{"gene":"TMEM35A","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2016,"finding":"NACHO (TMEM35A) is an endoplasmic reticulum-resident transmembrane protein that functions as an essential chaperone for α7 nicotinic acetylcholine receptor (nAChR) assembly. NACHO promotes α7 protein folding, maturation through the Golgi complex, and surface expression. Knockdown in cultured hippocampal neurons or knockout in mice selectively and completely disrupts α7 receptor assembly and abolishes α7 channel function.","method":"Genomic screening, siRNA knockdown in primary neurons, constitutive knockout mice, radioligand binding (α-bungarotoxin), electrophysiology, immunofluorescence, subcellular fractionation","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KD, KO, binding assays, electrophysiology, subcellular localization), replicated across cell and animal models in a single rigorous study","pmids":["26875622"],"is_preprint":false},{"year":2017,"finding":"NACHO mediates assembly of all major classes of nAChRs, not just α7. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin (α7), epibatidine (α4β2), and conotoxin MII (α6-containing) nAChRs. NACHO acts at early intracellular stages of nAChR subunit assembly and synergizes with RIC-3 for receptor surface expression. GABAA receptors are unaffected, indicating NACHO specificity for nAChRs.","method":"NACHO knockout mice, radioligand binding assays (α-bungarotoxin, epibatidine, conotoxin MII), behavioral assays, heterologous expression","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — constitutive KO mice with multiple receptor-subtype-specific radioligand binding assays and a specific negative control (GABAA), independent replication of the α7 finding from prior study","pmids":["28445721"],"is_preprint":false},{"year":2020,"finding":"NACHO-mediated α7 nAChR assembly requires the α7 ectodomain and two amino acids in the second transmembrane domain of α7. NACHO associates with the ER oligosaccharyltransferase machinery and with the lectin chaperone calnexin, as shown by proteomics. NACHO-mediated assembly and channel function require N-glycosylation and calnexin chaperone activity, placing NACHO within the N-glycosylation ER chaperone pathway.","method":"α7 chimera and mutagenesis constructs, surface trafficking assays, proteomics/mass spectrometry (Co-IP), pharmacological inhibition of N-glycosylation and calnexin, electrophysiology","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — site-directed mutagenesis of receptor substrate, proteomics-based interactome, pharmacological pathway dissection, multiple orthogonal methods in one study","pmids":["32783947"],"is_preprint":false},{"year":2020,"finding":"NACHO selectively promotes expression of the (α4)2(β2)3 stoichiometry of α4β2 nAChRs, whereas the cytosolic chaperone 14-3-3η selectively promotes the (α4)3(β2)2 stoichiometry. These two ER- and cytosol-resident chaperones thus differentially regulate subunit stoichiometry.","method":"Heterologous expression in mammalian cells, single-channel patch-clamp electrophysiology, calcium imaging","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology and calcium imaging as orthogonal functional readouts, single lab, heterologous system","pmids":["32676916"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of an assembly intermediate reveals that NACHO binds two α1 subunits of GABAAR in the ER, shielding the principal (+) transmembrane interface of subunits containing an immature extracellular conformation. Crosslinking and structure prediction identified an adjacent surface on NACHO for β2 subunit interactions, suggesting stepwise oligomerization. Mutations of either subunit-interacting surface on NACHO impaired homopentameric α7 nAChR formation, demonstrating a generic mechanism for pLGIC assembly.","method":"Cryo-EM structure determination, crosslinking mass spectrometry, structure-prediction (AlphaFold), site-directed mutagenesis of NACHO, functional assembly assays","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with mutagenesis validation and crosslinking, multiple orthogonal methods, single preprint study","pmids":["39553992"],"is_preprint":true},{"year":2010,"finding":"TMEM35/TUF1 (NACHO) protein is expressed in rat adrenal zona glomerulosa and binds the low-affinity neurotrophin receptor p75NTR. This binding is competitively displaced by nerve growth factor but not by a TUF1 mutant lacking the p75NTR binding motif, indicating a specific binding interface. TMEM35 expression in ZG cells increases after angiotensin II exposure in vitro.","method":"Competitive binding displacement assay, co-immunoprecipitation, immunohistochemistry, in vitro cell treatment with angiotensin II, Western blot","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — competitive binding assay with mutant controls and Co-IP, single lab, adrenal/peripheral tissue context distinct from CNS nAChR function","pmids":["20685870"],"is_preprint":false},{"year":2016,"finding":"Deletion of tmem35 (NACHO) in mice results in elevated basal corticosterone, increased anxiety-like behavior, impairment of hippocampus-dependent fear and spatial memories, and loss of long-term potentiation at the Schaffer collateral-CA1 pathway. Proteomic analysis of synaptosomes revealed lower levels of PSD95 and NMDA receptors in KO hippocampus, indicating NACHO is required for synaptic plasticity machinery.","method":"Constitutive knockout mice, behavioral testing (fear conditioning, Morris water maze), electrophysiology (LTP), synaptosomal proteomics, corticosterone ELISA","journal":"American journal of physiology. Regulatory, integrative and comparative physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mice with electrophysiology, behavioral phenotyping, and proteomics as orthogonal readouts, single lab","pmids":["27170659"],"is_preprint":false},{"year":2021,"finding":"NACHO enables functional heterologous expression of an insect homomeric α6 nAChR (from Apis mellifera) in Xenopus laevis oocytes, demonstrating that NACHO chaperone activity extends to invertebrate nAChR subunits.","method":"Heterologous expression in Xenopus oocytes, two-electrode voltage-clamp electrophysiology, pharmacology (ACh EC50, α-bungarotoxin antagonism, spinosad agonism)","journal":"Pesticide biochemistry and physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional electrophysiology with pharmacological validation, single lab, demonstrates functional consequence of NACHO co-expression","pmids":["35082026"],"is_preprint":false},{"year":2020,"finding":"In mice, ric3 knockout produces only subtle changes in α-bungarotoxin binding across brain regions, whereas tmem35a (NACHO) knockout causes complete loss of α-bungarotoxin binding throughout the brain. This in vivo result is inconsistent with in vitro findings where RIC3 promotes α7 surface expression even without NACHO, indicating additional regulatory factors operate in vivo.","method":"Constitutive knockout mice (ric3 KO and tmem35a KO), autoradiographic α-bungarotoxin binding, Western blot","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo radioligand autoradiography comparing two KO lines, preliminary analysis noted by authors, single lab","pmids":["32204458"],"is_preprint":false},{"year":2021,"finding":"Loss of tmem35a (NACHO) in mice produces thermal hyperalgesia and mechanical allodynia, indicating neuronal nAChRs dependent on NACHO contribute to spinal cord pain processing. Spinal cord transcriptomics in KO mice revealed 72 differentially expressed genes with pathway analysis suggesting increased neuroinflammation as a contributing mechanism.","method":"Constitutive tmem35a knockout mice, behavioral pain testing (thermal and mechanical), intrathecal drug administration, spinal cord RNA-seq, pathway analysis","journal":"Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mice with behavioral and transcriptomic readouts, pharmacological rescue experiments, single lab","pmids":["33422618"],"is_preprint":false},{"year":2024,"finding":"In CHO cells expressing α7 nAChR and RIC3 but lacking NACHO, plasma membrane insertion of α7 nAChR is reduced and N-glycosylation of α7 nAChR is absent or deficient (no appreciable N-glycosylation product detected by glycosylation digest), indicating NACHO is required for correct N-glycosylation and Golgi-dependent maturation of α7 nAChR.","method":"Stable CHO cell line with transgene α7 nAChR + RIC3 (without NACHO), Western blot, in/on-cell Western, glycosylation (PNGase F) digest, voltage clamp","journal":"The Journal of membrane biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical glycosylation assay and surface expression quantification in a defined cell system without NACHO, single lab","pmids":["38967800"],"is_preprint":false},{"year":2025,"finding":"TMEM35A (NACHO) and RIC-3 chaperones promote cell surface localization of α7 nAChR in airway smooth muscle (ASM) cells. In ASM, TMEM35A and RIC-3 regulate nicotine-induced ER stress, intracellular Ca2+ regulation, and ASM cell proliferation downstream of α7 nAChR signaling.","method":"Human ASM cells (asthma vs. non-asthma), siRNA knockdown of RIC-3 and TMEM35A, Ca2+ imaging, ER stress markers, cell proliferation assays, immunofluorescence","journal":"American journal of respiratory cell and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function knockdown with multiple downstream readouts (Ca2+, ER stress, proliferation), single lab, non-neuronal cell type","pmids":["39236288"],"is_preprint":false},{"year":2025,"finding":"Drosophila melanogaster NACHO (DmNACHO) markedly enhances the ACh- and neonicotinoid-induced response amplitude of Dα1/Dβ1 nAChRs co-expressed with three other cofactors (DmRIC-3, DmTMX3, DmUNC-50) in Xenopus oocytes, without appreciably influencing ligand affinity, demonstrating that NACHO enhances channel activity/expression but does not alter binding site pharmacology.","method":"Heterologous expression in Xenopus laevis oocytes, two-electrode voltage-clamp electrophysiology, site-directed mutagenesis of nAChR binding site loops","journal":"Pesticide biochemistry and physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional electrophysiology with defined mutagenesis controls, single lab, insect ortholog/heterologous system","pmids":["40082022"],"is_preprint":false},{"year":2025,"finding":"Data from α7-5HT3 chimeric receptor studies do not support direct binding of RIC3 or NACHO to the α7 nAChR TM4 (M4) transmembrane region. Instead, the conserved SAP motif in the C-terminal tail is functionally important for chaperone-dependent surface expression.","method":"Chimeric receptor constructs (α7-5HT3 with domain swaps), heterologous expression in HEK293/Bosc23 cells and Xenopus oocytes, α-bungarotoxin surface binding, electrophysiology","journal":"Molecules (Basel, Switzerland)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect inference from chimera expression data; negative result regarding TM4 binding and positive inference on SAP motif, no direct binding assay performed","pmids":["41226195"],"is_preprint":false}],"current_model":"NACHO (TMEM35A) is an ER-resident transmembrane chaperone that binds unassembled pentameric ligand-gated ion channel (pLGIC) subunits — including all major nAChR subtypes (α7, α4β2, α6-containing, α3-containing) and GABAAR subunits — at their principal transmembrane interface in the ER, shields them from premature interactions, engages the N-glycosylation machinery (oligosaccharyltransferase complex) and calnexin to promote correct folding, and then facilitates stepwise oligomerization, Golgi maturation, and cell-surface expression; its loss in mice completely abolishes α7 nAChR assembly, profoundly reduces other nAChR subtypes, impairs synaptic plasticity and memory, and produces hyperalgesia, while structurally NACHO contains distinct binding surfaces for different subunits to guide sequential assembly."},"narrative":{"mechanistic_narrative":"TMEM35A (NACHO) is an endoplasmic reticulum-resident transmembrane chaperone that drives the assembly and surface delivery of nicotinic acetylcholine receptors (nAChRs) and, more broadly, pentameric ligand-gated ion channels [PMID:26875622, PMID:28445721]. It is essential and selective for nAChRs: its loss in mice abolishes α7 receptor assembly entirely and profoundly reduces α4β2, α6-containing, and α3-containing subtypes, while leaving GABAA receptors unaffected, and it cooperates with RIC-3 to achieve surface expression [PMID:28445721, PMID:32204458]. Mechanistically, NACHO engages unassembled subunits in the ER, recruits the oligosaccharyltransferase machinery and the lectin chaperone calnexin, and requires N-glycosylation and calnexin activity to promote correct folding and Golgi-dependent maturation [PMID:32783947, PMID:38967800]. A cryo-EM structure of an assembly intermediate shows NACHO binding the principal (+) transmembrane interface of immature subunits with a second adjacent surface for the next subunit, accounting for stepwise oligomerization, and mutation of either surface impairs α7 pentamer formation [PMID:39553992]. NACHO can also dictate receptor stoichiometry, favoring the (α4)2(β2)3 form of α4β2 [PMID:32676916]. Loss of NACHO in mice impairs hippocampal long-term potentiation, fear and spatial memory, and reduces PSD95 and NMDA receptor levels, and produces thermal hyperalgesia and mechanical allodynia [PMID:27170659, PMID:33422618].","teleology":[{"year":2010,"claim":"Before its chaperone role was known, TMEM35/TUF1 was characterized as a specific binding partner of a neurotrophin receptor in adrenal tissue, the first functional handle on the protein.","evidence":"Competitive binding displacement and Co-IP in rat adrenal zona glomerulosa cells with mutant controls","pmids":["20685870"],"confidence":"Medium","gaps":["Relationship between p75NTR binding and the later-established nAChR chaperone function unclear","Peripheral/adrenal context distinct from CNS role","No structural mapping of the binding interface"]},{"year":2016,"claim":"Established that NACHO is an essential ER chaperone for α7 nAChR assembly, identifying the long-sought factor required for α7 folding, maturation, and surface expression.","evidence":"Genomic screening, siRNA knockdown in primary neurons, knockout mice, α-bungarotoxin binding, electrophysiology, and fractionation","pmids":["26875622"],"confidence":"High","gaps":["Molecular interaction surface with α7 not defined","Whether function extends beyond α7 untested","Folding/glycosylation machinery involved unknown"]},{"year":2016,"claim":"Linked NACHO loss to circuit- and behavior-level consequences, showing the chaperone is required for synaptic plasticity and memory.","evidence":"Knockout mice with fear conditioning, Morris water maze, CA1 LTP, and synaptosomal proteomics","pmids":["27170659"],"confidence":"Medium","gaps":["Causal chain from nAChR loss to reduced PSD95/NMDA receptors not established","Elevated corticosterone vs. direct synaptic effects not separated","Single lab"]},{"year":2017,"claim":"Broadened NACHO's role from α7 to all major nAChR classes and demonstrated subtype specificity, establishing it as a general nAChR assembly factor acting early and synergizing with RIC-3.","evidence":"Knockout mice with subtype-specific radioligand binding (α-bungarotoxin, epibatidine, conotoxin MII) and GABAA negative control","pmids":["28445721"],"confidence":"High","gaps":["Mechanistic basis of subtype selectivity unresolved","Nature of synergy with RIC-3 undefined","Why GABAA is spared not explained at this stage"]},{"year":2020,"claim":"Placed NACHO within the N-glycosylation ER chaperone pathway and identified its requirement for the α7 ectodomain and specific TM2 residues, defining how NACHO recognizes its substrate.","evidence":"α7 chimera/mutagenesis, surface trafficking assays, Co-IP proteomics identifying OST and calnexin, pharmacological N-glycosylation/calnexin inhibition, electrophysiology","pmids":["32783947"],"confidence":"High","gaps":["Direct vs. indirect association with OST/calnexin not fully resolved","Order of calnexin vs. NACHO engagement unclear","Whether TM2 residues form the binding site or stabilize folding"]},{"year":2020,"claim":"Showed NACHO controls receptor stoichiometry, not just assembly, distinguishing its output from a cytosolic chaperone.","evidence":"Heterologous expression with single-channel patch clamp and calcium imaging","pmids":["32676916"],"confidence":"Medium","gaps":["Structural basis for stoichiometry selection unknown","Single heterologous system","In vivo relevance of stoichiometry control untested"]},{"year":2020,"claim":"Demonstrated in vivo that NACHO, not RIC3, is the dominant determinant of brain α7 expression, refining the relative roles of the two cofactors beyond cell-culture inferences.","evidence":"Comparison of ric3 KO and tmem35a KO mice by α-bungarotoxin autoradiography and Western blot","pmids":["32204458"],"confidence":"Medium","gaps":["Additional in vivo regulatory factors implied but unidentified","Reconciliation with in vitro RIC3 sufficiency incomplete","Preliminary analysis noted by authors"]},{"year":2024,"claim":"Provided the first structural mechanism, showing NACHO shields the principal transmembrane interface of immature subunits and uses two distinct surfaces to template stepwise pentamer assembly.","evidence":"Cryo-EM of a GABAAR α1 assembly intermediate, crosslinking MS, AlphaFold prediction, and NACHO mutagenesis with functional α7 assembly assays (preprint)","pmids":["39553992"],"confidence":"High","gaps":["Preprint; not peer-reviewed","Structure captured on GABAAR α1 while functional generality inferred","Dynamics of sequential subunit hand-off not directly observed"]},{"year":2024,"claim":"Confirmed biochemically that NACHO is required for α7 N-glycosylation and Golgi maturation, separating its glycosylation-dependent role from RIC3.","evidence":"CHO cells expressing α7+RIC3 without NACHO, PNGase F digest, surface Western, voltage clamp","pmids":["38967800"],"confidence":"Medium","gaps":["Whether NACHO directly recruits the glycosylation machinery to α7 not shown here","Single cell system","Step at which glycosylation fails not pinpointed"]},{"year":2025,"claim":"Extended NACHO function to non-neuronal physiology, linking it to nicotine-induced ER stress, calcium handling, and airway smooth muscle proliferation.","evidence":"siRNA knockdown of TMEM35A and RIC-3 in human ASM cells with Ca2+ imaging, ER stress markers, and proliferation assays","pmids":["39236288"],"confidence":"Medium","gaps":["Direct chaperone role vs. signaling effect in ASM not separated","Disease relevance to asthma correlative","Single lab"]},{"year":2025,"claim":"Conservation studies in insect orthologs established that NACHO enhances channel expression/activity without altering ligand-binding pharmacology, clarifying that its action is on assembly rather than the binding site.","evidence":"Xenopus oocyte expression of insect nAChRs with two-electrode voltage clamp and binding-loop mutagenesis","pmids":["35082026","40082022"],"confidence":"Medium","gaps":["Mechanism of activity enhancement vs. pure surface expression not distinguished","Heterologous systems only","Cofactor interplay in insects not mapped to mammalian context"]},{"year":2025,"claim":"Chimeric-receptor mapping challenged the TM4-binding model and pointed to a C-terminal SAP motif as functionally important for chaperone-dependent surface expression.","evidence":"α7-5HT3 chimera domain swaps in HEK/oocytes with α-bungarotoxin surface binding and electrophysiology","pmids":["41226195"],"confidence":"Low","gaps":["No direct binding assay performed; inference is indirect","Negative result on TM4 binding not independently confirmed","SAP motif role not structurally validated"]},{"year":null,"claim":"How NACHO's distinct binding surfaces are coordinated with calnexin/OST engagement to enforce subtype-specific assembly and stoichiometry in vivo, and the basis of its restriction to nAChRs versus other pLGICs, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model spanning glycosylation, calnexin, and sequential subunit binding","Determinants of nAChR-versus-GABAAR selectivity unexplained","Substrate hand-off timing to downstream trafficking factors unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,1,2,4]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,4]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,2,4]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,10]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,10]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[1,6]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,4]}],"complexes":[],"partners":["CHRNA7","CHRNA4","CHRNB2","CANX","RIC3","GABRA1","P75NTR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q53FP2","full_name":"Novel acetylcholine receptor chaperone","aliases":[],"length_aa":167,"mass_kda":18.4,"function":"Molecular chaperone which mediates the proper assembly and functional expression of the nicotinic acetylcholine receptors (nAChRs) throughout the brain (PubMed:26875622, PubMed:27789755, PubMed:28445721, PubMed:32204458, PubMed:32783947). Essential for the proper folding, assembly, function and surface trafficking of alpha-7 (CHRNA7), alpha-4-beta-2, alpha-3-beta-2 and alpha-3-beta-4 receptors (PubMed:26875622, PubMed:27789755, PubMed:28445721, PubMed:32204458, PubMed:32783947). Stably associates with ribophorin-1 (RPN1) and ribophorin-2 (RPN2) (components of the oligosaccharyl transferase (OST) complex) and with calnexin (CANX), both of which are critical for NACHO-mediated effects on CHRNA7 assembly and function (By similarity). Facilitates the proper folding and assembly of alpha-6-beta-2 and alpha-6-beta-2-beta-3 receptors and acts at early stages of the nAChRs subunit assembly (PubMed:28445721). Promotes the expression of the alpha-4(2):beta-2(3) stoichiometric form over the alpha-4(3):beta-2(2) form (PubMed:32676916)","subcellular_location":"Peroxisome membrane; Cytoplasmic vesicle; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q53FP2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TMEM35A","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/TMEM35A","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Focal adhesion sites","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":55.8},{"tissue":"intestine","ntpm":37.3},{"tissue":"seminal vesicle","ntpm":48.0}],"url":"https://www.proteinatlas.org/search/TMEM35A"},"hgnc":{"alias_symbol":["FLJ14084","TUF-1","NACHO"],"prev_symbol":["TMEM35"]},"alphafold":{"accession":"Q53FP2","domains":[{"cath_id":"1.20.920","chopping":"3-132","consensus_level":"high","plddt":92.0062,"start":3,"end":132}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q53FP2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q53FP2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q53FP2-F1-predicted_aligned_error_v6.png","plddt_mean":81.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TMEM35A","jax_strain_url":"https://www.jax.org/strain/search?query=TMEM35A"},"sequence":{"accession":"Q53FP2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q53FP2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q53FP2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q53FP2"}},"corpus_meta":[{"pmid":"26875622","id":"PMC_26875622","title":"Brain α7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO.","date":"2016","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/26875622","citation_count":139,"is_preprint":false},{"pmid":"28445721","id":"PMC_28445721","title":"NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain.","date":"2017","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/28445721","citation_count":74,"is_preprint":false},{"pmid":"32783947","id":"PMC_32783947","title":"NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly.","date":"2020","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32783947","citation_count":20,"is_preprint":false},{"pmid":"38687639","id":"PMC_38687639","title":"Langerhans cell histiocytosis: NACHO update on progress, chaos, and opportunity on the path to rational cures.","date":"2024","source":"Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/38687639","citation_count":19,"is_preprint":false},{"pmid":"31504159","id":"PMC_31504159","title":"NACHO: an R package for quality control of NanoString nCounter data.","date":"2020","source":"Bioinformatics (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/31504159","citation_count":19,"is_preprint":false},{"pmid":"34593235","id":"PMC_34593235","title":"Anti-inflammation of epicatechin mediated by TMEM35A and TMPO in bovine mammary epithelial cell line cells and mouse mammary gland.","date":"2021","source":"Journal of dairy science","url":"https://pubmed.ncbi.nlm.nih.gov/34593235","citation_count":18,"is_preprint":false},{"pmid":"32676916","id":"PMC_32676916","title":"NACHO and 14-3-3 promote expression of distinct subunit stoichiometries of the α4β2 acetylcholine receptor.","date":"2020","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/32676916","citation_count":17,"is_preprint":false},{"pmid":"20685870","id":"PMC_20685870","title":"Sodium depletion increases sympathetic neurite outgrowth and expression of a novel TMEM35 gene-derived protein (TUF1) in the rat adrenal zona glomerulosa.","date":"2010","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/20685870","citation_count":17,"is_preprint":false},{"pmid":"27170659","id":"PMC_27170659","title":"Deletion of novel protein TMEM35 alters stress-related functions and impairs long-term memory in mice.","date":"2016","source":"American journal of physiology. Regulatory, integrative and comparative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/27170659","citation_count":14,"is_preprint":false},{"pmid":"32204458","id":"PMC_32204458","title":"Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?","date":"2020","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/32204458","citation_count":14,"is_preprint":false},{"pmid":"35082026","id":"PMC_35082026","title":"NACHO permits functional heterologous expression of an insect homomeric α6 nicotinic acetylcholine receptor.","date":"2021","source":"Pesticide biochemistry and physiology","url":"https://pubmed.ncbi.nlm.nih.gov/35082026","citation_count":9,"is_preprint":false},{"pmid":"39553992","id":"PMC_39553992","title":"Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39553992","citation_count":7,"is_preprint":false},{"pmid":"33422618","id":"PMC_33422618","title":"The nAChR Chaperone TMEM35a (NACHO) Contributes to the Development of Hyperalgesia in Mice.","date":"2021","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/33422618","citation_count":5,"is_preprint":false},{"pmid":"39236288","id":"PMC_39236288","title":"Nicotine-Induced Endoplasmic Reticulum Stress and Airway Smooth Muscle Cell Proliferation Is Mediated by α7nAChR and Chaperones-RIC-3 and TMEM35.","date":"2025","source":"American journal of respiratory cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/39236288","citation_count":5,"is_preprint":false},{"pmid":"28131622","id":"PMC_28131622","title":"Perinatal nicotine treatment induces transient increases in NACHO protein levels in the rat frontal cortex.","date":"2017","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/28131622","citation_count":4,"is_preprint":false},{"pmid":"38967800","id":"PMC_38967800","title":"N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.","date":"2024","source":"The Journal of membrane biology","url":"https://pubmed.ncbi.nlm.nih.gov/38967800","citation_count":3,"is_preprint":false},{"pmid":"19876762","id":"PMC_19876762","title":"Sodium choleate (NaCho) effects on Candida albicans: implications for its role as a gastrointestinal tract inhabitant.","date":"2009","source":"Mycopathologia","url":"https://pubmed.ncbi.nlm.nih.gov/19876762","citation_count":3,"is_preprint":false},{"pmid":"40082022","id":"PMC_40082022","title":"Binding site loops D and G make a stronger contribution than loop C to the actions of neonicotinoids on the NACHO-assisted, robustly expressed Drosophila melanogaster Dα1/Dβ1 nicotinic acetylcholine receptor.","date":"2025","source":"Pesticide biochemistry and physiology","url":"https://pubmed.ncbi.nlm.nih.gov/40082022","citation_count":3,"is_preprint":false},{"pmid":"41226195","id":"PMC_41226195","title":"Effects of Swapping 5HT3 and α7 Residues in Chimeric Receptor Proteins on RIC3 and NACHO Chaperone Actions.","date":"2025","source":"Molecules (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/41226195","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11504,"output_tokens":3972,"usd":0.047046,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11868,"output_tokens":3978,"usd":0.079395,"stage2_stop_reason":"end_turn"},"total_usd":0.126441,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"NACHO (TMEM35A) is an endoplasmic reticulum-resident transmembrane protein that functions as an essential chaperone for α7 nicotinic acetylcholine receptor (nAChR) assembly. NACHO promotes α7 protein folding, maturation through the Golgi complex, and surface expression. Knockdown in cultured hippocampal neurons or knockout in mice selectively and completely disrupts α7 receptor assembly and abolishes α7 channel function.\",\n      \"method\": \"Genomic screening, siRNA knockdown in primary neurons, constitutive knockout mice, radioligand binding (α-bungarotoxin), electrophysiology, immunofluorescence, subcellular fractionation\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KD, KO, binding assays, electrophysiology, subcellular localization), replicated across cell and animal models in a single rigorous study\",\n      \"pmids\": [\"26875622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NACHO mediates assembly of all major classes of nAChRs, not just α7. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin (α7), epibatidine (α4β2), and conotoxin MII (α6-containing) nAChRs. NACHO acts at early intracellular stages of nAChR subunit assembly and synergizes with RIC-3 for receptor surface expression. GABAA receptors are unaffected, indicating NACHO specificity for nAChRs.\",\n      \"method\": \"NACHO knockout mice, radioligand binding assays (α-bungarotoxin, epibatidine, conotoxin MII), behavioral assays, heterologous expression\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — constitutive KO mice with multiple receptor-subtype-specific radioligand binding assays and a specific negative control (GABAA), independent replication of the α7 finding from prior study\",\n      \"pmids\": [\"28445721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NACHO-mediated α7 nAChR assembly requires the α7 ectodomain and two amino acids in the second transmembrane domain of α7. NACHO associates with the ER oligosaccharyltransferase machinery and with the lectin chaperone calnexin, as shown by proteomics. NACHO-mediated assembly and channel function require N-glycosylation and calnexin chaperone activity, placing NACHO within the N-glycosylation ER chaperone pathway.\",\n      \"method\": \"α7 chimera and mutagenesis constructs, surface trafficking assays, proteomics/mass spectrometry (Co-IP), pharmacological inhibition of N-glycosylation and calnexin, electrophysiology\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — site-directed mutagenesis of receptor substrate, proteomics-based interactome, pharmacological pathway dissection, multiple orthogonal methods in one study\",\n      \"pmids\": [\"32783947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NACHO selectively promotes expression of the (α4)2(β2)3 stoichiometry of α4β2 nAChRs, whereas the cytosolic chaperone 14-3-3η selectively promotes the (α4)3(β2)2 stoichiometry. These two ER- and cytosol-resident chaperones thus differentially regulate subunit stoichiometry.\",\n      \"method\": \"Heterologous expression in mammalian cells, single-channel patch-clamp electrophysiology, calcium imaging\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology and calcium imaging as orthogonal functional readouts, single lab, heterologous system\",\n      \"pmids\": [\"32676916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of an assembly intermediate reveals that NACHO binds two α1 subunits of GABAAR in the ER, shielding the principal (+) transmembrane interface of subunits containing an immature extracellular conformation. Crosslinking and structure prediction identified an adjacent surface on NACHO for β2 subunit interactions, suggesting stepwise oligomerization. Mutations of either subunit-interacting surface on NACHO impaired homopentameric α7 nAChR formation, demonstrating a generic mechanism for pLGIC assembly.\",\n      \"method\": \"Cryo-EM structure determination, crosslinking mass spectrometry, structure-prediction (AlphaFold), site-directed mutagenesis of NACHO, functional assembly assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with mutagenesis validation and crosslinking, multiple orthogonal methods, single preprint study\",\n      \"pmids\": [\"39553992\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TMEM35/TUF1 (NACHO) protein is expressed in rat adrenal zona glomerulosa and binds the low-affinity neurotrophin receptor p75NTR. This binding is competitively displaced by nerve growth factor but not by a TUF1 mutant lacking the p75NTR binding motif, indicating a specific binding interface. TMEM35 expression in ZG cells increases after angiotensin II exposure in vitro.\",\n      \"method\": \"Competitive binding displacement assay, co-immunoprecipitation, immunohistochemistry, in vitro cell treatment with angiotensin II, Western blot\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — competitive binding assay with mutant controls and Co-IP, single lab, adrenal/peripheral tissue context distinct from CNS nAChR function\",\n      \"pmids\": [\"20685870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Deletion of tmem35 (NACHO) in mice results in elevated basal corticosterone, increased anxiety-like behavior, impairment of hippocampus-dependent fear and spatial memories, and loss of long-term potentiation at the Schaffer collateral-CA1 pathway. Proteomic analysis of synaptosomes revealed lower levels of PSD95 and NMDA receptors in KO hippocampus, indicating NACHO is required for synaptic plasticity machinery.\",\n      \"method\": \"Constitutive knockout mice, behavioral testing (fear conditioning, Morris water maze), electrophysiology (LTP), synaptosomal proteomics, corticosterone ELISA\",\n      \"journal\": \"American journal of physiology. Regulatory, integrative and comparative physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mice with electrophysiology, behavioral phenotyping, and proteomics as orthogonal readouts, single lab\",\n      \"pmids\": [\"27170659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NACHO enables functional heterologous expression of an insect homomeric α6 nAChR (from Apis mellifera) in Xenopus laevis oocytes, demonstrating that NACHO chaperone activity extends to invertebrate nAChR subunits.\",\n      \"method\": \"Heterologous expression in Xenopus oocytes, two-electrode voltage-clamp electrophysiology, pharmacology (ACh EC50, α-bungarotoxin antagonism, spinosad agonism)\",\n      \"journal\": \"Pesticide biochemistry and physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional electrophysiology with pharmacological validation, single lab, demonstrates functional consequence of NACHO co-expression\",\n      \"pmids\": [\"35082026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In mice, ric3 knockout produces only subtle changes in α-bungarotoxin binding across brain regions, whereas tmem35a (NACHO) knockout causes complete loss of α-bungarotoxin binding throughout the brain. This in vivo result is inconsistent with in vitro findings where RIC3 promotes α7 surface expression even without NACHO, indicating additional regulatory factors operate in vivo.\",\n      \"method\": \"Constitutive knockout mice (ric3 KO and tmem35a KO), autoradiographic α-bungarotoxin binding, Western blot\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo radioligand autoradiography comparing two KO lines, preliminary analysis noted by authors, single lab\",\n      \"pmids\": [\"32204458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of tmem35a (NACHO) in mice produces thermal hyperalgesia and mechanical allodynia, indicating neuronal nAChRs dependent on NACHO contribute to spinal cord pain processing. Spinal cord transcriptomics in KO mice revealed 72 differentially expressed genes with pathway analysis suggesting increased neuroinflammation as a contributing mechanism.\",\n      \"method\": \"Constitutive tmem35a knockout mice, behavioral pain testing (thermal and mechanical), intrathecal drug administration, spinal cord RNA-seq, pathway analysis\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mice with behavioral and transcriptomic readouts, pharmacological rescue experiments, single lab\",\n      \"pmids\": [\"33422618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In CHO cells expressing α7 nAChR and RIC3 but lacking NACHO, plasma membrane insertion of α7 nAChR is reduced and N-glycosylation of α7 nAChR is absent or deficient (no appreciable N-glycosylation product detected by glycosylation digest), indicating NACHO is required for correct N-glycosylation and Golgi-dependent maturation of α7 nAChR.\",\n      \"method\": \"Stable CHO cell line with transgene α7 nAChR + RIC3 (without NACHO), Western blot, in/on-cell Western, glycosylation (PNGase F) digest, voltage clamp\",\n      \"journal\": \"The Journal of membrane biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical glycosylation assay and surface expression quantification in a defined cell system without NACHO, single lab\",\n      \"pmids\": [\"38967800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM35A (NACHO) and RIC-3 chaperones promote cell surface localization of α7 nAChR in airway smooth muscle (ASM) cells. In ASM, TMEM35A and RIC-3 regulate nicotine-induced ER stress, intracellular Ca2+ regulation, and ASM cell proliferation downstream of α7 nAChR signaling.\",\n      \"method\": \"Human ASM cells (asthma vs. non-asthma), siRNA knockdown of RIC-3 and TMEM35A, Ca2+ imaging, ER stress markers, cell proliferation assays, immunofluorescence\",\n      \"journal\": \"American journal of respiratory cell and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function knockdown with multiple downstream readouts (Ca2+, ER stress, proliferation), single lab, non-neuronal cell type\",\n      \"pmids\": [\"39236288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Drosophila melanogaster NACHO (DmNACHO) markedly enhances the ACh- and neonicotinoid-induced response amplitude of Dα1/Dβ1 nAChRs co-expressed with three other cofactors (DmRIC-3, DmTMX3, DmUNC-50) in Xenopus oocytes, without appreciably influencing ligand affinity, demonstrating that NACHO enhances channel activity/expression but does not alter binding site pharmacology.\",\n      \"method\": \"Heterologous expression in Xenopus laevis oocytes, two-electrode voltage-clamp electrophysiology, site-directed mutagenesis of nAChR binding site loops\",\n      \"journal\": \"Pesticide biochemistry and physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional electrophysiology with defined mutagenesis controls, single lab, insect ortholog/heterologous system\",\n      \"pmids\": [\"40082022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Data from α7-5HT3 chimeric receptor studies do not support direct binding of RIC3 or NACHO to the α7 nAChR TM4 (M4) transmembrane region. Instead, the conserved SAP motif in the C-terminal tail is functionally important for chaperone-dependent surface expression.\",\n      \"method\": \"Chimeric receptor constructs (α7-5HT3 with domain swaps), heterologous expression in HEK293/Bosc23 cells and Xenopus oocytes, α-bungarotoxin surface binding, electrophysiology\",\n      \"journal\": \"Molecules (Basel, Switzerland)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect inference from chimera expression data; negative result regarding TM4 binding and positive inference on SAP motif, no direct binding assay performed\",\n      \"pmids\": [\"41226195\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NACHO (TMEM35A) is an ER-resident transmembrane chaperone that binds unassembled pentameric ligand-gated ion channel (pLGIC) subunits — including all major nAChR subtypes (α7, α4β2, α6-containing, α3-containing) and GABAAR subunits — at their principal transmembrane interface in the ER, shields them from premature interactions, engages the N-glycosylation machinery (oligosaccharyltransferase complex) and calnexin to promote correct folding, and then facilitates stepwise oligomerization, Golgi maturation, and cell-surface expression; its loss in mice completely abolishes α7 nAChR assembly, profoundly reduces other nAChR subtypes, impairs synaptic plasticity and memory, and produces hyperalgesia, while structurally NACHO contains distinct binding surfaces for different subunits to guide sequential assembly.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TMEM35A (NACHO) is an endoplasmic reticulum-resident transmembrane chaperone that drives the assembly and surface delivery of nicotinic acetylcholine receptors (nAChRs) and, more broadly, pentameric ligand-gated ion channels [#0, #1]. It is essential and selective for nAChRs: its loss in mice abolishes α7 receptor assembly entirely and profoundly reduces α4β2, α6-containing, and α3-containing subtypes, while leaving GABAA receptors unaffected, and it cooperates with RIC-3 to achieve surface expression [#1, #8]. Mechanistically, NACHO engages unassembled subunits in the ER, recruits the oligosaccharyltransferase machinery and the lectin chaperone calnexin, and requires N-glycosylation and calnexin activity to promote correct folding and Golgi-dependent maturation [#2, #10]. A cryo-EM structure of an assembly intermediate shows NACHO binding the principal (+) transmembrane interface of immature subunits with a second adjacent surface for the next subunit, accounting for stepwise oligomerization, and mutation of either surface impairs α7 pentamer formation [#4]. NACHO can also dictate receptor stoichiometry, favoring the (α4)2(β2)3 form of α4β2 [#3]. Loss of NACHO in mice impairs hippocampal long-term potentiation, fear and spatial memory, and reduces PSD95 and NMDA receptor levels, and produces thermal hyperalgesia and mechanical allodynia [#6, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Before its chaperone role was known, TMEM35/TUF1 was characterized as a specific binding partner of a neurotrophin receptor in adrenal tissue, the first functional handle on the protein.\",\n      \"evidence\": \"Competitive binding displacement and Co-IP in rat adrenal zona glomerulosa cells with mutant controls\",\n      \"pmids\": [\"20685870\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship between p75NTR binding and the later-established nAChR chaperone function unclear\", \"Peripheral/adrenal context distinct from CNS role\", \"No structural mapping of the binding interface\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established that NACHO is an essential ER chaperone for α7 nAChR assembly, identifying the long-sought factor required for α7 folding, maturation, and surface expression.\",\n      \"evidence\": \"Genomic screening, siRNA knockdown in primary neurons, knockout mice, α-bungarotoxin binding, electrophysiology, and fractionation\",\n      \"pmids\": [\"26875622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular interaction surface with α7 not defined\", \"Whether function extends beyond α7 untested\", \"Folding/glycosylation machinery involved unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked NACHO loss to circuit- and behavior-level consequences, showing the chaperone is required for synaptic plasticity and memory.\",\n      \"evidence\": \"Knockout mice with fear conditioning, Morris water maze, CA1 LTP, and synaptosomal proteomics\",\n      \"pmids\": [\"27170659\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from nAChR loss to reduced PSD95/NMDA receptors not established\", \"Elevated corticosterone vs. direct synaptic effects not separated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Broadened NACHO's role from α7 to all major nAChR classes and demonstrated subtype specificity, establishing it as a general nAChR assembly factor acting early and synergizing with RIC-3.\",\n      \"evidence\": \"Knockout mice with subtype-specific radioligand binding (α-bungarotoxin, epibatidine, conotoxin MII) and GABAA negative control\",\n      \"pmids\": [\"28445721\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic basis of subtype selectivity unresolved\", \"Nature of synergy with RIC-3 undefined\", \"Why GABAA is spared not explained at this stage\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed NACHO within the N-glycosylation ER chaperone pathway and identified its requirement for the α7 ectodomain and specific TM2 residues, defining how NACHO recognizes its substrate.\",\n      \"evidence\": \"α7 chimera/mutagenesis, surface trafficking assays, Co-IP proteomics identifying OST and calnexin, pharmacological N-glycosylation/calnexin inhibition, electrophysiology\",\n      \"pmids\": [\"32783947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs. indirect association with OST/calnexin not fully resolved\", \"Order of calnexin vs. NACHO engagement unclear\", \"Whether TM2 residues form the binding site or stabilize folding\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed NACHO controls receptor stoichiometry, not just assembly, distinguishing its output from a cytosolic chaperone.\",\n      \"evidence\": \"Heterologous expression with single-channel patch clamp and calcium imaging\",\n      \"pmids\": [\"32676916\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for stoichiometry selection unknown\", \"Single heterologous system\", \"In vivo relevance of stoichiometry control untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated in vivo that NACHO, not RIC3, is the dominant determinant of brain α7 expression, refining the relative roles of the two cofactors beyond cell-culture inferences.\",\n      \"evidence\": \"Comparison of ric3 KO and tmem35a KO mice by α-bungarotoxin autoradiography and Western blot\",\n      \"pmids\": [\"32204458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Additional in vivo regulatory factors implied but unidentified\", \"Reconciliation with in vitro RIC3 sufficiency incomplete\", \"Preliminary analysis noted by authors\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided the first structural mechanism, showing NACHO shields the principal transmembrane interface of immature subunits and uses two distinct surfaces to template stepwise pentamer assembly.\",\n      \"evidence\": \"Cryo-EM of a GABAAR α1 assembly intermediate, crosslinking MS, AlphaFold prediction, and NACHO mutagenesis with functional α7 assembly assays (preprint)\",\n      \"pmids\": [\"39553992\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Preprint; not peer-reviewed\", \"Structure captured on GABAAR α1 while functional generality inferred\", \"Dynamics of sequential subunit hand-off not directly observed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Confirmed biochemically that NACHO is required for α7 N-glycosylation and Golgi maturation, separating its glycosylation-dependent role from RIC3.\",\n      \"evidence\": \"CHO cells expressing α7+RIC3 without NACHO, PNGase F digest, surface Western, voltage clamp\",\n      \"pmids\": [\"38967800\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NACHO directly recruits the glycosylation machinery to α7 not shown here\", \"Single cell system\", \"Step at which glycosylation fails not pinpointed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended NACHO function to non-neuronal physiology, linking it to nicotine-induced ER stress, calcium handling, and airway smooth muscle proliferation.\",\n      \"evidence\": \"siRNA knockdown of TMEM35A and RIC-3 in human ASM cells with Ca2+ imaging, ER stress markers, and proliferation assays\",\n      \"pmids\": [\"39236288\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct chaperone role vs. signaling effect in ASM not separated\", \"Disease relevance to asthma correlative\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Conservation studies in insect orthologs established that NACHO enhances channel expression/activity without altering ligand-binding pharmacology, clarifying that its action is on assembly rather than the binding site.\",\n      \"evidence\": \"Xenopus oocyte expression of insect nAChRs with two-electrode voltage clamp and binding-loop mutagenesis\",\n      \"pmids\": [\"35082026\", \"40082022\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of activity enhancement vs. pure surface expression not distinguished\", \"Heterologous systems only\", \"Cofactor interplay in insects not mapped to mammalian context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Chimeric-receptor mapping challenged the TM4-binding model and pointed to a C-terminal SAP motif as functionally important for chaperone-dependent surface expression.\",\n      \"evidence\": \"α7-5HT3 chimera domain swaps in HEK/oocytes with α-bungarotoxin surface binding and electrophysiology\",\n      \"pmids\": [\"41226195\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct binding assay performed; inference is indirect\", \"Negative result on TM4 binding not independently confirmed\", \"SAP motif role not structurally validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NACHO's distinct binding surfaces are coordinated with calnexin/OST engagement to enforce subtype-specific assembly and stoichiometry in vivo, and the basis of its restriction to nAChRs versus other pLGICs, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model spanning glycosylation, calnexin, and sequential subunit binding\", \"Determinants of nAChR-versus-GABAAR selectivity unexplained\", \"Substrate hand-off timing to downstream trafficking factors unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 1, 2, 4]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 2, 4]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 10]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CHRNA7\", \"CHRNA4\", \"CHRNB2\", \"CANX\", \"RIC3\", \"GABRA1\", \"p75NTR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}