP mutant strains, kinetic analysis, plasma membrane expression, tyrosine phosphatase inhibitors\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple bacterial mutants plus pharmacological inhibitors with functional and expression readouts; single lab\",\n      \"pmids\": [\"22403793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The lipid flippase heterodimer ATP8B1-CDC50A is essential for apical membrane insertion and surface expression of SLC10A2/ASBT in intestinal Caco-2 cells. ATP8B1 depletion strongly impairs SLC10A2-mediated bile salt uptake and reduces apical membrane localization of SLC10A2, caused by impaired apical membrane insertion (not reduced synthesis). Endogenous ATP8B1 exists in a functional heterodimer with CDC50A in Caco-2 cells.\",\n      \"method\": \"siRNA depletion of ATP8B1 in Caco-2 cells, bile salt uptake assay, apical membrane biotinylation, co-immunoprecipitation of ATP8B1-CDC50A, fecal bile salt analysis from PFIC1 patients\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — siRNA KD with functional readout, membrane localization by biotinylation, reciprocal Co-IP of heterodimer, human patient data correlation\",\n      \"pmids\": [\"25239307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Resveratrol promotes ASBT protein degradation via the ubiquitin-proteasome pathway in a SIRT1-independent manner. Proteasome inhibitors MG132 and lactacystin, and ubiquitin inhibitor LDN57444, reverse RSV-mediated ASBT inhibition. Immunoprecipitation revealed high levels of ubiquitinated ASBT after RSV treatment. The effect is specific to ASBT; NTCP, OSTα, and ABCG1 are unaffected. Phosphorylation at the corresponding residues of rat Ser335/Thr339 does not contribute to RSV-mediated degradation.\",\n      \"method\": \"Proteasome inhibitor rescue assays, ubiquitin inhibitor, co-immunoprecipitation for ubiquitinated ASBT, site-directed mutagenesis of phosphorylation sites, transporter expression/function assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological rescue with multiple inhibitors, immunoprecipitation of ubiquitinated ASBT, mutagenesis of phosphorylation sites; single lab\",\n      \"pmids\": [\"24498857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"N-glycosylation of ASBT is essential for function and protein stability but not for targeting to the plasma membrane. Fully mature N-acetylglucosamine-rich ASBT (41 kDa band) and core mannose-rich glycoprotein (35 kDa band) are present. The N10Q glycosylation-deficient mutant reaches the plasma membrane but has reduced half-life and increased trypsin susceptibility. High glucose increases mature glycosylated ASBT along with increased ASBT function.\",\n      \"method\": \"Tunicamycin treatment, PNGase F and Endo H glycosidase treatment, N10Q mutant expression, pulse-chase/half-life analysis, protease susceptibility assay, taurocholate uptake\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — glycosidase mapping, glycosylation site mutation, protein stability analysis; single lab\",\n      \"pmids\": [\"25855079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"hASBT forms functional non-covalent homodimers and higher-order oligomers, independent of endogenous cysteine residues. Chemical cross-linking and co-immunoprecipitation of differentially tagged (HA, FLAG) wild-type and cysteine-less hASBT confirmed dimerization. Cysteine-less hASBT showed dominant-negative effect when co-expressed with wild-type hASBT, validating functional heterodimerization/oligomerization.\",\n      \"method\": \"Site-directed mutagenesis (all 13 cysteines mutated stepwise), chemical cross-linking, co-immunoprecipitation of HA-/FLAG-tagged species, dominant-negative functional assay\",\n      \"journal\": \"Biochimica et biophysica acta. Biomembranes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP with differentially tagged constructs, cross-linking, dominant-negative functional validation; multiple orthogonal approaches in one study\",\n      \"pmids\": [\"29198943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Tyrosine phosphorylation mediated by Src family kinases (SFKs) regulates surface expression, function, and stability of hASBT. SFK inhibition (PP2) reduces hASBT function and surface expression, rescued by proteasome inhibitor MG132, indicating dephosphorylation leads to proteasome-dependent degradation. Five intracellular tyrosine residues (Y148, Y216, Y308, Y311, Y337) individually alter ASBT function without changing total cellular expression; sequential mutation of all five renders ASBT nonfunctional with diminished expression.\",\n      \"method\": \"SFK inhibitor PP2, PTP inhibitor orthovanadate, proteasome inhibitor MG132, site-directed mutagenesis of five intracellular Tyr residues, taurocholate transport assays, surface expression measurements\",\n      \"journal\": \"Molecular pharmaceutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and mutagenesis approaches with functional and expression readouts; single lab\",\n      \"pmids\": [\"31194565\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PKC regulates hASBT activity by phosphorylation at Ser335, established as the predominant phosphosite. Parallel reaction monitoring targeted mass spectrometry identified phosphorylation at Thr330, Ser334, and Ser335, with Ser335 being dominant. A proportional relationship between Ser335 phosphorylation level and ASBT bile acid uptake activity was demonstrated.\",\n      \"method\": \"Parallel reaction monitoring (PRM) targeted mass spectrometry, kinase inhibitor/activator treatments, phosphorylation site-specific analysis, bile acid uptake activity assay\",\n      \"journal\": \"ACS omega\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — targeted mass spectrometry for site-specific phosphorylation occupancy with functional correlation; single study\",\n      \"pmids\": [\"39310206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ASBT-mediated bile salt (taurocholate) absorption in the distal ileum triggers CFTR activation and consequent Cl- secretion. TC-evoked Cl- secretion was abrogated in Cftr-null mice, blocked by a selective ASBT inhibitor, and was restricted to the distal ileum. The mechanism involves CFTR channel gating modulation rather than changes in driving force, and is independent of cAMP/cGMP signaling.\",\n      \"method\": \"Short circuit current measurements across ileal tissue, Cftr-null mice, cGMP-dependent kinase II-null mice, ASBT inhibitor, neurotransmitter/prostaglandin suppression, nystatin permeabilization, H89 (PKA inhibitor)\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout, pharmacological inhibition, electrophysiology in intact tissue; multiple orthogonal approaches\",\n      \"pmids\": [\"16037545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Gut microbiota suppresses intestinal Asbt expression via the transcription factor Gata4. In germ-free or antibiotic-treated mice, Asbt expression increased in the ileum and extended more proximally in the small intestine. Genetic inactivation of either Asbt or Gata4 prevented most metabolic effects of antibiotic treatment on bile acid homeostasis, establishing Gata4 as a mediator of microbiota-dependent Asbt suppression.\",\n      \"method\": \"Germ-free and antibiotic-treated mouse models, Asbt-KO mice, intestinal-specific Gata4-iKO mice, gene expression analysis, metabolic profiling\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis using Asbt-KO and Gata4-iKO mice with metabolic phenotyping; multiple mouse models\",\n      \"pmids\": [\"26022694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mutational analysis of conserved uncharged polar residues and proline in mouse Slc10a2 (Thr130-Pro142 region) revealed that T130A causes loss of cell surface localization, P142V abolishes almost all transport activity, and T134A affects taurocholic acid affinity. This region contains residues involved in substrate interaction, function, and cellular localization.\",\n      \"method\": \"Site-directed mutagenesis of conserved residues in mouse Slc10a2, taurocholic acid uptake assay, cell surface localization assessment\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro mutagenesis with transport and localization readouts, single lab, limited number of residues\",\n      \"pmids\": [\"19584562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mutational analysis of conserved proline and uncharged polar residues in mouse Slc10a2 (Pro107-Ser128 region) revealed that P107N/L impairs cell surface localization, S126A completely impairs cellular expression, S112A abolishes transport activity while maintaining surface expression, and T110A/S128A mutations enhance membrane expression. Tyr117 mutations show reduced activity proportional to side chain van der Waals volume.\",\n      \"method\": \"Site-directed mutagenesis, taurocholate uptake assay, cell surface localization assessment in heterologous expression system\",\n      \"journal\": \"BMC physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis with transport and localization readouts; extends prior work on conserved region\",\n      \"pmids\": [\"23374508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SLC10A2 coding region variants 292G>A and 431G>A show partially impaired taurocholate transport in vitro, and a novel variant 790A>G shows near complete loss of taurocholate transport, without affecting protein expression or cell surface trafficking.\",\n      \"method\": \"PCR-based temperature gradient capillary electrophoresis (TGCE) for SNP identification, heterologous expression, taurocholate transport assay, western blot, immunofluorescence confocal microscopy\",\n      \"journal\": \"Journal of gastroenterology and hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic SNP functional characterization with transport and trafficking readouts in heterologous system; single lab\",\n      \"pmids\": [\"21649730\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Systematic comparative transport experiments with NTCP, ASBT, and SOAT using stably transfected HEK293 cells identified taurolithocholic acid (TLC) as the first common substrate of all three SLC10 carriers (K_m values: NTCP 18.4 μM, ASBT 5.9 μM, SOAT 19.3 μM). Lithocholic acid was the only bile acid not transported by any. Troglitazone, BSP, and erythrosine B were pan-SLC10 inhibitors, while cyclosporine A, irbesartan, and others only inhibited NTCP and SOAT but not ASBT.\",\n      \"method\": \"Stably transfected HEK293 cells (NTCP-, ASBT-, SOAT-), transport assays with >20 substrates, inhibition experiments\",\n      \"journal\": \"Frontiers in molecular biosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic substrate and inhibitor profiling in stable cell lines; single lab but comprehensive substrate panel\",\n      \"pmids\": [\"34079822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The bacterial ASBT homologue ASBT_NM is a monomer (not a dimer or trimer as expected for elevator-type transporters). Na+ ions shift the conformational equilibrium toward the inward-facing state, facilitating cytoplasmic uptake of substrate. Site-directed alkylation monitored by in-gel fluorescence (SDAF) and DEER EPR spectroscopy validated the conformational states.\",\n      \"method\": \"Site-directed alkylation with in-gel fluorescence (SDAF), crosslinking experiments, DEER (double electron-electron resonance) EPR spectroscopy\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — two orthogonal biophysical methods (SDAF and DEER) on bacterial homologue; sodium-dependent conformational change demonstrated\",\n      \"pmids\": [\"33359100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Ileal SLC10A2 mRNA and protein levels are significantly increased by antibiotic-mediated reduction of enterobacteria in mice (FXR-independent), with increased bile acid concentrations in portal blood. Re-administration of taurodeoxycholic acid or cholic acid (enterobacteria-biotransformed bile acids) to antibiotic-treated mice decreased ileal SLC10A2 expression, suggesting that enterobacteria-mediated bile acid biotransformation modulates SLC10A2 expression.\",\n      \"method\": \"Antibiotic treatment of C57BL/6N and FXR-null mice, ileal SLC10A2 mRNA/protein measurement, portal blood bile acid measurement, bile acid re-administration experiments\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FXR-null mouse epistasis, bile acid rescue experiment, protein and mRNA analysis; single lab\",\n      \"pmids\": [\"20884752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ASBT expression and promoter activity are significantly decreased by insulin in Caco-2 cells. In streptozotocin-induced diabetic rats, ASBT mRNA and protein are significantly elevated, and ileal Na+-dependent taurocholate uptake is increased. Insulin treatment of diabetic rats reversed increased ASBT protein expression to control levels.\",\n      \"method\": \"STZ-induced diabetes in rats, Western blot, real-time qRT-PCR, isolated intestinal epithelial cell taurocholate uptake, Caco-2 promoter activity assays with insulin treatment\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo model plus in vitro mechanistic studies; promoter and expression analysis; single lab\",\n      \"pmids\": [\"20651004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KDM6B histone demethylase directly transcriptionally activates SLC10A2; KDM6B loss increases H3K27me3 repression at the SLC10A2 promoter, activating the ERK/AP-1 pathway and CXCL/CXCR2-dependent MDSC recruitment in colorectal cancer.\",\n      \"method\": \"Intestinal epithelial-specific KDM6B deletion mouse model, ChIP for H3K27me3 at SLC10A2 promoter, ERK/AP-1 pathway analysis, MDSC recruitment assays\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for histone modification at SLC10A2 promoter, genetic KO with downstream signaling analysis; single study\",\n      \"pmids\": [\"41387297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Transcription factor KLF9 promotes intestinal Asbt expression in the terminal ileum to enhance bile acid absorption. Klf9 knockout mice show increased bile acids in gallbladder and feces with decreased serum BA levels; intestine-specific Klf9 deletion recapitulates this phenotype. Klf9 transgenic mice show the opposite. Biochemical and molecular assays confirmed KLF9 directly promotes Asbt expression.\",\n      \"method\": \"Systemic Klf9-/- mice, intestine-specific Klf9vil-/- mice, intestinal Klf9 transgenic mice, biochemical/molecular/functional assays for Asbt expression and bile acid homeostasis\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple independent genetic mouse models (KO, tissue-specific KO, transgenic overexpression) with consistent phenotype\",\n      \"pmids\": [\"35105957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Apple-derived extracellular vesicles (APEVs) downregulate ASBT/SLC10A2 expression indirectly by reducing RARα/NR1B1 protein expression. APEVs decrease binding of RARα to the SLC10A2 promoter. MicroRNAs in APEVs reduce NR1B1 mRNA stability by targeting its 3'UTR, and apple microRNA mimics suppress NR1B1 mRNA expression.\",\n      \"method\": \"Proteomics, RARα binding to SLC10A2 promoter assay, NR1B1-3'UTR reporter assay, apple microRNA mimic transfection, ASBT expression and uptake activity\",\n      \"journal\": \"Drug metabolism and pharmacokinetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — mechanistic cascade proposed with promoter binding and miRNA mimic data but indirect mechanism; single lab\",\n      \"pmids\": [\"37517353\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SLC10A2 (ASBT) is an intestinal apical sodium-dependent bile acid cotransporter with a 7-transmembrane topology (N-terminus extracellular, C-terminus cytosolic) that functions as a non-covalent homodimer/oligomer; it uses two sodium ions (with Na+-sensing residues in EL1 and TM2/TM3) to drive uphill transport of bile acids via an elevator-like mechanism involving a core domain and a panel domain (established by bacterial homologue crystal structure), with specific transmembrane helices (TM1, TM3, TM4, TM7) lining the substrate translocation pathway; its activity and surface expression are acutely regulated by PKCζ-dependent vesicular recycling, Src family kinase-mediated tyrosine phosphorylation, PKC-mediated Ser335 phosphorylation, ubiquitin-proteasome degradation (promoted by resveratrol), N-glycosylation (required for stability/protease protection), and lipid raft association; its gene expression is transcriptionally controlled by HNF1α, PPARα, glucocorticoid receptor (via IR3 elements), CDX1/CDX2 homeobox factors, KLF9, FXR-SHP-FTF cascade (bile acid feedback), KDM6B epigenetic activation, and microbiota-dependent suppression via GATA4; loss-of-function mutations cause primary bile acid malabsorption, and ASBT-mediated bile acid uptake also activates CFTR-dependent Cl- secretion in the ileum.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SLC10A2 (ASBT) is the apical sodium-dependent bile acid cotransporter that drives reclamation of bile acids from the intestinal lumen, and loss-of-function mutations in it cause primary bile acid malabsorption [#0]. The transporter adopts a 7-transmembrane topology with an exofacial N-terminus and cytosolic C-terminus [#3], and homology to a high-resolution bacterial structure organizes the protein into a sodium-bearing core domain and a panel domain between which substrate binds in an inward-facing cavity [#1]; sodium binding shifts the conformational equilibrium toward this inward-facing state to favor cytoplasmic substrate release [#32]. Systematic cysteine-scanning of individual helices has mapped function onto specific helices: TM1, TM3, TM4, and TM7 line the substrate translocation pathway and undergo substrate-dependent conformational changes [#4, #7, #8, #9], while TM2 and TM3 residues form the sodium binding/translocation route [#10], with EL1 residues Asp-122 sensing Na+ and Asp-124 recognizing the 7\\u03b1-OH of bile acids [#6]. ASBT functions as a non-covalent homodimer/oligomer [#23] and transports a defined panel of bile acids, including taurolithocholic acid shared across the SLC10 family [#31]. Surface expression and activity are tuned post-translationally by N-glycosylation required for stability [#22], lipid raft association [#17], PKC-mediated Ser335 phosphorylation [#25], Src family kinase tyrosine phosphorylation that protects against proteasomal degradation [#24], ubiquitin-proteasome turnover [#21], and apical insertion dependent on the ATP8B1-CDC50A flippase heterodimer [#20]. Gene expression is governed by a transcriptional network including HNF1\\u03b1 and PPAR\\u03b1 [#12], the glucocorticoid receptor [#13], CDX1/CDX2 [#15], KLF9 [#36], the FXR\\u2192SHP\\u2192FTF bile acid feedback cascade [#14], KDM6B-mediated chromatin activation [#35], and microbiota-dependent suppression through GATA4 [#27]. Functionally, ASBT-mediated bile acid uptake in the distal ileum activates CFTR-dependent Cl- secretion [#26].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that SLC10A2 dysfunction directly causes a human disease by showing that specific missense mutations abolish bile acid transport without disrupting protein expression or membrane trafficking.\",\n      \"evidence\": \"Site-directed mutagenesis and taurocholate uptake assays in transfected COS cells with disease-associated alleles\",\n      \"pmids\": [\"9109432\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of the affected residues\", \"Did not define the transport mechanism or sodium stoichiometry\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Resolved the membrane architecture of the transporter, supporting a 7-transmembrane topology and an initial 3D model of the bile acid binding domain.\",\n      \"evidence\": \"N-glycosylation scanning mutagenesis, homology modeling, and cholic acid docking\",\n      \"pmids\": [\"15350125\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Model not experimentally confirmed by structure\", \"Topology of cytosolic loops not fully resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Definitively settled the topology debate by confirming a 7TM model and refuting the alternative 9TM configuration, and identified TM7 as part of the substrate translocation pathway.\",\n      \"evidence\": \"Dual epitope-insertion mutagenesis with confocal imaging, plus cysteine-scanning SCAM of TM7 with substrate protection in COS-1 cells\",\n      \"pmids\": [\"16411770\", \"16899538\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not localize sodium binding sites\", \"Functional roles of other helices unaddressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Dissected which helices and residues form the substrate path versus the sodium path, identifying Asp-122 as a Na+ sensor and Asp-124 as a 7\\u03b1-OH bile acid recognition residue.\",\n      \"evidence\": \"Cysteine-scanning/SCAM of EL1 and TM4 with substrate-specificity and sodium-sensitivity kinetics\",\n      \"pmids\": [\"18508772\", \"18311924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not provide a holistic structural model integrating all helices\", \"Number and exact coordination of both Na+ ions not fully defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended the translocation-pathway map to TM3 and identified residues forming the substrate exit route, and demonstrated PKC\\u03b6-dependent post-translational control of surface expression.\",\n      \"evidence\": \"SCAM of TM3 with kinetic analysis; PKC isoform-selective pharmacology and membrane fractionation in Caco-2 cells\",\n      \"pmids\": [\"19653651\", \"19571234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phosphorylation target of PKC\\u03b6 not defined here\", \"Vesicular recycling machinery not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided the structural framework for the elevator mechanism via a substrate-bound bacterial homologue crystal structure, while helix-level mutagenesis assigned TM1 to both sodium co-transport and folding.\",\n      \"evidence\": \"X-ray crystallography of ASBT_NM with taurocholate; SCAM of TM1 with sodium/substrate kinetics and proteasome-inhibitor folding rescue\",\n      \"pmids\": [\"21976025\", \"21646357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure is of a bacterial monomer, not human ASBT\", \"Oligomeric state of human transporter unresolved at this point\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Completed the helix-by-helix functional map by assigning TM2 to the sodium pathway and TM5 to helix-helix packing rather than direct translocation.\",\n      \"evidence\": \"SCAM/mutagenesis of TM2 and TM5 with sodium kinetics, conservative substitutions, and 3D modeling\",\n      \"pmids\": [\"24045943\", \"23815591\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TM5 not tested by direct SCAM accessibility\", \"Inter-helix dynamics during transport not directly observed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the oligomeric organization of the human transporter, demonstrating functional non-covalent homodimers/oligomers independent of cysteine residues.\",\n      \"evidence\": \"Cross-linking, reciprocal co-IP of differentially tagged constructs, and dominant-negative functional assays\",\n      \"pmids\": [\"29198943\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dimer interface residues not mapped\", \"Apparent conflict with monomeric bacterial homologue not reconciled\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the sodium-driven conformational mechanism of the homologue and benchmarked human ASBT substrate/inhibitor selectivity within the SLC10 family.\",\n      \"evidence\": \"SDAF and DEER EPR on ASBT_NM; comparative substrate/inhibitor profiling of NTCP, ASBT, SOAT in stable HEK293 cells\",\n      \"pmids\": [\"33359100\", \"34079822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational dynamics not measured on human ASBT directly\", \"Structural basis of inhibitor selectivity unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified Ser335 as the predominant PKC phosphosite controlling ASBT activity, providing site-level resolution of post-translational regulation.\",\n      \"evidence\": \"Parallel reaction monitoring targeted mass spectrometry with kinase modulation and uptake assays\",\n      \"pmids\": [\"39310206\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Responsible PKC isoform at Ser335 not definitively assigned\", \"Single study, mechanism linking phosphorylation to activity unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse post-translational inputs (glycosylation, raft association, tyrosine/serine phosphorylation, ubiquitination, flippase-dependent insertion) are coordinated in vivo, and a high-resolution structure of human ASBT, remain to be established.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No human ASBT structure in the corpus\", \"Integration of competing regulatory signals not modeled\", \"Dimer interface and physiological role of oligomerization undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 1, 6, 31]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [6, 10, 31]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [6, 31]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 17, 20, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 1, 26]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [14, 27, 31]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ATP8B1\", \"CDC50A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}