{"gene":"FKBP2","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1991,"finding":"FKBP-13 (FKBP2) is a membrane-associated 13-kDa FK506- and rapamycin-binding protein with a 21-amino acid signal peptide and a C-terminal ER retention sequence (Arg-Thr-Glu-Leu), lacking a transmembrane domain but appearing to localize to the ER membrane. The conserved residues comprising the drug-binding site and rotamase (peptidylprolyl cis-trans isomerase) active site of FKBP-12 are completely conserved in FKBP-13.","method":"Molecular cloning, sequence analysis, subcellular fractionation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — molecular cloning with sequence-based inference of active-site conservation; localization by fractionation; single lab but multiple methods","pmids":["1713687"],"is_preprint":false},{"year":1992,"finding":"Yeast FKBP-13 (encoded by FKB2) binds FK506 with 15-fold lower affinity than FKBP-12 and possesses peptidylprolyl cis-trans isomerase (PPIase/rotamase) activity with a similar substrate profile. It is membrane-associated and has a hydrophobic signal sequence. fkb2 and fkb1/fkb2 double mutants are viable and show no altered sensitivity to FK506 or rapamycin, indicating distinct functions from FKBP-12.","method":"Protein purification, FK506-binding assay, PPIase activity assay, genetic epistasis (null mutants), N-terminal sequencing, gene cloning","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic activity and binding assays with purified protein plus genetic epistasis; replicated across yeast and mammalian systems in multiple papers","pmids":["1380159"],"is_preprint":false},{"year":1993,"finding":"FKBP13 localizes to the lumen of the rough endoplasmic reticulum (ER), not the cytosol. By immunoblotting of subcellular fractions from canine pancreatic homogenate, FKBP13 co-fractionated exclusively with rough microsomal ER markers (BiP, grp94, ribophorin I) and co-banded with them on isopycnic sucrose gradients. Immunofluorescence of overexpressed FKBP13 cDNA in HeLa cells showed an ER pattern, and membrane/lumen separation confirmed it as a luminal ER protein.","method":"Subcellular fractionation, isopycnic sucrose gradient, immunoblotting, immunofluorescence microscopy, membrane/lumen separation","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal localization methods (fractionation, gradient, immunofluorescence), replicated in two species","pmids":["8373365"],"is_preprint":false},{"year":1993,"finding":"In Saccharomyces cerevisiae, FKB2 (encoding FKBP-13) is transcriptionally induced by accumulation of unfolded proteins in the ER (unfolded protein response, UPR). The induction is mediated through a 21-bp UPR element in the 5' noncoding region of FKB2, analogous to UPR elements in yeast KAR2 (BiP) and mammalian GRP78/GRP94. Mutations blocking N-glycosylation (tunicamycin treatment, sec53-6 mutant) elevated FKB2 mRNA; blocks in other secretory steps did not, indicating specificity to ER unfolded protein accumulation.","method":"Northern blot, genetic epistasis (secretory pathway mutants), promoter element analysis (UPR element identification)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple secretory mutants, promoter element mapping, replicated with chemical (tunicamycin) and genetic perturbations","pmids":["7685904"],"is_preprint":false},{"year":1994,"finding":"FKBP13 mRNA is induced in mammalian (MDCK) cells by agents that cause protein misfolding in the ER: tunicamycin causes ~2-fold induction and Ca2+ ionophores (ionomycin) cause up to 5-fold induction. The increase is due to transcriptional upregulation rather than mRNA stabilization. The 5' flanking region of murine FKBP13 contains a ~37 bp sequence with ~50% identity to the BiP unfolded protein response element, supporting a role as an ER chaperone.","method":"Northern blot, actinomycin D mRNA stability assay, 5' flanking region sequencing and comparison","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Northern blot with mRNA stability controls and promoter sequence analysis; single lab, two orthogonal methods","pmids":["7526846"],"is_preprint":false},{"year":1998,"finding":"FKBP13 physically interacts with the COOH-terminal domain (CTD) of a novel broadly expressed protein 4.1 homologue (4.1G). The interaction requires the histidyl-proline moiety of 4.1G-CTD. FKBP13 co-fractionates with the red blood cell protein 4.1R in erythrocyte ghost, inside-out vesicle, and Triton shell preparations, indicating a role as a component of membrane cytoskeletal scaffolds in addition to its ER chaperone function.","method":"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation, truncation/mutation analysis of 4.1G-CTD, subcellular fractionation of erythrocyte membranes","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — three orthogonal binding methods (yeast two-hybrid, in vitro binding, Co-IP) plus mutagenesis defining the binding requirement, single lab","pmids":["9531554"],"is_preprint":false},{"year":2004,"finding":"FKBP13 specifically interacts with the C-chain of complement C1q (C1q-C). The interaction was detected by yeast two-hybrid screening and confirmed by a protein complementation assay. FKBP12, FKBP25, FKBP52, and CypA did not interact with C1q-C, demonstrating specificity. FK506 did not prevent the interaction, suggesting that regions outside the drug-binding pocket mediate the C1q-C interaction.","method":"Yeast two-hybrid, protein complementation assay, competition with FK506","journal":"BMC pharmacology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — two yeast-based assays with specificity controls; no Co-IP or in vitro biochemical validation; single lab","pmids":["15353007"],"is_preprint":false},{"year":1996,"finding":"The solution structure of FK506 bound to a Q50R/A95H/K98I triple mutant of FKBP-13 was determined by NMR (HSQC-NOESY with 13C-labeled FK506, 87 NOE distance restraints). The conformation of FK506 in complex with mutant FKBP-13 is similar to that in wild-type FKBP-12, consistent with conservation of the drug-binding pocket.","method":"NMR (HSQC-NOESY), time-averaged restrained molecular dynamics (TARMD) structure calculation","journal":"Journal of biomolecular NMR","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structure determination is Tier 1, but performed on a mutant form only in a single study focused on methodology","pmids":["21136324"],"is_preprint":false},{"year":2021,"finding":"FKBP2 (FKBP13) inhibits Bax-induced apoptosis in yeast. When co-expressed with the pro-apoptotic protein Bax in a yeast model, FKBP2 protected cells from Bax-induced cell death, identified via human hippocampal cDNA library screening in a yeast-based system.","method":"Yeast-based cDNA library screen, co-expression of Bax and HA-tagged FKBP2, cell viability assay","journal":"Cell biology and toxicology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single method (yeast co-expression viability), single lab, no mechanistic dissection of how protection occurs","pmids":["34342774"],"is_preprint":false},{"year":2025,"finding":"FKBP2 functions as a critical ER-localized cis-trans prolyl isomerase required for proinsulin processing and β cell differentiation. Loss of FKBP2 in human pluripotent stem cell-derived β cells impairs insulin granule morphology and reduces insulin secretion. FKBP2 deficiency causes sustained ER stress, elevated intracellular calcium, and activation of the NFAT2–HDAC9 signaling axis, shifting endocrine lineage allocation from β cells to α cells. Pharmacological inhibition of HDAC class IIa partially rescues β cell differentiation, supporting a causal role for this pathway downstream of FKBP2 loss.","method":"Loss- and gain-of-function FKBP2 models in hPSCs, single-cell RNA sequencing, insulin secretion assay, electron microscopy of granule morphology, pharmacological HDAC inhibition, intracellular calcium measurement","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (scRNA-seq, functional assays, pharmacological rescue) in single lab; preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.09.01.672695"],"is_preprint":true}],"current_model":"FKBP2 (FKBP13) is an ER-luminal cis-trans prolyl isomerase (PPIase) that binds FK506 and rapamycin, is transcriptionally induced by ER unfolded protein stress via a UPR promoter element, serves as an ER chaperone for protein folding (including proinsulin), interacts with cytoskeletal protein 4.1G and complement C1q-C, can protect against Bax-induced apoptosis, and regulates pancreatic β cell differentiation through an NFAT2–HDAC9 axis downstream of ER stress."},"narrative":{"mechanistic_narrative":"FKBP2 (FKBP13) is an ER-luminal peptidylprolyl cis-trans isomerase (PPIase/rotamase) that binds the immunosuppressants FK506 and rapamycin and functions in folding and quality control of secretory proteins [PMID:1380159, PMID:8373365]. The protein carries an N-terminal signal peptide and a C-terminal ER retention sequence, and orthogonal fractionation and immunofluorescence place it in the lumen of the rough ER alongside BiP, grp94, and ribophorin I [PMID:1713687, PMID:8373365]; its drug-binding pocket is structurally conserved with FKBP-12 [PMID:21136324]. Consistent with a chaperone role, FKBP2/FKB2 transcription is induced by accumulation of unfolded protein in the ER through a UPR promoter element, in both yeast and mammalian cells [PMID:7685904, PMID:7526846]. In human stem-cell-derived β cells, FKBP2 is required for proinsulin processing and insulin granule formation; its loss provokes sustained ER stress and elevated intracellular calcium that activate an NFAT2–HDAC9 axis and divert endocrine lineage allocation away from β cells [PMID:bio_10.1101_2025.09.01.672695]. Beyond its ER chaperone function, FKBP2 physically associates with the C-terminal domain of protein 4.1G and co-fractionates with the membrane cytoskeletal protein 4.1R, and it binds the C-chain of complement C1q through a surface outside the FK506 pocket [PMID:9531554, PMID:15353007]. It also protects yeast from Bax-induced cell death [PMID:34342774].","teleology":[{"year":1991,"claim":"Established FKBP2 as a distinct FK506/rapamycin-binding immunophilin localized to the ER membrane rather than the cytosol, raising the question of a secretory-pathway-specific FKBP.","evidence":"Molecular cloning and sequence analysis with subcellular fractionation","pmids":["1713687"],"confidence":"Medium","gaps":["Active-site conservation inferred from sequence, not measured enzymatically","Luminal vs membrane topology not yet resolved"]},{"year":1992,"claim":"Demonstrated that FKBP2 is a catalytically active rotamase with FK506-binding capacity but functions distinctly from FKBP-12, since null mutants are viable and drug-insensitive.","evidence":"Purified-protein PPIase and FK506-binding assays plus genetic epistasis in yeast","pmids":["1380159"],"confidence":"High","gaps":["Physiological folding substrates not identified","Lower FK506 affinity than FKBP-12 leaves the in vivo drug relevance unclear"]},{"year":1993,"claim":"Confirmed FKBP2 resides in the rough ER lumen, defining its compartment for chaperone function.","evidence":"Subcellular fractionation, isopycnic gradients, and immunofluorescence across two species","pmids":["8373365"],"confidence":"High","gaps":["Does not identify luminal client proteins","Mechanism of luminal retention not dissected"]},{"year":1993,"claim":"Linked FKBP2 to the unfolded protein response by showing transcriptional induction through a defined UPR promoter element specific to ER unfolded-protein accumulation.","evidence":"Northern blot, secretory-pathway mutant epistasis, and UPR element mapping in yeast","pmids":["7685904"],"confidence":"High","gaps":["Functional consequence of induction (which clients require FKBP2) not shown"]},{"year":1994,"claim":"Extended UPR-driven induction to mammalian cells, supporting a conserved role as a stress-inducible ER chaperone.","evidence":"Northern blot with actinomycin D stability controls and promoter comparison in MDCK cells","pmids":["7526846"],"confidence":"Medium","gaps":["UPR element only inferred by sequence similarity, not functionally mapped in mammalian cells","Chaperone clients not identified"]},{"year":1996,"claim":"Showed the FKBP2 drug-binding pocket adopts an FKBP-12-like FK506 conformation, structurally rationalizing immunophilin classification.","evidence":"NMR (HSQC-NOESY) on a triple mutant of FKBP-13 bound to 13C-FK506","pmids":["21136324"],"confidence":"Medium","gaps":["Structure determined only for a triple mutant, not wild type","No structure of a physiological substrate complex"]},{"year":1998,"claim":"Identified a cytoskeletal-scaffolding role by showing FKBP2 binds protein 4.1G via its histidyl-proline-containing C-terminal domain and co-fractionates with 4.1R in erythrocyte membranes.","evidence":"Yeast two-hybrid, in vitro binding, Co-IP, and erythrocyte membrane fractionation","pmids":["9531554"],"confidence":"High","gaps":["How an ER-luminal protein engages cytoplasmic 4.1 proteins is unresolved","Functional consequence of the 4.1 interaction not established"]},{"year":2004,"claim":"Defined a specific interaction with complement C1q C-chain mediated by a surface outside the drug-binding pocket, distinguishing FKBP2 from related immunophilins.","evidence":"Yeast two-hybrid and protein complementation assay with FK506 competition and FKBP/Cyp specificity controls","pmids":["15353007"],"confidence":"Medium","gaps":["No Co-IP or in vitro biochemical confirmation","Physiological setting of the C1q-C interaction unknown"]},{"year":2021,"claim":"Implicated FKBP2 in cell survival by showing it suppresses Bax-induced death in a yeast model.","evidence":"Yeast cDNA-library screen with Bax/FKBP2 co-expression viability assay","pmids":["34342774"],"confidence":"Low","gaps":["Single-method yeast viability assay with no mechanistic dissection","Not validated in mammalian apoptosis systems"]},{"year":2025,"claim":"Connected FKBP2 PPIase function to a physiological program—proinsulin processing and β cell fate—via an ER stress→calcium→NFAT2–HDAC9 axis that controls endocrine lineage allocation.","evidence":"Loss/gain-of-function in hPSC-derived β cells, scRNA-seq, insulin secretion and granule EM, calcium measurement, and pharmacological HDAC IIa rescue (preprint)","pmids":["bio_10.1101_2025.09.01.672695"],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Direct proinsulin–FKBP2 isomerase substrate relationship not biochemically demonstrated","Whether NFAT2–HDAC9 activation is a direct consequence of lost PPIase activity is unresolved"]},{"year":null,"claim":"The direct physiological folding substrates of FKBP2's rotamase activity and how its ER chaperone function mechanistically links to its cytoskeletal, complement, and apoptotic associations remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No defined endogenous PPIase substrate beyond the proinsulin functional link","Topological reconciliation of luminal ER residence with 4.1 cytoskeletal binding is unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,9]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[3,4,9]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,2]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,9]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[3,4]}],"complexes":[],"partners":["EPB41L2","EPB41","C1QC"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P26885","full_name":"Peptidyl-prolyl cis-trans isomerase FKBP2","aliases":["13 kDa FK506-binding protein","13 kDa FKBP","FKBP-13","FK506-binding protein 2","FKBP-2","Immunophilin FKBP13","Rotamase"],"length_aa":142,"mass_kda":15.6,"function":"PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/P26885/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FKBP2","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FKBP2","total_profiled":1310},"omim":[{"mim_id":"608455","title":"GLYCOGEN PHOSPHORYLASE, MUSCLE; PYGM","url":"https://www.omim.org/entry/608455"},{"mim_id":"601516","title":"SPLICING FACTOR 1; SF1","url":"https://www.omim.org/entry/601516"},{"mim_id":"601442","title":"COFILIN 1; CFL1","url":"https://www.omim.org/entry/601442"},{"mim_id":"600526","title":"MAP/MICROTUBULE AFFINITY-REGULATING KINASE 2; MARK2","url":"https://www.omim.org/entry/600526"},{"mim_id":"600230","title":"PHOSPHOLIPASE C, BETA-3; PLCB3","url":"https://www.omim.org/entry/600230"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/FKBP2"},"hgnc":{"alias_symbol":["FKBP-13","FKBP13","PPIase"],"prev_symbol":[]},"alphafold":{"accession":"P26885","domains":[{"cath_id":"3.10.50.40","chopping":"31-136","consensus_level":"high","plddt":98.0948,"start":31,"end":136}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P26885","model_url":"https://alphafold.ebi.ac.uk/files/AF-P26885-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P26885-F1-predicted_aligned_error_v6.png","plddt_mean":89.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FKBP2","jax_strain_url":"https://www.jax.org/strain/search?query=FKBP2"},"sequence":{"accession":"P26885","fasta_url":"https://rest.uniprot.org/uniprotkb/P26885.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P26885/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P26885"}},"corpus_meta":[{"pmid":"1713687","id":"PMC_1713687","title":"Molecular cloning of a membrane-associated human FK506- and rapamycin-binding protein, FKBP-13.","date":"1991","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/1713687","citation_count":180,"is_preprint":false},{"pmid":"1371107","id":"PMC_1371107","title":"The Hsp56 component of steroid receptor complexes binds to immobilized FK506 and shows homology to FKBP-12 and FKBP-13.","date":"1992","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1371107","citation_count":169,"is_preprint":false},{"pmid":"9531554","id":"PMC_9531554","title":"The 13-kD FK506 binding protein, FKBP13, interacts with a novel homologue of the erythrocyte membrane cytoskeletal protein 4.1.","date":"1998","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/9531554","citation_count":100,"is_preprint":false},{"pmid":"7685904","id":"PMC_7685904","title":"The FKB2 gene of Saccharomyces cerevisiae, encoding the immunosuppressant-binding protein FKBP-13, is regulated in response to accumulation of unfolded proteins in the endoplasmic reticulum.","date":"1993","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/7685904","citation_count":79,"is_preprint":false},{"pmid":"1380159","id":"PMC_1380159","title":"Yeast FKBP-13 is a membrane-associated FK506-binding protein encoded by the nonessential gene FKB2.","date":"1992","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/1380159","citation_count":57,"is_preprint":false},{"pmid":"8373365","id":"PMC_8373365","title":"Localization of the FK506-binding protein, FKBP 13, to the lumen of the endoplasmic reticulum.","date":"1993","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/8373365","citation_count":56,"is_preprint":false},{"pmid":"7526846","id":"PMC_7526846","title":"Induction of the FK506-binding protein, FKBP13, under conditions which misfold proteins in the endoplasmic reticulum.","date":"1994","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/7526846","citation_count":51,"is_preprint":false},{"pmid":"1279908","id":"PMC_1279908","title":"Saccharomyces cerevisiae contains a homolog of human FKBP-13, a membrane-associated FK506/rapamycin binding protein.","date":"1992","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/1279908","citation_count":21,"is_preprint":false},{"pmid":"7535744","id":"PMC_7535744","title":"Exclusion of the 13-kDa rapamycin binding protein gene (FKBP2) as a candidate gene for multiple endocrine neoplasia type 1.","date":"1995","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/7535744","citation_count":20,"is_preprint":false},{"pmid":"7505249","id":"PMC_7505249","title":"Structural organization of the genes encoding human and murine FK506-binding protein (FKBP) 13 and comparison to FKBP1.","date":"1993","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/7505249","citation_count":15,"is_preprint":false},{"pmid":"1281998","id":"PMC_1281998","title":"Chromosomal band assignments of the genes encoding human FKBP12 and FKBP13.","date":"1992","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/1281998","citation_count":10,"is_preprint":false},{"pmid":"34342774","id":"PMC_34342774","title":"FK506-binding protein 2 (FKBP13) inhibit Bax-induced apoptosis in Saccharomyces cerevisiae (yeast).","date":"2021","source":"Cell biology and toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/34342774","citation_count":3,"is_preprint":false},{"pmid":"15353007","id":"PMC_15353007","title":"The FK506 binding protein 13 kDa (FKBP13) interacts with the C-chain of complement C1q.","date":"2004","source":"BMC pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/15353007","citation_count":2,"is_preprint":false},{"pmid":"21136324","id":"PMC_21136324","title":"Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13.","date":"1996","source":"Journal of biomolecular NMR","url":"https://pubmed.ncbi.nlm.nih.gov/21136324","citation_count":2,"is_preprint":false},{"pmid":"41645412","id":"PMC_41645412","title":"Geminivirus V2-mediated inhibition of plant FKBP13 PPIase activity activates UPR signaling to enhance viral pathogenicity.","date":"2026","source":"Journal of integrative plant biology","url":"https://pubmed.ncbi.nlm.nih.gov/41645412","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.01.672695","title":"ER stress and cell fate: FKBP2 regulates proinsulin folding and α- vs. β cell differentiation via NFAT and HDAC9","date":"2025-09-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.01.672695","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9243,"output_tokens":3044,"usd":0.036694,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10275,"output_tokens":3131,"usd":0.064825,"stage2_stop_reason":"end_turn"},"total_usd":0.101519,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"FKBP-13 (FKBP2) is a membrane-associated 13-kDa FK506- and rapamycin-binding protein with a 21-amino acid signal peptide and a C-terminal ER retention sequence (Arg-Thr-Glu-Leu), lacking a transmembrane domain but appearing to localize to the ER membrane. The conserved residues comprising the drug-binding site and rotamase (peptidylprolyl cis-trans isomerase) active site of FKBP-12 are completely conserved in FKBP-13.\",\n      \"method\": \"Molecular cloning, sequence analysis, subcellular fractionation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — molecular cloning with sequence-based inference of active-site conservation; localization by fractionation; single lab but multiple methods\",\n      \"pmids\": [\"1713687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Yeast FKBP-13 (encoded by FKB2) binds FK506 with 15-fold lower affinity than FKBP-12 and possesses peptidylprolyl cis-trans isomerase (PPIase/rotamase) activity with a similar substrate profile. It is membrane-associated and has a hydrophobic signal sequence. fkb2 and fkb1/fkb2 double mutants are viable and show no altered sensitivity to FK506 or rapamycin, indicating distinct functions from FKBP-12.\",\n      \"method\": \"Protein purification, FK506-binding assay, PPIase activity assay, genetic epistasis (null mutants), N-terminal sequencing, gene cloning\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic activity and binding assays with purified protein plus genetic epistasis; replicated across yeast and mammalian systems in multiple papers\",\n      \"pmids\": [\"1380159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"FKBP13 localizes to the lumen of the rough endoplasmic reticulum (ER), not the cytosol. By immunoblotting of subcellular fractions from canine pancreatic homogenate, FKBP13 co-fractionated exclusively with rough microsomal ER markers (BiP, grp94, ribophorin I) and co-banded with them on isopycnic sucrose gradients. Immunofluorescence of overexpressed FKBP13 cDNA in HeLa cells showed an ER pattern, and membrane/lumen separation confirmed it as a luminal ER protein.\",\n      \"method\": \"Subcellular fractionation, isopycnic sucrose gradient, immunoblotting, immunofluorescence microscopy, membrane/lumen separation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal localization methods (fractionation, gradient, immunofluorescence), replicated in two species\",\n      \"pmids\": [\"8373365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"In Saccharomyces cerevisiae, FKB2 (encoding FKBP-13) is transcriptionally induced by accumulation of unfolded proteins in the ER (unfolded protein response, UPR). The induction is mediated through a 21-bp UPR element in the 5' noncoding region of FKB2, analogous to UPR elements in yeast KAR2 (BiP) and mammalian GRP78/GRP94. Mutations blocking N-glycosylation (tunicamycin treatment, sec53-6 mutant) elevated FKB2 mRNA; blocks in other secretory steps did not, indicating specificity to ER unfolded protein accumulation.\",\n      \"method\": \"Northern blot, genetic epistasis (secretory pathway mutants), promoter element analysis (UPR element identification)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple secretory mutants, promoter element mapping, replicated with chemical (tunicamycin) and genetic perturbations\",\n      \"pmids\": [\"7685904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"FKBP13 mRNA is induced in mammalian (MDCK) cells by agents that cause protein misfolding in the ER: tunicamycin causes ~2-fold induction and Ca2+ ionophores (ionomycin) cause up to 5-fold induction. The increase is due to transcriptional upregulation rather than mRNA stabilization. The 5' flanking region of murine FKBP13 contains a ~37 bp sequence with ~50% identity to the BiP unfolded protein response element, supporting a role as an ER chaperone.\",\n      \"method\": \"Northern blot, actinomycin D mRNA stability assay, 5' flanking region sequencing and comparison\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Northern blot with mRNA stability controls and promoter sequence analysis; single lab, two orthogonal methods\",\n      \"pmids\": [\"7526846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"FKBP13 physically interacts with the COOH-terminal domain (CTD) of a novel broadly expressed protein 4.1 homologue (4.1G). The interaction requires the histidyl-proline moiety of 4.1G-CTD. FKBP13 co-fractionates with the red blood cell protein 4.1R in erythrocyte ghost, inside-out vesicle, and Triton shell preparations, indicating a role as a component of membrane cytoskeletal scaffolds in addition to its ER chaperone function.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation, truncation/mutation analysis of 4.1G-CTD, subcellular fractionation of erythrocyte membranes\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three orthogonal binding methods (yeast two-hybrid, in vitro binding, Co-IP) plus mutagenesis defining the binding requirement, single lab\",\n      \"pmids\": [\"9531554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"FKBP13 specifically interacts with the C-chain of complement C1q (C1q-C). The interaction was detected by yeast two-hybrid screening and confirmed by a protein complementation assay. FKBP12, FKBP25, FKBP52, and CypA did not interact with C1q-C, demonstrating specificity. FK506 did not prevent the interaction, suggesting that regions outside the drug-binding pocket mediate the C1q-C interaction.\",\n      \"method\": \"Yeast two-hybrid, protein complementation assay, competition with FK506\",\n      \"journal\": \"BMC pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — two yeast-based assays with specificity controls; no Co-IP or in vitro biochemical validation; single lab\",\n      \"pmids\": [\"15353007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The solution structure of FK506 bound to a Q50R/A95H/K98I triple mutant of FKBP-13 was determined by NMR (HSQC-NOESY with 13C-labeled FK506, 87 NOE distance restraints). The conformation of FK506 in complex with mutant FKBP-13 is similar to that in wild-type FKBP-12, consistent with conservation of the drug-binding pocket.\",\n      \"method\": \"NMR (HSQC-NOESY), time-averaged restrained molecular dynamics (TARMD) structure calculation\",\n      \"journal\": \"Journal of biomolecular NMR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structure determination is Tier 1, but performed on a mutant form only in a single study focused on methodology\",\n      \"pmids\": [\"21136324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FKBP2 (FKBP13) inhibits Bax-induced apoptosis in yeast. When co-expressed with the pro-apoptotic protein Bax in a yeast model, FKBP2 protected cells from Bax-induced cell death, identified via human hippocampal cDNA library screening in a yeast-based system.\",\n      \"method\": \"Yeast-based cDNA library screen, co-expression of Bax and HA-tagged FKBP2, cell viability assay\",\n      \"journal\": \"Cell biology and toxicology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single method (yeast co-expression viability), single lab, no mechanistic dissection of how protection occurs\",\n      \"pmids\": [\"34342774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FKBP2 functions as a critical ER-localized cis-trans prolyl isomerase required for proinsulin processing and β cell differentiation. Loss of FKBP2 in human pluripotent stem cell-derived β cells impairs insulin granule morphology and reduces insulin secretion. FKBP2 deficiency causes sustained ER stress, elevated intracellular calcium, and activation of the NFAT2–HDAC9 signaling axis, shifting endocrine lineage allocation from β cells to α cells. Pharmacological inhibition of HDAC class IIa partially rescues β cell differentiation, supporting a causal role for this pathway downstream of FKBP2 loss.\",\n      \"method\": \"Loss- and gain-of-function FKBP2 models in hPSCs, single-cell RNA sequencing, insulin secretion assay, electron microscopy of granule morphology, pharmacological HDAC inhibition, intracellular calcium measurement\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (scRNA-seq, functional assays, pharmacological rescue) in single lab; preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.09.01.672695\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"FKBP2 (FKBP13) is an ER-luminal cis-trans prolyl isomerase (PPIase) that binds FK506 and rapamycin, is transcriptionally induced by ER unfolded protein stress via a UPR promoter element, serves as an ER chaperone for protein folding (including proinsulin), interacts with cytoskeletal protein 4.1G and complement C1q-C, can protect against Bax-induced apoptosis, and regulates pancreatic β cell differentiation through an NFAT2–HDAC9 axis downstream of ER stress.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FKBP2 (FKBP13) is an ER-luminal peptidylprolyl cis-trans isomerase (PPIase/rotamase) that binds the immunosuppressants FK506 and rapamycin and functions in folding and quality control of secretory proteins [#1, #2]. The protein carries an N-terminal signal peptide and a C-terminal ER retention sequence, and orthogonal fractionation and immunofluorescence place it in the lumen of the rough ER alongside BiP, grp94, and ribophorin I [#0, #2]; its drug-binding pocket is structurally conserved with FKBP-12 [#7]. Consistent with a chaperone role, FKBP2/FKB2 transcription is induced by accumulation of unfolded protein in the ER through a UPR promoter element, in both yeast and mammalian cells [#3, #4]. In human stem-cell-derived β cells, FKBP2 is required for proinsulin processing and insulin granule formation; its loss provokes sustained ER stress and elevated intracellular calcium that activate an NFAT2–HDAC9 axis and divert endocrine lineage allocation away from β cells [#9]. Beyond its ER chaperone function, FKBP2 physically associates with the C-terminal domain of protein 4.1G and co-fractionates with the membrane cytoskeletal protein 4.1R, and it binds the C-chain of complement C1q through a surface outside the FK506 pocket [#5, #6]. It also protects yeast from Bax-induced cell death [#8].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Established FKBP2 as a distinct FK506/rapamycin-binding immunophilin localized to the ER membrane rather than the cytosol, raising the question of a secretory-pathway-specific FKBP.\",\n      \"evidence\": \"Molecular cloning and sequence analysis with subcellular fractionation\",\n      \"pmids\": [\"1713687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Active-site conservation inferred from sequence, not measured enzymatically\", \"Luminal vs membrane topology not yet resolved\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Demonstrated that FKBP2 is a catalytically active rotamase with FK506-binding capacity but functions distinctly from FKBP-12, since null mutants are viable and drug-insensitive.\",\n      \"evidence\": \"Purified-protein PPIase and FK506-binding assays plus genetic epistasis in yeast\",\n      \"pmids\": [\"1380159\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological folding substrates not identified\", \"Lower FK506 affinity than FKBP-12 leaves the in vivo drug relevance unclear\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Confirmed FKBP2 resides in the rough ER lumen, defining its compartment for chaperone function.\",\n      \"evidence\": \"Subcellular fractionation, isopycnic gradients, and immunofluorescence across two species\",\n      \"pmids\": [\"8373365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify luminal client proteins\", \"Mechanism of luminal retention not dissected\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Linked FKBP2 to the unfolded protein response by showing transcriptional induction through a defined UPR promoter element specific to ER unfolded-protein accumulation.\",\n      \"evidence\": \"Northern blot, secretory-pathway mutant epistasis, and UPR element mapping in yeast\",\n      \"pmids\": [\"7685904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of induction (which clients require FKBP2) not shown\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Extended UPR-driven induction to mammalian cells, supporting a conserved role as a stress-inducible ER chaperone.\",\n      \"evidence\": \"Northern blot with actinomycin D stability controls and promoter comparison in MDCK cells\",\n      \"pmids\": [\"7526846\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"UPR element only inferred by sequence similarity, not functionally mapped in mammalian cells\", \"Chaperone clients not identified\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Showed the FKBP2 drug-binding pocket adopts an FKBP-12-like FK506 conformation, structurally rationalizing immunophilin classification.\",\n      \"evidence\": \"NMR (HSQC-NOESY) on a triple mutant of FKBP-13 bound to 13C-FK506\",\n      \"pmids\": [\"21136324\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structure determined only for a triple mutant, not wild type\", \"No structure of a physiological substrate complex\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identified a cytoskeletal-scaffolding role by showing FKBP2 binds protein 4.1G via its histidyl-proline-containing C-terminal domain and co-fractionates with 4.1R in erythrocyte membranes.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, Co-IP, and erythrocyte membrane fractionation\",\n      \"pmids\": [\"9531554\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How an ER-luminal protein engages cytoplasmic 4.1 proteins is unresolved\", \"Functional consequence of the 4.1 interaction not established\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined a specific interaction with complement C1q C-chain mediated by a surface outside the drug-binding pocket, distinguishing FKBP2 from related immunophilins.\",\n      \"evidence\": \"Yeast two-hybrid and protein complementation assay with FK506 competition and FKBP/Cyp specificity controls\",\n      \"pmids\": [\"15353007\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No Co-IP or in vitro biochemical confirmation\", \"Physiological setting of the C1q-C interaction unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Implicated FKBP2 in cell survival by showing it suppresses Bax-induced death in a yeast model.\",\n      \"evidence\": \"Yeast cDNA-library screen with Bax/FKBP2 co-expression viability assay\",\n      \"pmids\": [\"34342774\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single-method yeast viability assay with no mechanistic dissection\", \"Not validated in mammalian apoptosis systems\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected FKBP2 PPIase function to a physiological program—proinsulin processing and β cell fate—via an ER stress→calcium→NFAT2–HDAC9 axis that controls endocrine lineage allocation.\",\n      \"evidence\": \"Loss/gain-of-function in hPSC-derived β cells, scRNA-seq, insulin secretion and granule EM, calcium measurement, and pharmacological HDAC IIa rescue (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.09.01.672695\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"Direct proinsulin–FKBP2 isomerase substrate relationship not biochemically demonstrated\", \"Whether NFAT2–HDAC9 activation is a direct consequence of lost PPIase activity is unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct physiological folding substrates of FKBP2's rotamase activity and how its ER chaperone function mechanistically links to its cytoskeletal, complement, and apoptotic associations remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No defined endogenous PPIase substrate beyond the proinsulin functional link\", \"Topological reconciliation of luminal ER residence with 4.1 cytoskeletal binding is unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 9]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [3, 4, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 9]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EPB41L2\", \"EPB41\", \"C1QC\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}