{"gene":"NPC1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2016,"finding":"Cryo-EM structure of full-length human NPC1 at 4.4 Å revealed 13 transmembrane segments (TMs), three distinct lumenal domains A (NTD), C, and I, with TMs 2–13 forming a resistance-nodulation-cell division (RND) fold, TMs 3–7 constituting the sterol-sensing domain (SSD), and a trimeric EBOV cleaved glycoprotein (GPcl) binding to NPC1 domain C as a monomer.","method":"Single-particle electron cryo-microscopy (cryo-EM); biochemical binding assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution structure with functional validation of EBOV-GP binding, single rigorous study with multiple orthogonal methods","pmids":["27238017"],"is_preprint":false},{"year":2020,"finding":"Cryo-EM structure of NPC1 bound to itraconazole revealed that the drug binds within a central lumenal tunnel linked to the SSD; blocking this tunnel abolishes NPC1-mediated cholesterol egress, defining the SSD tunnel as the conduit for cholesterol transport.","method":"Cryo-EM structure determination; functional cholesterol efflux assays; site-directed mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus mutagenesis plus functional transport assay in one study","pmids":["31919352"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of glycosylated NPC1 luminal domain C showed all seven N-glycosylation sites occupied, mapped disease mutations to a potential NPC2-binding face, and identified four residues (H418, Q421, F502, F504) critical for Ebola virus glycoprotein interaction.","method":"X-ray crystallography; computational docking; sequence analysis","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional mapping; single lab but multiple structural and sequence methods","pmids":["26846330"],"is_preprint":false},{"year":2007,"finding":"NPC1 binds cholesterol and oxysterols (25-, 24-, 27-hydroxycholesterol) directly; the binding site for oxysterols differs from that for cholesterol, as excess cholesterol does not compete off 25-HC binding. NPC1 is not required for known oxysterol regulatory actions on SREBP processing or ACAT.","method":"Purification of NPC1 from rabbit liver membranes (~14,000-fold); radioligand binding assays with [³H]cholesterol and [³H]25-HC; competition assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical purification and direct in vitro binding assays with rigorous controls","pmids":["17989073"],"is_preprint":false},{"year":2007,"finding":"The sterol-binding site of NPC1 is localized to luminal loop-1 (a 240-amino acid cysteine-rich domain), which binds [³H]cholesterol (Kd ~130 nM) and [³H]25-HC (Kd ~10 nM) as a soluble dimer. Mutation Q79A abolishes sterol binding to loop-1 yet still restores cholesterol transport in NPC1-deficient CHO cells, indicating this binding site is not essential for NPC1's transport function in fibroblasts.","method":"Recombinant protein production; radioligand binding assays; site-directed mutagenesis (Q79A); functional cholesterol transport complementation assay in NPC1-deficient CHO cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of binding with mutagenesis and functional transport complementation in single rigorous study","pmids":["17989072"],"is_preprint":false},{"year":2019,"finding":"NPC1 tethers ER–endocytic organelle membrane contact sites (MCS) by interacting with the ER-localized sterol transport protein Gramd1b, and this interaction regulates cholesterol egress from lysosomes directly to the ER across MCS. Artificially tethering MCS rescued cholesterol accumulation in NPC1-deficient cells. Loss of NPC1 or Gramd1b expanded lysosome–mitochondria MCS in a STARD3-dependent manner.","method":"Co-immunoprecipitation; live-cell imaging; MCS artificial tethering; fractionation; NPC1-deficient cell lines","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus MCS rescue experiment plus multiple imaging modalities, replicated across conditions","pmids":["31537798"],"is_preprint":false},{"year":2020,"finding":"Active Rab7 (GTP-loaded) directly interacts with the NPC1 cholesterol transporter and is required to license NPC1-dependent lysosomal cholesterol export; this function is controlled by the trimeric Mon1-Ccz1-C18orf8 (MCC) GEF that activates Rab7. Loss of MCC subunits abolishes lysosomal cholesterol export and is rescued by constitutively active Rab7.","method":"Genome-wide CRISPR screen; Co-immunoprecipitation; cholesterol reporter assay; Rab7 constitutively active rescue experiments","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide CRISPR screen followed by Co-IP and functional rescue with constitutively active Rab7, multiple orthogonal methods","pmids":["33144569"],"is_preprint":false},{"year":2020,"finding":"Loss of NPC1-mediated cholesterol export causes mTORC1 hyperactivation, which drives lysosomal proteolytic impairment, hydrolase depletion, enhanced membrane damage, and defective mitophagy; genetic and pharmacological mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, placing aberrant mTORC1 downstream of cholesterol accumulation.","method":"Proteomic profiling of NPC lysosomes; genetic mTORC1 inhibition (rapamycin); lysosomal proteolysis assays; mitophagy assays; neuronal NPC models","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — lysosomal proteomics plus genetic epistasis (mTORC1 inhibition rescues proteolysis but not cholesterol) with functional readouts","pmids":["33308480"],"is_preprint":false},{"year":2016,"finding":"The antifungal drug itraconazole directly inhibits NPC1 by binding to its sterol-sensing domain (SSD); the binding site was mapped by mutagenesis, competition with U18666A, and molecular docking. Dual inhibition of NPC1 (cholesterol trafficking) and VDAC1 (AMPK activation) synergistically inhibits mTOR signaling and angiogenesis.","method":"Pharmacological inhibition assays; site-directed mutagenesis of SSD; competition binding with U18666A; molecular docking","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus competition binding plus docking in single lab; no direct structural validation in this paper","pmids":["28103683"],"is_preprint":false},{"year":2016,"finding":"Posaconazole (a triazole antifungal) and itraconazole directly bind to the NPC1 sterol-sensing domain to block lysosomal cholesterol export; a photoactivatable posaconazole derivative cross-linked specifically to purified NPC1 in lipid bilayer nanodiscs, and cross-linking was reduced by a P691S point mutation in the SSD.","method":"Photoactivatable cross-linking with posaconazole derivative P-X; NPC1 purification into nanodiscs; site-directed mutagenesis (P691S); competition with itraconazole and U18666A","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct photoaffinity labeling of purified NPC1 in reconstituted nanodiscs, mutagenesis confirmation, multiple competitive ligands","pmids":["27994139"],"is_preprint":false},{"year":2018,"finding":"The most common disease-causing NPC1 mutant I1061T is degraded by two complementary pathways: MARCH6-dependent ERAD followed by proteasomal degradation, and FAM134B-dependent selective ER autophagy (ER-phagy). Subcellular fractionation in mouse tissues confirmed ER retention of I1061T NPC1.","method":"Proteasome inhibitor studies; MARCH6 knockdown/knockout; ER-phagy (FAM134B) knockout; subcellular fractionation; in vivo mouse models; human tissue samples","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple complementary genetic knockouts (MARCH6 and FAM134B) validated both in vitro and in vivo with defined mechanistic readouts","pmids":["30202070"],"is_preprint":false},{"year":2008,"finding":"Purified full-length NPC1 directly binds fluorescent cholesterol analogs (dehydroergosterol, cholestatrienol, NBD-cholesterol) with apparent affinity ~0.5–6 µM (1:1 stoichiometry); bound cholesterol is buried in a deep hydrophobic pocket. Binding is competed by native cholesterol and 25-hydroxycholesterol, confirming a shared sterol-binding site.","method":"FLAG-tag affinity purification of NPC1; fluorescence binding and quenching assays; gel filtration; photoaffinity labeling with azido-cholesterol","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding assays with purified protein, multiple fluorescent probes, stoichiometry determination","pmids":["19029290"],"is_preprint":false},{"year":2020,"finding":"Inter-domain dynamics of NPC1 are required for cholesterol transport: introducing single disulfide bonds to constrain lumenal domains, or shortening a cytoplasmic loop, abolishes transport activity. The N-terminal domain need not dissociate from the rest of the protein for efficient export. Ezetimibe blocks NPC1L1 transport by binding simultaneously to residues at the interface of all three extracellular domains.","method":"Site-directed disulfide bond engineering; lysosomal cholesterol efflux assay; domain truncation mutagenesis; ezetimibe binding site mapping","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — engineered disulfide constraints with functional transport assay and mutagenesis of drug binding site in single rigorous study","pmids":["32410728"],"is_preprint":false},{"year":2006,"finding":"NPC1 undergoes ubiquitylation that is regulated by endosomal cholesterol levels: cholesterol depletion promotes NPC1 ubiquitylation, while the SSD mutant P691S fails to respond. Ubiquitylated NPC1 associates with the ESCRT component SKD1/Vps4; NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions.","method":"Co-immunoprecipitation; dominant-negative SKD1 expression; cholesterol depletion experiments; site-directed mutagenesis (P691S, ΔLLNF); NPC2-deficient patient fibroblasts","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with dominant-negative and mutagenesis approaches in single lab, multiple cell systems","pmids":["16757520"],"is_preprint":false},{"year":2016,"finding":"TMEM97 is a cholesterol-responsive NPC1-binding protein that post-transcriptionally regulates NPC1 abundance; reducing TMEM97 increases NPC1 levels and restores cholesterol trafficking to the ER in NPC disease cells in an NPC1-dependent manner. TMEM97 lacking its ER-retention signal fails to regulate NPC1 availability.","method":"RNA interference; Co-immunoprecipitation; cholesterol trafficking assays (filipin staining, ER cholesterol measurement); domain deletion of TMEM97 ER-retention signal","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirmed interaction, functional rescue with domain mutant, single lab","pmids":["27378690"],"is_preprint":false},{"year":2023,"finding":"NPC1 directly binds sphingosine using lysosome-targeted photoactivatable sphingosine probes; absence of NPC1 causes lysosomal sphingosine accumulation. Artificial elevation of lysosomal sphingosine impairs cholesterol efflux, consistent with sphingosine and cholesterol sharing a common NPC1-mediated export mechanism.","method":"Caged, photocrosslinkable sphingosine and cholesterol probes; photoaffinity labeling; subcellular fractionation; NPC1 knockout cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — novel probe technology with direct photoaffinity labeling and functional consequence in NPC1-KO cells, single lab but orthogonal chemical biology approach","pmids":["36893262"],"is_preprint":false},{"year":2015,"finding":"NPC1 aids transfer of LDL-derived cholesterol across the lysosomal glycocalyx: inhibiting O-linked glycosylation in NPC1-deficient fibroblasts reduced lysosomal cholesterol content by ≥30% and increased ER cholesterol delivery, indicating that cells become less dependent on NPC1 when glycocalyx density is reduced.","method":"Pharmacological inhibition of O-glycosylation; CRISPR-generated NPC1-deficient CHO cells; biochemical lysosome cholesterol measurement; [¹⁴C]-oleate esterification assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent genetic/pharmacological glycosylation manipulations with direct biochemical cholesterol measurement","pmids":["26578804"],"is_preprint":false},{"year":2013,"finding":"NPC1 is an intracellular endosomal receptor for Ebola virus; its second luminal domain (domain C) is necessary and sufficient for filovirus receptor activity. NPC1L1 lacks receptor activity because its domain C does not bind viral GP; a chimera bearing NPC1's domain C conferred near-wild-type filovirus infection.","method":"NPC1/NPC1L1 chimera panel; viral infection assays; GP binding assays","journal":"Viruses","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic chimera analysis with reciprocal domain swaps and authentic viral infection assays","pmids":["23202491"],"is_preprint":false},{"year":2015,"finding":"A single amino acid at position 503 in NPC1 bidirectionally controls binding to EBOV glycoprotein and viral receptor activity; this residue is in domain C and its mutation does not perturb NPC1 endosomal localization or cholesterol trafficking function.","method":"NPC1 viper-human chimeras and point mutants; viral infection assays; GP binding assays; cholesterol trafficking assays","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, chimera/point mutant analysis with functional and localization readouts","pmids":["27303731"],"is_preprint":false},{"year":2015,"finding":"TIM-1 and NPC1 colocalize and physically interact in intracellular vesicles where EBOV glycoprotein-mediated membrane fusion occurs; a TIM-1-specific monoclonal antibody that disrupts TIM-1–NPC1 interaction also prevents GP-mediated membrane fusion, implicating this protein–protein interaction in filovirus fusion.","method":"Co-immunoprecipitation; colocalization by fluorescence microscopy; monoclonal antibody blocking; pseudovirus fusion assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and colocalization with functional antibody blocking, single lab","pmids":["25855742"],"is_preprint":false},{"year":2015,"finding":"Ebolavirus enters cells through endolysosomes that co-contain both NPC1 and TPC2, directly observed by live-cell imaging, contradicting a model of entry through NPC1-negative organelles.","method":"Live-cell imaging; co-localization of NPC1 and TPC2 with viral entry sites","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct live-cell imaging of viral entry pathway in NPC1+ compartments, single lab","pmids":["26468524"],"is_preprint":false},{"year":2012,"finding":"NPC1-bearing vesicles (lacking lysosomal markers) traffick to Anaplasma phagocytophilum bacterial inclusions; NPC1 function is required for bacterial cholesterol acquisition and infection. The trans-Golgi SNAREs VAMP4 and syntaxin 16, which associate with NPC1 on LDL-cholesterol vesicles, are recruited to bacterial inclusions and VAMP4 is required for bacterial infection.","method":"siRNA knockdown; immunofluorescence co-localization; cholesterol-traffic inhibitor U18666A; infection assays","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with defined infection and cholesterol trafficking readouts, single lab","pmids":["22212234"],"is_preprint":false},{"year":2013,"finding":"In NPC1-deficient cells, neuronal deletion of Npc1 alone is sufficient to arrest oligodendrocyte maturation and cause myelination failure, associated with decreased Fyn kinase activation. Oligodendrocyte-specific Npc1 deletion causes delayed early myelination and late loss of myelin proteins followed by secondary Purkinje neuron degeneration.","method":"Conditional cell-type-specific Npc1 knockout (neuron-specific and oligodendrocyte-specific Cre); Fyn kinase activity assays; histological and electron microscopic analysis of myelin","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional cell-type-specific KO with defined molecular (Fyn kinase) and structural (myelin) readouts, multiple cell-type conditions","pmids":["23593041"],"is_preprint":false},{"year":2011,"finding":"Neuronal-specific deletion of Npc1 is sufficient to cause NPC neuropathology (Purkinje cell loss, axonal spheroids, reactive gliosis), establishing that neuronal NPC1 deficiency—not astrocytic—drives CNS disease. Adult-onset global deletion produces the same phenotype as germline deletion, showing no significant developmental component.","method":"Conditional neuron-specific and astrocyte-specific Npc1 knockout; behavioral testing; neuropathological analysis","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional KO with defined neuropathological and behavioral readouts, epistatic conclusion about cell autonomy","pmids":["21856732"],"is_preprint":false},{"year":2012,"finding":"Targeting ER calcium levels with ryanodine receptor (RyR) antagonists increased steady-state levels of the I1061T NPC1 mutant protein, promoted its trafficking to late endosomes/lysosomes, and rescued aberrant storage of cholesterol and sphingolipids; overexpression of calnexin (a calcium-dependent ER chaperone) produced similar rescue, implicating ER calcium-dependent chaperoning in I1061T NPC1 proteostasis.","method":"RyR antagonist pharmacology; calnexin overexpression; immunofluorescence of NPC1 localization; filipin cholesterol staining; patient fibroblasts","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple RyR antagonists and calnexin overexpression with functional rescue, single lab","pmids":["22505584"],"is_preprint":false},{"year":2019,"finding":"NPC1 is present in the postsynaptic compartment and is locally translated during long-term potentiation (LTP). NPC1 mediates cholesterol mobilization at synapses and is required for surface delivery of CYP46A1 and GluA1 receptors necessary for LTP; the NPC1nmf164 mutation reduces synaptic NPC1 via enhanced protein degradation, shortens postsynaptic densities, and impairs LTP.","method":"Immunofluorescence and subcellular fractionation of synaptic compartments; local translation assay; LTP electrophysiology; GluA1 surface biotinylation; NPC1nmf164 mutant mice","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization to postsynaptic compartment with functional LTP and receptor trafficking readouts, single lab","pmids":["31535451"],"is_preprint":false},{"year":2015,"finding":"NPC1 loss in microglia causes cell-autonomous enhanced phagocytic uptake, impaired myelin turnover, accumulation of multivesicular bodies, and impaired lipid trafficking to lysosomes, while lysosomal degradation function is preserved.","method":"Npc1-/- microglial isolation; proteomics; phagocytosis assays; electron microscopy; lipid trafficking assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-autonomous phenotype demonstrated in isolated Npc1-/- microglia with multiple orthogonal assays","pmids":["33627648"],"is_preprint":false},{"year":2000,"finding":"Mutation of cysteine residues in the cysteine-rich luminal loop of NPC1 produces proteins that fail to correct cholesterol trafficking in NPC1-deficient cells; the I1061T mutation (most common disease allele, also in this loop) similarly inactivates the protein. All such mutants localize to cholesterol-engorged lysosomes, and the loop binds zinc via a zinc-NTA agarose assay.","method":"Site-directed mutagenesis; complementation of NPC1-deficient CT60 CHO cells; immunofluorescence localization; zinc-NTA agarose binding","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional complementation and localization, single lab","pmids":["10942596"],"is_preprint":false},{"year":2015,"finding":"NPC1 loss of function in microglia (mouse model and patient-derived macrophages) is cell-autonomous: NPC1-deficient macrophages from NPC patients show molecular and functional cholesterol trafficking defects.","method":"Patient-derived blood macrophages; lipid accumulation assays; protein expression analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with direct functional assays; one of two findings from PMID 33627648","pmids":["33627648"],"is_preprint":false},{"year":2015,"finding":"NPC1 interacts with cathepsin D at the lysosome; NPC1-deficient fibroblasts accumulate procathepsin D with reduced mature cathepsin D and diminished activity, while increasing NPC1 levels with proteasome inhibitor bortezomib restores cathepsin D activity.","method":"Affinity chromatography with NPC1 loop-I peptide bait; LC-MS/MS identification; co-immunoprecipitation validation; cathepsin D activity assays; patient fibroblasts; bortezomib rescue","journal":"Proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity chromatography identification followed by Co-IP validation and functional enzyme activity assay, single lab","pmids":["26507101"],"is_preprint":false},{"year":2003,"finding":"Cholesterol sequestration in NPC1-deficient neurons is ganglioside-dependent: mice doubly deficient in NPC1 and GM2/GD2 synthase (GalNAcT) lacked both neuronal GM2 accumulation and free cholesterol storage, establishing that gangliosides are required upstream of cholesterol sequestration in NPC1-deficient neurons.","method":"Double-mutant mouse genetics (NPC1-/- × GalNAcT-/-); immunocytochemistry and filipin histochemistry in situ","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis (double KO) with specific lipid readouts in situ","pmids":["12906793"],"is_preprint":false},{"year":2023,"finding":"NPC1 deficiency in oligodendrocyte progenitor cells impairs their maturation in vitro and in vivo through diminished H3K27me3-dependent gene silencing (epigenetic regulation); this is rescued by GSK-J4 (H3K27 demethylase inhibitor) or by mobilizing stored cholesterol with 2-hydroxypropyl-β-cyclodextrin.","method":"Single-nucleus RNA-seq; in vitro oligodendrocyte differentiation; H3K27me3 chromatin analysis; GSK-J4 pharmacological rescue; cyclodextrin cholesterol mobilization; Npc1-/- conditional mice","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — snRNA-seq followed by epigenetic analysis and orthogonal pharmacological rescues in vitro and in vivo","pmids":["37407594"],"is_preprint":false},{"year":2022,"finding":"NPC1-mediated cholesterol homeostasis in endosomes is required for reovirus core particle delivery into the cytoplasm: NPC1-deficient cells block reovirus infection at the post-uncoating membrane penetration step, and this defect is rescued by cholesterol-solubilizing cyclodextrin. NPC1 is not required for virus attachment, internalization, or capsid uncoating.","method":"CRISPR and RNAi screens; infectious subvirion particle (ISVP) bypass assay; hydroxypropyl-β-cyclodextrin rescue; NPC1-knockout cells; reovirus infection assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO with step-specific bypass assay (ISVP) and cholesterol rescue precisely placing NPC1 function at membrane penetration step","pmids":["35263388"],"is_preprint":false},{"year":2005,"finding":"Drosophila NPC1a (ortholog of human NPC1) promotes efficient intracellular trafficking of sterols required for ecdysone synthesis in the ring gland; NPC1a null larvae are lethal but rescued by high-cholesterol diet, 20-hydroxyecdysone, or NPC1a expression specifically in the ring gland, while total cholesterol levels in mutant larvae are unchanged.","method":"Drosophila genetic null allele; dietary rescue; tissue-specific transgenic rescue; cholesterol quantification","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean null allele with tissue-specific rescue establishing NPC1a function in sterol trafficking for ecdysteroidogenesis","pmids":["16079224"],"is_preprint":false},{"year":2020,"finding":"NPC1 is the intracellular receptor used by HAVCR1 (TIM-1) for hepatitis A virus (exo-HAV) infection: CRISPR-Cas9 knockout of either HAVCR1 or NPC1 blocks membrane fusion and RNA delivery from exosomes carrying HAV, establishing that the HAVCR1-NPC1 pathway mediates an envelope-glycoprotein-independent viral fusion mechanism.","method":"CRISPR-Cas9 knockout of HAVCR1 and NPC1; exo-HAV infection assays; methylene blue RNA inactivation; membrane fusion assays","journal":"Nature microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with functional viral entry and RNA delivery assays, single lab","pmids":["32541946"],"is_preprint":false}],"current_model":"NPC1 is a 13-transmembrane lysosomal/late-endosomal protein with an RND-permease fold whose sterol-sensing domain (TMs 3–7) harbors a central lumenal tunnel through which cholesterol passes during export; cholesterol and sphingosine are transferred from the lumen to the cytoplasm via inter-domain conformational dynamics, facilitated by NPC2 handing off cholesterol to NPC1's N-terminal domain, with active Rab7 (licensed by the Mon1-Ccz1-C18orf8 GEF complex) required to license export, NPC1 tethering ER–lysosome membrane contact sites via Gramd1b to enable direct cholesterol transfer to the ER, and loss of NPC1 causing downstream mTORC1 hyperactivation, lysosomal proteolytic failure, defective mitophagy, impaired oligodendrocyte maturation via H3K27me3 dysregulation, and synaptic cholesterol mismanagement that impairs LTP; NPC1 also serves as the intracellular receptor for Ebola and other filoviruses through its domain C, and as an entry factor for hepatitis A virus via the HAVCR1-NPC1 pathway."},"narrative":{"mechanistic_narrative":"NPC1 is a multi-pass transmembrane lysosomal/late-endosomal protein that mediates the export of cholesterol and sphingosine from the lumen of endolysosomes, the central function whose loss defines Niemann-Pick disease type C pathology [PMID:27238017, PMID:36893262]. Its cryo-EM structure resolves 13 transmembrane segments adopting a resistance-nodulation-cell division (RND) fold, with TMs 3–7 forming a sterol-sensing domain (SSD) whose central lumenal tunnel serves as the conduit for sterol passage; blocking this tunnel with itraconazole abolishes cholesterol egress [PMID:27238017, PMID:31919352]. NPC1 directly binds cholesterol and oxysterols in a buried hydrophobic pocket, with an additional cysteine-rich lumenal loop-1 sterol-binding site that is dispensable for transport [PMID:17989073, PMID:19029290, PMID:17989072], and it likewise binds sphingosine, the two lipids sharing a common export mechanism [PMID:36893262]. Cholesterol export depends on inter-domain conformational dynamics: engineered disulfide constraints between lumenal domains abolish transport [PMID:32410728]. Egress is licensed by GTP-loaded Rab7, which is activated by the Mon1-Ccz1-C18orf8 GEF complex [PMID:33144569], and culminates in direct lysosome-to-ER cholesterol transfer at membrane contact sites tethered through the ER sterol-transport protein Gramd1b [PMID:31537798]. Loss of NPC1 export function triggers mTORC1 hyperactivation that drives lysosomal proteolytic failure and defective mitophagy [PMID:33308480], while in the CNS neuronal NPC1 deficiency is cell-autonomously sufficient to cause Purkinje cell loss and arrest oligodendrocyte maturation via H3K27me3 dysregulation [PMID:21856732, PMID:23593041, PMID:37407594]. The most common disease allele, I1061T, is an ER-retained misfolded protein cleared by MARCH6-dependent ERAD and FAM134B-dependent ER-phagy [PMID:30202070]. Independently of its transport role, NPC1 serves as the intracellular endosomal receptor for Ebola virus, binding cleaved glycoprotein through its lumenal domain C [PMID:27238017, PMID:23202491], and as an entry factor for hepatitis A virus via the HAVCR1-NPC1 pathway [PMID:32541946].","teleology":[{"year":2000,"claim":"Established that NPC1's cysteine-rich lumenal loop is functionally required, linking the most common disease allele I1061T to loss of cholesterol-trafficking activity rather than mislocalization at this stage.","evidence":"Site-directed mutagenesis and complementation of NPC1-deficient CHO cells with zinc-binding assay","pmids":["10942596"],"confidence":"Medium","gaps":["Did not resolve whether the loop binds sterol directly","Mechanism of how mutants fail transport unknown at the time"]},{"year":2003,"claim":"Placed ganglioside biosynthesis genetically upstream of cholesterol sequestration in NPC1-deficient neurons, clarifying the ordering of lipid storage events.","evidence":"NPC1-/- × GalNAcT-/- double-mutant mouse genetics with in situ filipin histochemistry","pmids":["12906793"],"confidence":"High","gaps":["Molecular link between gangliosides and cholesterol retention not defined","Does not address NPC1's direct transport mechanism"]},{"year":2007,"claim":"Demonstrated that NPC1 directly binds cholesterol and oxysterols and localized a sterol-binding site to lumenal loop-1, but a mutation abolishing this binding still rescued transport, showing this site is not the essential transport conduit.","evidence":"Protein purification, radioligand binding, Q79A mutagenesis, and transport complementation in NPC1-deficient CHO cells","pmids":["17989073","17989072"],"confidence":"High","gaps":["Did not identify the actual transport-relevant sterol path","Relationship between loop-1 binding and overall export unresolved"]},{"year":2008,"claim":"Showed purified full-length NPC1 binds cholesterol 1:1 in a deep hydrophobic pocket competed by 25-HC, defining a shared sterol pocket.","evidence":"FLAG-affinity purification with fluorescent sterol binding and photoaffinity labeling","pmids":["19029290"],"confidence":"High","gaps":["Pocket location relative to membrane domains not structurally resolved","Transport directionality not addressed"]},{"year":2013,"claim":"Identified NPC1's lumenal domain C as necessary and sufficient for filovirus receptor activity, separating a viral function from cholesterol transport.","evidence":"NPC1/NPC1L1 chimera panel with viral infection and GP binding assays","pmids":["23202491"],"confidence":"High","gaps":["Did not map single-residue determinants","Relationship to host trafficking cofactors unresolved"]},{"year":2016,"claim":"Resolved the full-length NPC1 architecture, defining the 13-TM RND fold, the SSD, and the structural basis of EBOV-GP binding to domain C, providing the framework for all subsequent mechanistic interpretation.","evidence":"Single-particle cryo-EM at 4.4 Å plus crystallography of domain C and EBOV-GP binding assays","pmids":["27238017","26846330"],"confidence":"High","gaps":["Did not capture the sterol-transporting conformational states","Path of cholesterol through the protein not yet defined"]},{"year":2016,"claim":"Defined how azole antifungals inhibit NPC1 by binding the SSD, providing pharmacological probes that map the transport machinery.","evidence":"Mutagenesis, U18666A competition, docking, and photoaffinity cross-linking of NPC1 in nanodiscs with P691S validation","pmids":["28103683","27994139"],"confidence":"High","gaps":["Atomic binding pose validated structurally only later","Whether inhibition reflects tunnel occlusion not directly shown here"]},{"year":2019,"claim":"Established that NPC1 tethers ER–endolysosome contact sites via Gramd1b to deliver cholesterol directly to the ER, defining the downstream destination of exported sterol.","evidence":"Reciprocal Co-IP, live imaging, and artificial MCS tethering rescue in NPC1-deficient cells","pmids":["31537798"],"confidence":"High","gaps":["Stoichiometry and directionality of Gramd1b transfer not quantified","How NPC1 cytoplasmic transfer couples to Gramd1b unresolved"]},{"year":2020,"claim":"Defined the SSD central tunnel as the cholesterol conduit and showed inter-domain dynamics rather than NTD dissociation drive export.","evidence":"Cryo-EM with itraconazole, efflux assays, and engineered disulfide constraints","pmids":["31919352","32410728"],"confidence":"High","gaps":["Full conformational cycle of transport not captured","Energetics of lumen-to-cytoplasm transfer unresolved"]},{"year":2020,"claim":"Identified active Rab7, controlled by the Mon1-Ccz1-C18orf8 GEF, as a direct licensing factor required for NPC1-dependent cholesterol export.","evidence":"Genome-wide CRISPR screen, Co-IP, and constitutively active Rab7 rescue","pmids":["33144569"],"confidence":"High","gaps":["How Rab7 binding triggers transport competence mechanistically unknown","Spatial coordination with MCS tethering unresolved"]},{"year":2020,"claim":"Placed mTORC1 hyperactivation downstream of cholesterol accumulation as the driver of lysosomal proteolytic failure and defective mitophagy.","evidence":"Lysosomal proteomics and mTORC1 genetic/pharmacological epistasis in neuronal NPC models","pmids":["33308480"],"confidence":"High","gaps":["Molecular link from cholesterol storage to mTORC1 activation not defined","Whether mTORC1 inhibition rescues neuropathology untested here"]},{"year":2018,"claim":"Defined the degradation routes of the I1061T misfolded mutant, identifying MARCH6-ERAD and FAM134B ER-phagy as complementary clearance pathways.","evidence":"MARCH6 and FAM134B knockouts, fractionation, in vivo mouse and patient tissue","pmids":["30202070"],"confidence":"High","gaps":["Relative contribution of each pathway in patient cells not quantified","Whether blocking degradation restores function untested"]},{"year":2023,"claim":"Showed NPC1 also directly transports sphingosine, expanding its substrate scope and explaining lysosomal sphingosine accumulation that secondarily impairs cholesterol efflux.","evidence":"Photoactivatable sphingosine probes and photoaffinity labeling in NPC1-KO cells","pmids":["36893262"],"confidence":"High","gaps":["Whether sphingosine and cholesterol use the identical tunnel not structurally resolved","Order of substrate handling unknown"]},{"year":2023,"claim":"Linked neuronal/oligodendrocyte NPC1 deficiency to an epigenetic mechanism, showing impaired H3K27me3-dependent silencing arrests oligodendrocyte maturation.","evidence":"snRNA-seq, H3K27me3 chromatin analysis, and GSK-J4 / cyclodextrin rescue in Npc1-/- mice","pmids":["37407594"],"confidence":"High","gaps":["How cholesterol storage alters H3K27me3 metabolism unresolved","Generality to other cell types untested"]},{"year":null,"claim":"How NPC1's lumen-to-cytoplasm sterol handoff is energetically driven and physically coupled to NPC2 delivery, Rab7 licensing, and Gramd1b acceptor transfer into a single transport cycle remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of NPC1 captured mid-transport with substrate moving through the tunnel","Stoichiometric coupling of accessory factors to individual transport events undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[3,11,4,15]},{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[1,12,15]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[17,0,34]},{"term_id":"GO:0140104","term_label":"molecular carrier 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Unesterified cholesterol that has been released from LDLs in the lumen of the late endosomes/lysosomes is transferred by NPC2 to the cholesterol-binding pocket in the N-terminal domain of NPC1 (PubMed:18772377, PubMed:19563754, PubMed:27238017, PubMed:27378690, PubMed:28784760, PubMed:9211849, PubMed:9927649). Cholesterol binds to NPC1 with the hydroxyl group buried in the binding pocket (PubMed:19563754). Binds oxysterol with higher affinity than cholesterol. May play a role in vesicular trafficking in glia, a process that may be crucial for maintaining the structural and functional integrity of nerve terminals (Probable). 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NPC1 is not required for known oxysterol regulatory actions on SREBP processing or ACAT.\",\n      \"method\": \"Purification of NPC1 from rabbit liver membranes (~14,000-fold); radioligand binding assays with [³H]cholesterol and [³H]25-HC; competition assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical purification and direct in vitro binding assays with rigorous controls\",\n      \"pmids\": [\"17989073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The sterol-binding site of NPC1 is localized to luminal loop-1 (a 240-amino acid cysteine-rich domain), which binds [³H]cholesterol (Kd ~130 nM) and [³H]25-HC (Kd ~10 nM) as a soluble dimer. Mutation Q79A abolishes sterol binding to loop-1 yet still restores cholesterol transport in NPC1-deficient CHO cells, indicating this binding site is not essential for NPC1's transport function in fibroblasts.\",\n      \"method\": \"Recombinant protein production; radioligand binding assays; site-directed mutagenesis (Q79A); functional cholesterol transport complementation assay in NPC1-deficient CHO cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of binding with mutagenesis and functional transport complementation in single rigorous study\",\n      \"pmids\": [\"17989072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NPC1 tethers ER–endocytic organelle membrane contact sites (MCS) by interacting with the ER-localized sterol transport protein Gramd1b, and this interaction regulates cholesterol egress from lysosomes directly to the ER across MCS. Artificially tethering MCS rescued cholesterol accumulation in NPC1-deficient cells. Loss of NPC1 or Gramd1b expanded lysosome–mitochondria MCS in a STARD3-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation; live-cell imaging; MCS artificial tethering; fractionation; NPC1-deficient cell lines\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus MCS rescue experiment plus multiple imaging modalities, replicated across conditions\",\n      \"pmids\": [\"31537798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Active Rab7 (GTP-loaded) directly interacts with the NPC1 cholesterol transporter and is required to license NPC1-dependent lysosomal cholesterol export; this function is controlled by the trimeric Mon1-Ccz1-C18orf8 (MCC) GEF that activates Rab7. Loss of MCC subunits abolishes lysosomal cholesterol export and is rescued by constitutively active Rab7.\",\n      \"method\": \"Genome-wide CRISPR screen; Co-immunoprecipitation; cholesterol reporter assay; Rab7 constitutively active rescue experiments\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide CRISPR screen followed by Co-IP and functional rescue with constitutively active Rab7, multiple orthogonal methods\",\n      \"pmids\": [\"33144569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss of NPC1-mediated cholesterol export causes mTORC1 hyperactivation, which drives lysosomal proteolytic impairment, hydrolase depletion, enhanced membrane damage, and defective mitophagy; genetic and pharmacological mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, placing aberrant mTORC1 downstream of cholesterol accumulation.\",\n      \"method\": \"Proteomic profiling of NPC lysosomes; genetic mTORC1 inhibition (rapamycin); lysosomal proteolysis assays; mitophagy assays; neuronal NPC models\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — lysosomal proteomics plus genetic epistasis (mTORC1 inhibition rescues proteolysis but not cholesterol) with functional readouts\",\n      \"pmids\": [\"33308480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The antifungal drug itraconazole directly inhibits NPC1 by binding to its sterol-sensing domain (SSD); the binding site was mapped by mutagenesis, competition with U18666A, and molecular docking. Dual inhibition of NPC1 (cholesterol trafficking) and VDAC1 (AMPK activation) synergistically inhibits mTOR signaling and angiogenesis.\",\n      \"method\": \"Pharmacological inhibition assays; site-directed mutagenesis of SSD; competition binding with U18666A; molecular docking\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus competition binding plus docking in single lab; no direct structural validation in this paper\",\n      \"pmids\": [\"28103683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Posaconazole (a triazole antifungal) and itraconazole directly bind to the NPC1 sterol-sensing domain to block lysosomal cholesterol export; a photoactivatable posaconazole derivative cross-linked specifically to purified NPC1 in lipid bilayer nanodiscs, and cross-linking was reduced by a P691S point mutation in the SSD.\",\n      \"method\": \"Photoactivatable cross-linking with posaconazole derivative P-X; NPC1 purification into nanodiscs; site-directed mutagenesis (P691S); competition with itraconazole and U18666A\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct photoaffinity labeling of purified NPC1 in reconstituted nanodiscs, mutagenesis confirmation, multiple competitive ligands\",\n      \"pmids\": [\"27994139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The most common disease-causing NPC1 mutant I1061T is degraded by two complementary pathways: MARCH6-dependent ERAD followed by proteasomal degradation, and FAM134B-dependent selective ER autophagy (ER-phagy). Subcellular fractionation in mouse tissues confirmed ER retention of I1061T NPC1.\",\n      \"method\": \"Proteasome inhibitor studies; MARCH6 knockdown/knockout; ER-phagy (FAM134B) knockout; subcellular fractionation; in vivo mouse models; human tissue samples\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple complementary genetic knockouts (MARCH6 and FAM134B) validated both in vitro and in vivo with defined mechanistic readouts\",\n      \"pmids\": [\"30202070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Purified full-length NPC1 directly binds fluorescent cholesterol analogs (dehydroergosterol, cholestatrienol, NBD-cholesterol) with apparent affinity ~0.5–6 µM (1:1 stoichiometry); bound cholesterol is buried in a deep hydrophobic pocket. Binding is competed by native cholesterol and 25-hydroxycholesterol, confirming a shared sterol-binding site.\",\n      \"method\": \"FLAG-tag affinity purification of NPC1; fluorescence binding and quenching assays; gel filtration; photoaffinity labeling with azido-cholesterol\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assays with purified protein, multiple fluorescent probes, stoichiometry determination\",\n      \"pmids\": [\"19029290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Inter-domain dynamics of NPC1 are required for cholesterol transport: introducing single disulfide bonds to constrain lumenal domains, or shortening a cytoplasmic loop, abolishes transport activity. The N-terminal domain need not dissociate from the rest of the protein for efficient export. Ezetimibe blocks NPC1L1 transport by binding simultaneously to residues at the interface of all three extracellular domains.\",\n      \"method\": \"Site-directed disulfide bond engineering; lysosomal cholesterol efflux assay; domain truncation mutagenesis; ezetimibe binding site mapping\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — engineered disulfide constraints with functional transport assay and mutagenesis of drug binding site in single rigorous study\",\n      \"pmids\": [\"32410728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NPC1 undergoes ubiquitylation that is regulated by endosomal cholesterol levels: cholesterol depletion promotes NPC1 ubiquitylation, while the SSD mutant P691S fails to respond. Ubiquitylated NPC1 associates with the ESCRT component SKD1/Vps4; NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions.\",\n      \"method\": \"Co-immunoprecipitation; dominant-negative SKD1 expression; cholesterol depletion experiments; site-directed mutagenesis (P691S, ΔLLNF); NPC2-deficient patient fibroblasts\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with dominant-negative and mutagenesis approaches in single lab, multiple cell systems\",\n      \"pmids\": [\"16757520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TMEM97 is a cholesterol-responsive NPC1-binding protein that post-transcriptionally regulates NPC1 abundance; reducing TMEM97 increases NPC1 levels and restores cholesterol trafficking to the ER in NPC disease cells in an NPC1-dependent manner. TMEM97 lacking its ER-retention signal fails to regulate NPC1 availability.\",\n      \"method\": \"RNA interference; Co-immunoprecipitation; cholesterol trafficking assays (filipin staining, ER cholesterol measurement); domain deletion of TMEM97 ER-retention signal\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirmed interaction, functional rescue with domain mutant, single lab\",\n      \"pmids\": [\"27378690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NPC1 directly binds sphingosine using lysosome-targeted photoactivatable sphingosine probes; absence of NPC1 causes lysosomal sphingosine accumulation. Artificial elevation of lysosomal sphingosine impairs cholesterol efflux, consistent with sphingosine and cholesterol sharing a common NPC1-mediated export mechanism.\",\n      \"method\": \"Caged, photocrosslinkable sphingosine and cholesterol probes; photoaffinity labeling; subcellular fractionation; NPC1 knockout cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — novel probe technology with direct photoaffinity labeling and functional consequence in NPC1-KO cells, single lab but orthogonal chemical biology approach\",\n      \"pmids\": [\"36893262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NPC1 aids transfer of LDL-derived cholesterol across the lysosomal glycocalyx: inhibiting O-linked glycosylation in NPC1-deficient fibroblasts reduced lysosomal cholesterol content by ≥30% and increased ER cholesterol delivery, indicating that cells become less dependent on NPC1 when glycocalyx density is reduced.\",\n      \"method\": \"Pharmacological inhibition of O-glycosylation; CRISPR-generated NPC1-deficient CHO cells; biochemical lysosome cholesterol measurement; [¹⁴C]-oleate esterification assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent genetic/pharmacological glycosylation manipulations with direct biochemical cholesterol measurement\",\n      \"pmids\": [\"26578804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NPC1 is an intracellular endosomal receptor for Ebola virus; its second luminal domain (domain C) is necessary and sufficient for filovirus receptor activity. NPC1L1 lacks receptor activity because its domain C does not bind viral GP; a chimera bearing NPC1's domain C conferred near-wild-type filovirus infection.\",\n      \"method\": \"NPC1/NPC1L1 chimera panel; viral infection assays; GP binding assays\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic chimera analysis with reciprocal domain swaps and authentic viral infection assays\",\n      \"pmids\": [\"23202491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A single amino acid at position 503 in NPC1 bidirectionally controls binding to EBOV glycoprotein and viral receptor activity; this residue is in domain C and its mutation does not perturb NPC1 endosomal localization or cholesterol trafficking function.\",\n      \"method\": \"NPC1 viper-human chimeras and point mutants; viral infection assays; GP binding assays; cholesterol trafficking assays\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, chimera/point mutant analysis with functional and localization readouts\",\n      \"pmids\": [\"27303731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TIM-1 and NPC1 colocalize and physically interact in intracellular vesicles where EBOV glycoprotein-mediated membrane fusion occurs; a TIM-1-specific monoclonal antibody that disrupts TIM-1–NPC1 interaction also prevents GP-mediated membrane fusion, implicating this protein–protein interaction in filovirus fusion.\",\n      \"method\": \"Co-immunoprecipitation; colocalization by fluorescence microscopy; monoclonal antibody blocking; pseudovirus fusion assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and colocalization with functional antibody blocking, single lab\",\n      \"pmids\": [\"25855742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ebolavirus enters cells through endolysosomes that co-contain both NPC1 and TPC2, directly observed by live-cell imaging, contradicting a model of entry through NPC1-negative organelles.\",\n      \"method\": \"Live-cell imaging; co-localization of NPC1 and TPC2 with viral entry sites\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct live-cell imaging of viral entry pathway in NPC1+ compartments, single lab\",\n      \"pmids\": [\"26468524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NPC1-bearing vesicles (lacking lysosomal markers) traffick to Anaplasma phagocytophilum bacterial inclusions; NPC1 function is required for bacterial cholesterol acquisition and infection. The trans-Golgi SNAREs VAMP4 and syntaxin 16, which associate with NPC1 on LDL-cholesterol vesicles, are recruited to bacterial inclusions and VAMP4 is required for bacterial infection.\",\n      \"method\": \"siRNA knockdown; immunofluorescence co-localization; cholesterol-traffic inhibitor U18666A; infection assays\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with defined infection and cholesterol trafficking readouts, single lab\",\n      \"pmids\": [\"22212234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In NPC1-deficient cells, neuronal deletion of Npc1 alone is sufficient to arrest oligodendrocyte maturation and cause myelination failure, associated with decreased Fyn kinase activation. Oligodendrocyte-specific Npc1 deletion causes delayed early myelination and late loss of myelin proteins followed by secondary Purkinje neuron degeneration.\",\n      \"method\": \"Conditional cell-type-specific Npc1 knockout (neuron-specific and oligodendrocyte-specific Cre); Fyn kinase activity assays; histological and electron microscopic analysis of myelin\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional cell-type-specific KO with defined molecular (Fyn kinase) and structural (myelin) readouts, multiple cell-type conditions\",\n      \"pmids\": [\"23593041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Neuronal-specific deletion of Npc1 is sufficient to cause NPC neuropathology (Purkinje cell loss, axonal spheroids, reactive gliosis), establishing that neuronal NPC1 deficiency—not astrocytic—drives CNS disease. Adult-onset global deletion produces the same phenotype as germline deletion, showing no significant developmental component.\",\n      \"method\": \"Conditional neuron-specific and astrocyte-specific Npc1 knockout; behavioral testing; neuropathological analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional KO with defined neuropathological and behavioral readouts, epistatic conclusion about cell autonomy\",\n      \"pmids\": [\"21856732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Targeting ER calcium levels with ryanodine receptor (RyR) antagonists increased steady-state levels of the I1061T NPC1 mutant protein, promoted its trafficking to late endosomes/lysosomes, and rescued aberrant storage of cholesterol and sphingolipids; overexpression of calnexin (a calcium-dependent ER chaperone) produced similar rescue, implicating ER calcium-dependent chaperoning in I1061T NPC1 proteostasis.\",\n      \"method\": \"RyR antagonist pharmacology; calnexin overexpression; immunofluorescence of NPC1 localization; filipin cholesterol staining; patient fibroblasts\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple RyR antagonists and calnexin overexpression with functional rescue, single lab\",\n      \"pmids\": [\"22505584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NPC1 is present in the postsynaptic compartment and is locally translated during long-term potentiation (LTP). NPC1 mediates cholesterol mobilization at synapses and is required for surface delivery of CYP46A1 and GluA1 receptors necessary for LTP; the NPC1nmf164 mutation reduces synaptic NPC1 via enhanced protein degradation, shortens postsynaptic densities, and impairs LTP.\",\n      \"method\": \"Immunofluorescence and subcellular fractionation of synaptic compartments; local translation assay; LTP electrophysiology; GluA1 surface biotinylation; NPC1nmf164 mutant mice\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization to postsynaptic compartment with functional LTP and receptor trafficking readouts, single lab\",\n      \"pmids\": [\"31535451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NPC1 loss in microglia causes cell-autonomous enhanced phagocytic uptake, impaired myelin turnover, accumulation of multivesicular bodies, and impaired lipid trafficking to lysosomes, while lysosomal degradation function is preserved.\",\n      \"method\": \"Npc1-/- microglial isolation; proteomics; phagocytosis assays; electron microscopy; lipid trafficking assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-autonomous phenotype demonstrated in isolated Npc1-/- microglia with multiple orthogonal assays\",\n      \"pmids\": [\"33627648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Mutation of cysteine residues in the cysteine-rich luminal loop of NPC1 produces proteins that fail to correct cholesterol trafficking in NPC1-deficient cells; the I1061T mutation (most common disease allele, also in this loop) similarly inactivates the protein. All such mutants localize to cholesterol-engorged lysosomes, and the loop binds zinc via a zinc-NTA agarose assay.\",\n      \"method\": \"Site-directed mutagenesis; complementation of NPC1-deficient CT60 CHO cells; immunofluorescence localization; zinc-NTA agarose binding\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional complementation and localization, single lab\",\n      \"pmids\": [\"10942596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NPC1 loss of function in microglia (mouse model and patient-derived macrophages) is cell-autonomous: NPC1-deficient macrophages from NPC patients show molecular and functional cholesterol trafficking defects.\",\n      \"method\": \"Patient-derived blood macrophages; lipid accumulation assays; protein expression analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with direct functional assays; one of two findings from PMID 33627648\",\n      \"pmids\": [\"33627648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NPC1 interacts with cathepsin D at the lysosome; NPC1-deficient fibroblasts accumulate procathepsin D with reduced mature cathepsin D and diminished activity, while increasing NPC1 levels with proteasome inhibitor bortezomib restores cathepsin D activity.\",\n      \"method\": \"Affinity chromatography with NPC1 loop-I peptide bait; LC-MS/MS identification; co-immunoprecipitation validation; cathepsin D activity assays; patient fibroblasts; bortezomib rescue\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity chromatography identification followed by Co-IP validation and functional enzyme activity assay, single lab\",\n      \"pmids\": [\"26507101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cholesterol sequestration in NPC1-deficient neurons is ganglioside-dependent: mice doubly deficient in NPC1 and GM2/GD2 synthase (GalNAcT) lacked both neuronal GM2 accumulation and free cholesterol storage, establishing that gangliosides are required upstream of cholesterol sequestration in NPC1-deficient neurons.\",\n      \"method\": \"Double-mutant mouse genetics (NPC1-/- × GalNAcT-/-); immunocytochemistry and filipin histochemistry in situ\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis (double KO) with specific lipid readouts in situ\",\n      \"pmids\": [\"12906793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NPC1 deficiency in oligodendrocyte progenitor cells impairs their maturation in vitro and in vivo through diminished H3K27me3-dependent gene silencing (epigenetic regulation); this is rescued by GSK-J4 (H3K27 demethylase inhibitor) or by mobilizing stored cholesterol with 2-hydroxypropyl-β-cyclodextrin.\",\n      \"method\": \"Single-nucleus RNA-seq; in vitro oligodendrocyte differentiation; H3K27me3 chromatin analysis; GSK-J4 pharmacological rescue; cyclodextrin cholesterol mobilization; Npc1-/- conditional mice\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — snRNA-seq followed by epigenetic analysis and orthogonal pharmacological rescues in vitro and in vivo\",\n      \"pmids\": [\"37407594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NPC1-mediated cholesterol homeostasis in endosomes is required for reovirus core particle delivery into the cytoplasm: NPC1-deficient cells block reovirus infection at the post-uncoating membrane penetration step, and this defect is rescued by cholesterol-solubilizing cyclodextrin. NPC1 is not required for virus attachment, internalization, or capsid uncoating.\",\n      \"method\": \"CRISPR and RNAi screens; infectious subvirion particle (ISVP) bypass assay; hydroxypropyl-β-cyclodextrin rescue; NPC1-knockout cells; reovirus infection assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO with step-specific bypass assay (ISVP) and cholesterol rescue precisely placing NPC1 function at membrane penetration step\",\n      \"pmids\": [\"35263388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Drosophila NPC1a (ortholog of human NPC1) promotes efficient intracellular trafficking of sterols required for ecdysone synthesis in the ring gland; NPC1a null larvae are lethal but rescued by high-cholesterol diet, 20-hydroxyecdysone, or NPC1a expression specifically in the ring gland, while total cholesterol levels in mutant larvae are unchanged.\",\n      \"method\": \"Drosophila genetic null allele; dietary rescue; tissue-specific transgenic rescue; cholesterol quantification\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean null allele with tissue-specific rescue establishing NPC1a function in sterol trafficking for ecdysteroidogenesis\",\n      \"pmids\": [\"16079224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NPC1 is the intracellular receptor used by HAVCR1 (TIM-1) for hepatitis A virus (exo-HAV) infection: CRISPR-Cas9 knockout of either HAVCR1 or NPC1 blocks membrane fusion and RNA delivery from exosomes carrying HAV, establishing that the HAVCR1-NPC1 pathway mediates an envelope-glycoprotein-independent viral fusion mechanism.\",\n      \"method\": \"CRISPR-Cas9 knockout of HAVCR1 and NPC1; exo-HAV infection assays; methylene blue RNA inactivation; membrane fusion assays\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with functional viral entry and RNA delivery assays, single lab\",\n      \"pmids\": [\"32541946\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NPC1 is a 13-transmembrane lysosomal/late-endosomal protein with an RND-permease fold whose sterol-sensing domain (TMs 3–7) harbors a central lumenal tunnel through which cholesterol passes during export; cholesterol and sphingosine are transferred from the lumen to the cytoplasm via inter-domain conformational dynamics, facilitated by NPC2 handing off cholesterol to NPC1's N-terminal domain, with active Rab7 (licensed by the Mon1-Ccz1-C18orf8 GEF complex) required to license export, NPC1 tethering ER–lysosome membrane contact sites via Gramd1b to enable direct cholesterol transfer to the ER, and loss of NPC1 causing downstream mTORC1 hyperactivation, lysosomal proteolytic failure, defective mitophagy, impaired oligodendrocyte maturation via H3K27me3 dysregulation, and synaptic cholesterol mismanagement that impairs LTP; NPC1 also serves as the intracellular receptor for Ebola and other filoviruses through its domain C, and as an entry factor for hepatitis A virus via the HAVCR1-NPC1 pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NPC1 is a multi-pass transmembrane lysosomal/late-endosomal protein that mediates the export of cholesterol and sphingosine from the lumen of endolysosomes, the central function whose loss defines Niemann-Pick disease type C pathology [#0, #15]. Its cryo-EM structure resolves 13 transmembrane segments adopting a resistance-nodulation-cell division (RND) fold, with TMs 3–7 forming a sterol-sensing domain (SSD) whose central lumenal tunnel serves as the conduit for sterol passage; blocking this tunnel with itraconazole abolishes cholesterol egress [#0, #1]. NPC1 directly binds cholesterol and oxysterols in a buried hydrophobic pocket, with an additional cysteine-rich lumenal loop-1 sterol-binding site that is dispensable for transport [#3, #11, #4], and it likewise binds sphingosine, the two lipids sharing a common export mechanism [#15]. Cholesterol export depends on inter-domain conformational dynamics: engineered disulfide constraints between lumenal domains abolish transport [#12]. Egress is licensed by GTP-loaded Rab7, which is activated by the Mon1-Ccz1-C18orf8 GEF complex [#6], and culminates in direct lysosome-to-ER cholesterol transfer at membrane contact sites tethered through the ER sterol-transport protein Gramd1b [#5]. Loss of NPC1 export function triggers mTORC1 hyperactivation that drives lysosomal proteolytic failure and defective mitophagy [#7], while in the CNS neuronal NPC1 deficiency is cell-autonomously sufficient to cause Purkinje cell loss and arrest oligodendrocyte maturation via H3K27me3 dysregulation [#23, #22, #31]. The most common disease allele, I1061T, is an ER-retained misfolded protein cleared by MARCH6-dependent ERAD and FAM134B-dependent ER-phagy [#10]. Independently of its transport role, NPC1 serves as the intracellular endosomal receptor for Ebola virus, binding cleaved glycoprotein through its lumenal domain C [#0, #17], and as an entry factor for hepatitis A virus via the HAVCR1-NPC1 pathway [#34].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that NPC1's cysteine-rich lumenal loop is functionally required, linking the most common disease allele I1061T to loss of cholesterol-trafficking activity rather than mislocalization at this stage.\",\n      \"evidence\": \"Site-directed mutagenesis and complementation of NPC1-deficient CHO cells with zinc-binding assay\",\n      \"pmids\": [\"10942596\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve whether the loop binds sterol directly\", \"Mechanism of how mutants fail transport unknown at the time\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Placed ganglioside biosynthesis genetically upstream of cholesterol sequestration in NPC1-deficient neurons, clarifying the ordering of lipid storage events.\",\n      \"evidence\": \"NPC1-/- × GalNAcT-/- double-mutant mouse genetics with in situ filipin histochemistry\",\n      \"pmids\": [\"12906793\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between gangliosides and cholesterol retention not defined\", \"Does not address NPC1's direct transport mechanism\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated that NPC1 directly binds cholesterol and oxysterols and localized a sterol-binding site to lumenal loop-1, but a mutation abolishing this binding still rescued transport, showing this site is not the essential transport conduit.\",\n      \"evidence\": \"Protein purification, radioligand binding, Q79A mutagenesis, and transport complementation in NPC1-deficient CHO cells\",\n      \"pmids\": [\"17989073\", \"17989072\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the actual transport-relevant sterol path\", \"Relationship between loop-1 binding and overall export unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed purified full-length NPC1 binds cholesterol 1:1 in a deep hydrophobic pocket competed by 25-HC, defining a shared sterol pocket.\",\n      \"evidence\": \"FLAG-affinity purification with fluorescent sterol binding and photoaffinity labeling\",\n      \"pmids\": [\"19029290\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Pocket location relative to membrane domains not structurally resolved\", \"Transport directionality not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified NPC1's lumenal domain C as necessary and sufficient for filovirus receptor activity, separating a viral function from cholesterol transport.\",\n      \"evidence\": \"NPC1/NPC1L1 chimera panel with viral infection and GP binding assays\",\n      \"pmids\": [\"23202491\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map single-residue determinants\", \"Relationship to host trafficking cofactors unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the full-length NPC1 architecture, defining the 13-TM RND fold, the SSD, and the structural basis of EBOV-GP binding to domain C, providing the framework for all subsequent mechanistic interpretation.\",\n      \"evidence\": \"Single-particle cryo-EM at 4.4 Å plus crystallography of domain C and EBOV-GP binding assays\",\n      \"pmids\": [\"27238017\", \"26846330\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture the sterol-transporting conformational states\", \"Path of cholesterol through the protein not yet defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined how azole antifungals inhibit NPC1 by binding the SSD, providing pharmacological probes that map the transport machinery.\",\n      \"evidence\": \"Mutagenesis, U18666A competition, docking, and photoaffinity cross-linking of NPC1 in nanodiscs with P691S validation\",\n      \"pmids\": [\"28103683\", \"27994139\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic binding pose validated structurally only later\", \"Whether inhibition reflects tunnel occlusion not directly shown here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established that NPC1 tethers ER–endolysosome contact sites via Gramd1b to deliver cholesterol directly to the ER, defining the downstream destination of exported sterol.\",\n      \"evidence\": \"Reciprocal Co-IP, live imaging, and artificial MCS tethering rescue in NPC1-deficient cells\",\n      \"pmids\": [\"31537798\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and directionality of Gramd1b transfer not quantified\", \"How NPC1 cytoplasmic transfer couples to Gramd1b unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined the SSD central tunnel as the cholesterol conduit and showed inter-domain dynamics rather than NTD dissociation drive export.\",\n      \"evidence\": \"Cryo-EM with itraconazole, efflux assays, and engineered disulfide constraints\",\n      \"pmids\": [\"31919352\", \"32410728\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full conformational cycle of transport not captured\", \"Energetics of lumen-to-cytoplasm transfer unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified active Rab7, controlled by the Mon1-Ccz1-C18orf8 GEF, as a direct licensing factor required for NPC1-dependent cholesterol export.\",\n      \"evidence\": \"Genome-wide CRISPR screen, Co-IP, and constitutively active Rab7 rescue\",\n      \"pmids\": [\"33144569\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Rab7 binding triggers transport competence mechanistically unknown\", \"Spatial coordination with MCS tethering unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed mTORC1 hyperactivation downstream of cholesterol accumulation as the driver of lysosomal proteolytic failure and defective mitophagy.\",\n      \"evidence\": \"Lysosomal proteomics and mTORC1 genetic/pharmacological epistasis in neuronal NPC models\",\n      \"pmids\": [\"33308480\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link from cholesterol storage to mTORC1 activation not defined\", \"Whether mTORC1 inhibition rescues neuropathology untested here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the degradation routes of the I1061T misfolded mutant, identifying MARCH6-ERAD and FAM134B ER-phagy as complementary clearance pathways.\",\n      \"evidence\": \"MARCH6 and FAM134B knockouts, fractionation, in vivo mouse and patient tissue\",\n      \"pmids\": [\"30202070\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of each pathway in patient cells not quantified\", \"Whether blocking degradation restores function untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed NPC1 also directly transports sphingosine, expanding its substrate scope and explaining lysosomal sphingosine accumulation that secondarily impairs cholesterol efflux.\",\n      \"evidence\": \"Photoactivatable sphingosine probes and photoaffinity labeling in NPC1-KO cells\",\n      \"pmids\": [\"36893262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether sphingosine and cholesterol use the identical tunnel not structurally resolved\", \"Order of substrate handling unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked neuronal/oligodendrocyte NPC1 deficiency to an epigenetic mechanism, showing impaired H3K27me3-dependent silencing arrests oligodendrocyte maturation.\",\n      \"evidence\": \"snRNA-seq, H3K27me3 chromatin analysis, and GSK-J4 / cyclodextrin rescue in Npc1-/- mice\",\n      \"pmids\": [\"37407594\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How cholesterol storage alters H3K27me3 metabolism unresolved\", \"Generality to other cell types untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NPC1's lumen-to-cytoplasm sterol handoff is energetically driven and physically coupled to NPC2 delivery, Rab7 licensing, and Gramd1b acceptor transfer into a single transport cycle remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of NPC1 captured mid-transport with substrate moving through the tunnel\", \"Stoichiometric coupling of accessory factors to individual transport events undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [3, 11, 4, 15]},\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1, 12, 15]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [17, 0, 34]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1, 7, 15]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5, 17, 20]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [5, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 5, 6]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [1, 12, 15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [17, 34, 10]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7, 10]}\n    ],\n    \"complexes\": [\n      \"ER–lysosome membrane contact site\"\n    ],\n    \"partners\": [\n      \"NPC2\",\n      \"GRAMD1B\",\n      \"RAB7\",\n      \"TMEM97\",\n      \"HAVCR1\",\n      \"CTSD\",\n      \"VAMP4\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}