{"gene":"ZFYVE27","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2006,"finding":"ZFYVE27 (protrudin) was identified as a specific spastin-binding protein via yeast two-hybrid screen, and the interaction was validated by in vivo co-immunoprecipitation and colocalization in mammalian cells. A disease-associated mutation in ZFYVE27 severely impairs its interaction with spastin and causes an aberrant intracellular tubular pattern.","method":"Yeast two-hybrid, co-immunoprecipitation, colocalization (fluorescence microscopy)","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and colocalization, yeast two-hybrid, plus functional consequence of mutant shown; single lab but multiple orthogonal methods","pmids":["16826525"],"is_preprint":false},{"year":2011,"finding":"ZFYVE27 (protrudin) self-interacts via its third hydrophobic region (HR3) and oligomerizes into dimer/tetramer forms as shown by sucrose gradient centrifugation. ZFYVE27 is a peripheral membrane protein that binds phosphatidylinositol 3-phosphate. Deletion of HR3 (ΔHR3) abolishes protrusion formation and acts as a dominant negative over wild-type ZFYVE27, demonstrating that oligomerization is required for neurite extension.","method":"Yeast two-hybrid, co-immunoprecipitation, sucrose gradient centrifugation, subcellular fractionation, Triton X-114 phase separation, lipid-binding assay, dominant-negative overexpression","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical and cell-biological methods in a single study establishing oligomerization mechanism and functional requirement","pmids":["22216323"],"is_preprint":false},{"year":2013,"finding":"Protrudin (ZFYVE27/SPG33) contains hydrophobic intramembrane hairpin domains, interacts with tubular ER proteins (atlastins and ER-shaping proteins), and functions in ER morphogenesis by regulating the sheet-to-tubule balance and tubule interconnection density. Protrudin also harbors a KIF5-interacting domain, a Rab-binding domain, a non-canonical FYVE domain, and an FFAT domain.","method":"Co-immunoprecipitation, domain analysis, ER morphology assays (overexpression/knockdown), live-cell imaging","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple domain-function experiments, binding partner identification by Co-IP, and direct morphological readout; single lab but multiple orthogonal methods","pmids":["23969831"],"is_preprint":false},{"year":2014,"finding":"Protrudin localizes predominantly to the tubular endoplasmic reticulum (ER) and forced expression promotes formation and stabilization of the tubular ER network. One of three hydrophobic segments forms a hydrophobic hairpin domain. Protrudin interacts with multiple HSP-related proteins (SPG2/PLP1, SPG3A/atlastin-1, SPG31/REEP1, REEP5, KIF5A/B/C, reticulon 1/3/4). The HSP-associated mutant protrudin(G191V) has increased intracellular stability and cells expressing it show increased susceptibility to ER stress.","method":"Proteomics (purified protrudin complexes from transgenic mouse brain), membrane topology analysis, ER morphology assay, ER stress assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vivo proteomic identification of interactors from transgenic brain tissue, membrane topology, and functional ER morphology/stress readouts in single study with multiple methods","pmids":["24668814"],"is_preprint":false},{"year":2017,"finding":"SRRM4 regulates alternative splicing of protrudin (ZFYVE27) pre-mRNA to include a neuron-specific microexon (exon L, encoding 7 amino acids) by recognizing a UGC motif upstream of exon L. The resulting long isoform (protrudin-L) is more effective at promoting neurite outgrowth than the short isoform (protrudin-S). Deletion of exon L impairs neurite outgrowth in Neuro2A cells and embryonic stem cells.","method":"RT-PCR/splicing assays, SRRM4 knockdown, exon deletion (CRISPR/genome editing), neurite outgrowth assays, motif mutation","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function of splicing regulator, exon deletion with defined cellular phenotype, motif mapping; multiple orthogonal methods in single study","pmids":["28106138"],"is_preprint":false},{"year":2019,"finding":"Protrudin functions as a tethering factor at membrane contact sites (MCSs) between the ER and other organelles. Its roles at MCSs involve inactivation of the small GTPase Rab11, bending of the ER membrane, and functional interactions with motor protein KIF5 and ER protein VAP.","method":"Review synthesizing experimental data (organelle fractionation, domain-function studies, MCS tethering assays cited within)","journal":"Proceedings of the Japan Academy. Series B, Physical and biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — review article synthesizing experimental findings from the same group; individual experiments support claims but this paper itself does not present primary data","pmids":["31406056"],"is_preprint":false},{"year":2020,"finding":"Protrudin-deficient mice show pleiotropic behavioral abnormalities (hyperactivity, depression-like behavior, attention deficits, impaired fear-conditioning memory) but no HSP signs, indicating that the HSP-associated mutations likely cause neural degeneration through a gain-of-toxic-function rather than loss-of-function mechanism.","method":"Knockout mouse generation, battery of behavioral tests","journal":"Molecular brain","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined behavioral phenotype, but pathway/molecular mechanism not directly resolved; single lab","pmids":["33172474"],"is_preprint":false},{"year":2021,"finding":"PDZD8 is a protrudin (ZFYVE27)-interacting protein that acts as a tether at ER–late endosome/lysosome membrane contact sites. PDZD8's SMP domain binds glycerophospholipids and ceramides and can transfer lipids between membranes in vitro, and this lipid transfer activity is required for late endosome/lysosome positioning and neurite outgrowth.","method":"Co-immunoprecipitation, in vitro lipid-binding and lipid-transfer assays, organelle positioning assays, knockdown with neurite outgrowth readout","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro reconstitution of lipid transfer, Co-IP for interaction, and defined cellular phenotype upon KD; multiple orthogonal methods","pmids":["33912962"],"is_preprint":false},{"year":2022,"finding":"Protrudin knockdown in endothelial cells inhibits FAK activation, disrupts polarized phospho-FAK distribution, causes perinuclear accumulation of mTOR, and decreases VEGF-mediated S6K activation, leading to impaired endothelial cell migration and angiogenesis. Mice with global protrudin deletion show reduced retinal vascular progression.","method":"siRNA knockdown, phospho-FAK immunofluorescence, mTOR/S6K immunoblotting, tube formation assay, in vivo retinal vascular analysis in KO mice","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with specific signaling readout (FAK phosphorylation) and in vivo KO vascular phenotype; single lab, multiple methods","pmids":["35368213"],"is_preprint":false}],"current_model":"ZFYVE27/protrudin is an ER-resident peripheral membrane protein with a non-canonical FYVE domain, FFAT domain, Rab-binding domain, and hydrophobic hairpin domains that localizes to tubular ER, where it tethers ER–endosome/lysosome membrane contact sites, regulates ER tubular network morphology, promotes directional endosome trafficking via KIF5, inactivates Rab11, and interacts with spastin and multiple other HSP-associated proteins; its oligomerization (via HR3) is required for neurite outgrowth, a neuron-specific long isoform is generated by SRRM4-dependent splicing, and HSP-associated mutations impair spastin binding and cause ER stress through a likely gain-of-function mechanism."},"narrative":{"mechanistic_narrative":"ZFYVE27 (protrudin) is an endoplasmic reticulum-resident peripheral membrane protein that shapes the tubular ER network and tethers ER membrane contact sites to drive directional membrane trafficking and neurite outgrowth [PMID:23969831, PMID:24668814, PMID:31406056]. Through hydrophobic intramembrane hairpin domains and interactions with ER-shaping atlastins and reticulons, it regulates the ER sheet-to-tubule balance and stabilizes the tubular ER network where it predominantly localizes [PMID:23969831, PMID:24668814]. As a multidomain scaffold bearing a non-canonical FYVE domain that binds phosphatidylinositol 3-phosphate, an FFAT motif, a Rab-binding domain, and a KIF5-interacting domain, protrudin acts as a tethering factor at ER–endosome/lysosome contact sites, inactivating the small GTPase Rab11 and coupling to the kinesin motor KIF5; it partners with the SMP-domain lipid-transfer protein PDZD8 at ER–late endosome/lysosome contacts to position endolysosomes and promote neurite outgrowth [PMID:31406056, PMID:33912962]. Oligomerization via its third hydrophobic region (HR3) is required for protrusion formation, and a neuron-specific microexon included by SRRM4-dependent alternative splicing yields a long isoform that more potently drives neurite outgrowth [PMID:22216323, PMID:28106138]. Protrudin was identified as a spastin-binding protein, and an HSP-associated mutation impairs spastin binding while increasing protein stability and rendering cells susceptible to ER stress; knockout mice lack HSP signs, indicating the disease mutations act by a gain-of-toxic-function mechanism [PMID:16826525, PMID:24668814, PMID:33172474]. Beyond the neural system, protrudin supports endothelial FAK and mTOR/S6K signaling required for cell migration and retinal angiogenesis [PMID:35368213].","teleology":[{"year":2006,"claim":"Establishing protrudin's first molecular partner answered how a disease-linked protein connects to HSP biology by showing it binds the microtubule-severing protein spastin and that a disease mutation disrupts this interaction.","evidence":"Yeast two-hybrid, reciprocal co-immunoprecipitation, and colocalization in mammalian cells with a disease-associated mutant","pmids":["16826525"],"confidence":"High","gaps":["Functional consequence of the spastin interaction not resolved","Mechanism linking mutation to the aberrant tubular pattern unclear"]},{"year":2011,"claim":"Defining protrudin as a PI3P-binding peripheral membrane protein that self-oligomerizes via HR3 answered how it acts mechanistically, showing oligomerization is functionally required for protrusion formation.","evidence":"Sucrose gradient centrifugation, Triton X-114 phase separation, lipid-binding assays, and dominant-negative ΔHR3 overexpression","pmids":["22216323"],"confidence":"High","gaps":["Structural basis of HR3-mediated oligomerization not determined","Stoichiometry in vivo unknown"]},{"year":2013,"claim":"Mapping protrudin's domain architecture and ER-shaping partners answered what cellular structure it builds, placing it as a regulator of tubular ER morphogenesis.","evidence":"Co-immunoprecipitation with atlastins/ER-shaping proteins, domain analysis, and ER morphology assays with live-cell imaging","pmids":["23969831"],"confidence":"High","gaps":["Quantitative contribution of each domain to morphology not separated","Direct membrane-bending activity not reconstituted"]},{"year":2014,"claim":"An in vivo interactome and topology study answered which HSP-network proteins protrudin engages and how a disease mutation alters protein behavior, revealing increased mutant stability and ER stress susceptibility.","evidence":"Proteomics of purified protrudin complexes from transgenic mouse brain, membrane topology mapping, and ER stress assays","pmids":["24668814"],"confidence":"High","gaps":["Causal link between increased stability and toxicity not established","ER stress mechanism not molecularly defined"]},{"year":2017,"claim":"Identifying SRRM4-dependent microexon inclusion answered how neurons tune protrudin function, generating a long isoform with enhanced neurite-promoting activity.","evidence":"Splicing assays, SRRM4 knockdown, CRISPR exon deletion, motif mutation, and neurite outgrowth assays in Neuro2A and embryonic stem cells","pmids":["28106138"],"confidence":"High","gaps":["Molecular basis for enhanced activity of the long isoform unknown","In vivo relevance of the microexon not tested"]},{"year":2019,"claim":"Synthesizing prior data framed protrudin as a membrane contact site tether, integrating Rab11 inactivation, ER membrane bending, and KIF5/VAP interactions into one mechanism.","evidence":"Review consolidating organelle fractionation, domain-function, and MCS tethering experiments","pmids":["31406056"],"confidence":"Medium","gaps":["Review presents no primary data","Direct ordering of tethering events not established"]},{"year":2020,"claim":"A knockout mouse answered whether HSP mutations act by loss or gain of function, showing behavioral abnormalities but no HSP phenotype and pointing to a gain-of-toxic-function disease mechanism.","evidence":"Protrudin knockout mice subjected to a behavioral test battery","pmids":["33172474"],"confidence":"Medium","gaps":["Molecular pathway underlying behavioral deficits not resolved","Toxic gain-of-function mechanism not directly demonstrated"]},{"year":2021,"claim":"Identifying PDZD8 as a partner answered how lipids are delivered at protrudin tethers, showing SMP-domain lipid transfer is required for endolysosome positioning and neurite outgrowth.","evidence":"Co-immunoprecipitation, in vitro lipid-binding and lipid-transfer assays, organelle positioning, and knockdown neurite outgrowth readouts","pmids":["33912962"],"confidence":"High","gaps":["Direction and selectivity of in vivo lipid flux unknown","Whether protrudin regulates PDZD8 transfer activity unclear"]},{"year":2022,"claim":"Extending protrudin beyond neurons answered whether it has vascular roles, linking it to FAK and mTOR/S6K signaling required for endothelial migration and retinal angiogenesis.","evidence":"siRNA knockdown with phospho-FAK imaging and mTOR/S6K immunoblotting, tube formation assays, and retinal vascular analysis in knockout mice","pmids":["35368213"],"confidence":"Medium","gaps":["Direct molecular link between protrudin and FAK activation not defined","Whether the contact-site/trafficking function underlies the signaling phenotype unknown"]},{"year":null,"claim":"How protrudin's gain-of-toxic-function HSP mutations mechanistically cause neurodegeneration, and how its membrane-bending and lipid-transfer tethering activities are coordinated in vivo, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the protrudin tethering complex","Causal chain from mutant stability/ER stress to neurodegeneration unestablished","Physiological substrates of lipid transfer at protrudin MCSs undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,5,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2,3]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[5,7]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[2,3]}],"complexes":[],"partners":["SPAST","KIF5A","ATL1","REEP1","VAPA","PDZD8","RAB11A","PLP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5T4F4","full_name":"Protrudin","aliases":["Spastic paraplegia 33 protein","Zinc finger FYVE domain-containing protein 27"],"length_aa":411,"mass_kda":45.8,"function":"Key regulator of RAB11-dependent vesicular trafficking during neurite extension through polarized membrane transport (PubMed:17082457). Promotes axonal elongation and contributes to the establishment of neuronal cell polarity (By similarity). Involved in nerve growth factor-induced neurite formation in VAPA-dependent manner (PubMed:19289470). Contributes to both the formation and stabilization of the tubular ER network (PubMed:24668814). Involved in ER morphogenesis by regulating the sheet-to-tubule balance and possibly the density of tubule interconnections (PubMed:23969831). Acts as an adapter protein and facilitates the interaction of KIF5A with VAPA, VAPB, SURF4, RAB11A, RAB11B and RTN3 and the ZFYVE27-KIF5A complex contributes to the transport of these proteins in neurons. Can induce formation of neurite-like membrane protrusions in non-neuronal cells in a KIF5A/B-dependent manner (PubMed:21976701)","subcellular_location":"Recycling endosome membrane; Endoplasmic reticulum membrane; Cell projection, growth cone membrane","url":"https://www.uniprot.org/uniprotkb/Q5T4F4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ZFYVE27","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"VAPA","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ZFYVE27","total_profiled":1310},"omim":[{"mim_id":"614235","title":"PDZ DOMAIN-CONTAINING PROTEIN 8; PDZD8","url":"https://www.omim.org/entry/614235"},{"mim_id":"610244","title":"SPASTIC PARAPLEGIA 33, AUTOSOMAL DOMINANT; SPG33","url":"https://www.omim.org/entry/610244"},{"mim_id":"610243","title":"ZINC FINGER FYVE DOMAIN-CONTAINING PROTEIN 27; ZFYVE27","url":"https://www.omim.org/entry/610243"},{"mim_id":"605570","title":"RAS-ASSOCIATED PROTEIN RAB11A; RAB11A","url":"https://www.omim.org/entry/605570"},{"mim_id":"182600","title":"SPASTIC PARAPLEGIA 3, AUTOSOMAL DOMINANT; SPG3A","url":"https://www.omim.org/entry/182600"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ZFYVE27"},"hgnc":{"alias_symbol":["FLJ32919","SPG33"],"prev_symbol":[]},"alphafold":{"accession":"Q5T4F4","domains":[{"cath_id":"-","chopping":"38-228","consensus_level":"medium","plddt":82.0758,"start":38,"end":228},{"cath_id":"3.30.40.10","chopping":"351-411","consensus_level":"medium","plddt":89.3002,"start":351,"end":411}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T4F4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T4F4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T4F4-F1-predicted_aligned_error_v6.png","plddt_mean":70.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ZFYVE27","jax_strain_url":"https://www.jax.org/strain/search?query=ZFYVE27"},"sequence":{"accession":"Q5T4F4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5T4F4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5T4F4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T4F4"}},"corpus_meta":[{"pmid":"22554690","id":"PMC_22554690","title":"Hereditary spastic paraplegias with autosomal dominant, recessive, X-linked, or maternal trait of inheritance.","date":"2012","source":"Journal of the neurological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/22554690","citation_count":229,"is_preprint":false},{"pmid":"16826525","id":"PMC_16826525","title":"ZFYVE27 (SPG33), a novel spastin-binding protein, is mutated in hereditary spastic paraplegia.","date":"2006","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/16826525","citation_count":113,"is_preprint":false},{"pmid":"29980238","id":"PMC_29980238","title":"Clinical spectrum and genetic landscape for hereditary spastic paraplegias in China.","date":"2018","source":"Molecular neurodegeneration","url":"https://pubmed.ncbi.nlm.nih.gov/29980238","citation_count":72,"is_preprint":false},{"pmid":"23969831","id":"PMC_23969831","title":"Protrudin binds atlastins and endoplasmic reticulum-shaping proteins and regulates network formation.","date":"2013","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/23969831","citation_count":56,"is_preprint":false},{"pmid":"24668814","id":"PMC_24668814","title":"Protrudin regulates endoplasmic reticulum morphology and function associated with the pathogenesis of hereditary spastic paraplegia.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24668814","citation_count":53,"is_preprint":false},{"pmid":"33912962","id":"PMC_33912962","title":"PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.","date":"2021","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/33912962","citation_count":46,"is_preprint":false},{"pmid":"28106138","id":"PMC_28106138","title":"SRRM4-dependent neuron-specific alternative splicing of protrudin transcripts regulates neurite outgrowth.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28106138","citation_count":39,"is_preprint":false},{"pmid":"18413476","id":"PMC_18413476","title":"Lack of spartin protein in Troyer syndrome: a loss-of-function disease mechanism?","date":"2008","source":"Archives of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/18413476","citation_count":33,"is_preprint":false},{"pmid":"30283000","id":"PMC_30283000","title":"Protein-protein interactions reveal key canonical pathways, upstream regulators, interactome domains, and novel targets in ALS.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30283000","citation_count":30,"is_preprint":false},{"pmid":"27117784","id":"PMC_27117784","title":"A Subpopulation of Label-Retaining Cells of the Kidney Papilla Regenerates Injured Kidney Medullary Tubules.","date":"2016","source":"Stem cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/27117784","citation_count":19,"is_preprint":false},{"pmid":"22216323","id":"PMC_22216323","title":"Oligomerization of ZFYVE27 (Protrudin) is necessary to promote neurite extension.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22216323","citation_count":18,"is_preprint":false},{"pmid":"35368213","id":"PMC_35368213","title":"Protrudin regulates FAK activation, endothelial cell migration and angiogenesis.","date":"2022","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/35368213","citation_count":18,"is_preprint":false},{"pmid":"18364116","id":"PMC_18364116","title":"A novel candidate locus on chromosome 11p14.1-p11.2 for autosomal dominant hereditary spastic paraplegia.","date":"2008","source":"Chinese medical journal","url":"https://pubmed.ncbi.nlm.nih.gov/18364116","citation_count":13,"is_preprint":false},{"pmid":"33172474","id":"PMC_33172474","title":"Protrudin-deficient mice manifest depression-like behavior with abnormalities in activity, attention, and cued fear-conditioning.","date":"2020","source":"Molecular brain","url":"https://pubmed.ncbi.nlm.nih.gov/33172474","citation_count":8,"is_preprint":false},{"pmid":"31406056","id":"PMC_31406056","title":"Roles of protrudin at interorganelle membrane contact sites.","date":"2019","source":"Proceedings of the Japan Academy. Series B, Physical and biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31406056","citation_count":6,"is_preprint":false},{"pmid":"36573383","id":"PMC_36573383","title":"Demographic and Genome Wide Association Analyses According to Muscle Mass Using Data of the Korean Genome and Epidemiology Study.","date":"2022","source":"Journal of Korean medical science","url":"https://pubmed.ncbi.nlm.nih.gov/36573383","citation_count":4,"is_preprint":false},{"pmid":"18394049","id":"PMC_18394049","title":"Refinement of the SPG9 locus on chromosome 10q23.3-24.2 and exclusion of candidate genes.","date":"2008","source":"European journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/18394049","citation_count":4,"is_preprint":false},{"pmid":"35704118","id":"PMC_35704118","title":"MYO1H is a novel candidate gene for autosomal dominant pure hereditary spastic paraplegia.","date":"2022","source":"Molecular genetics and genomics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/35704118","citation_count":2,"is_preprint":false},{"pmid":"41674924","id":"PMC_41674924","title":"Causal Association Between Plasma Proteins and Pericarditis: A Mendelian Randomization Study With Therapeutic Target Identification.","date":"2026","source":"Mediators of inflammation","url":"https://pubmed.ncbi.nlm.nih.gov/41674924","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11626,"output_tokens":2543,"usd":0.036512,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9719,"output_tokens":3240,"usd":0.064797,"stage2_stop_reason":"end_turn"},"total_usd":0.101309,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"ZFYVE27 (protrudin) was identified as a specific spastin-binding protein via yeast two-hybrid screen, and the interaction was validated by in vivo co-immunoprecipitation and colocalization in mammalian cells. A disease-associated mutation in ZFYVE27 severely impairs its interaction with spastin and causes an aberrant intracellular tubular pattern.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, colocalization (fluorescence microscopy)\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and colocalization, yeast two-hybrid, plus functional consequence of mutant shown; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16826525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ZFYVE27 (protrudin) self-interacts via its third hydrophobic region (HR3) and oligomerizes into dimer/tetramer forms as shown by sucrose gradient centrifugation. ZFYVE27 is a peripheral membrane protein that binds phosphatidylinositol 3-phosphate. Deletion of HR3 (ΔHR3) abolishes protrusion formation and acts as a dominant negative over wild-type ZFYVE27, demonstrating that oligomerization is required for neurite extension.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, sucrose gradient centrifugation, subcellular fractionation, Triton X-114 phase separation, lipid-binding assay, dominant-negative overexpression\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical and cell-biological methods in a single study establishing oligomerization mechanism and functional requirement\",\n      \"pmids\": [\"22216323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Protrudin (ZFYVE27/SPG33) contains hydrophobic intramembrane hairpin domains, interacts with tubular ER proteins (atlastins and ER-shaping proteins), and functions in ER morphogenesis by regulating the sheet-to-tubule balance and tubule interconnection density. Protrudin also harbors a KIF5-interacting domain, a Rab-binding domain, a non-canonical FYVE domain, and an FFAT domain.\",\n      \"method\": \"Co-immunoprecipitation, domain analysis, ER morphology assays (overexpression/knockdown), live-cell imaging\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple domain-function experiments, binding partner identification by Co-IP, and direct morphological readout; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"23969831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Protrudin localizes predominantly to the tubular endoplasmic reticulum (ER) and forced expression promotes formation and stabilization of the tubular ER network. One of three hydrophobic segments forms a hydrophobic hairpin domain. Protrudin interacts with multiple HSP-related proteins (SPG2/PLP1, SPG3A/atlastin-1, SPG31/REEP1, REEP5, KIF5A/B/C, reticulon 1/3/4). The HSP-associated mutant protrudin(G191V) has increased intracellular stability and cells expressing it show increased susceptibility to ER stress.\",\n      \"method\": \"Proteomics (purified protrudin complexes from transgenic mouse brain), membrane topology analysis, ER morphology assay, ER stress assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vivo proteomic identification of interactors from transgenic brain tissue, membrane topology, and functional ER morphology/stress readouts in single study with multiple methods\",\n      \"pmids\": [\"24668814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SRRM4 regulates alternative splicing of protrudin (ZFYVE27) pre-mRNA to include a neuron-specific microexon (exon L, encoding 7 amino acids) by recognizing a UGC motif upstream of exon L. The resulting long isoform (protrudin-L) is more effective at promoting neurite outgrowth than the short isoform (protrudin-S). Deletion of exon L impairs neurite outgrowth in Neuro2A cells and embryonic stem cells.\",\n      \"method\": \"RT-PCR/splicing assays, SRRM4 knockdown, exon deletion (CRISPR/genome editing), neurite outgrowth assays, motif mutation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function of splicing regulator, exon deletion with defined cellular phenotype, motif mapping; multiple orthogonal methods in single study\",\n      \"pmids\": [\"28106138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Protrudin functions as a tethering factor at membrane contact sites (MCSs) between the ER and other organelles. Its roles at MCSs involve inactivation of the small GTPase Rab11, bending of the ER membrane, and functional interactions with motor protein KIF5 and ER protein VAP.\",\n      \"method\": \"Review synthesizing experimental data (organelle fractionation, domain-function studies, MCS tethering assays cited within)\",\n      \"journal\": \"Proceedings of the Japan Academy. Series B, Physical and biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — review article synthesizing experimental findings from the same group; individual experiments support claims but this paper itself does not present primary data\",\n      \"pmids\": [\"31406056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Protrudin-deficient mice show pleiotropic behavioral abnormalities (hyperactivity, depression-like behavior, attention deficits, impaired fear-conditioning memory) but no HSP signs, indicating that the HSP-associated mutations likely cause neural degeneration through a gain-of-toxic-function rather than loss-of-function mechanism.\",\n      \"method\": \"Knockout mouse generation, battery of behavioral tests\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined behavioral phenotype, but pathway/molecular mechanism not directly resolved; single lab\",\n      \"pmids\": [\"33172474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PDZD8 is a protrudin (ZFYVE27)-interacting protein that acts as a tether at ER–late endosome/lysosome membrane contact sites. PDZD8's SMP domain binds glycerophospholipids and ceramides and can transfer lipids between membranes in vitro, and this lipid transfer activity is required for late endosome/lysosome positioning and neurite outgrowth.\",\n      \"method\": \"Co-immunoprecipitation, in vitro lipid-binding and lipid-transfer assays, organelle positioning assays, knockdown with neurite outgrowth readout\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro reconstitution of lipid transfer, Co-IP for interaction, and defined cellular phenotype upon KD; multiple orthogonal methods\",\n      \"pmids\": [\"33912962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Protrudin knockdown in endothelial cells inhibits FAK activation, disrupts polarized phospho-FAK distribution, causes perinuclear accumulation of mTOR, and decreases VEGF-mediated S6K activation, leading to impaired endothelial cell migration and angiogenesis. Mice with global protrudin deletion show reduced retinal vascular progression.\",\n      \"method\": \"siRNA knockdown, phospho-FAK immunofluorescence, mTOR/S6K immunoblotting, tube formation assay, in vivo retinal vascular analysis in KO mice\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with specific signaling readout (FAK phosphorylation) and in vivo KO vascular phenotype; single lab, multiple methods\",\n      \"pmids\": [\"35368213\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ZFYVE27/protrudin is an ER-resident peripheral membrane protein with a non-canonical FYVE domain, FFAT domain, Rab-binding domain, and hydrophobic hairpin domains that localizes to tubular ER, where it tethers ER–endosome/lysosome membrane contact sites, regulates ER tubular network morphology, promotes directional endosome trafficking via KIF5, inactivates Rab11, and interacts with spastin and multiple other HSP-associated proteins; its oligomerization (via HR3) is required for neurite outgrowth, a neuron-specific long isoform is generated by SRRM4-dependent splicing, and HSP-associated mutations impair spastin binding and cause ER stress through a likely gain-of-function mechanism.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ZFYVE27 (protrudin) is an endoplasmic reticulum-resident peripheral membrane protein that shapes the tubular ER network and tethers ER membrane contact sites to drive directional membrane trafficking and neurite outgrowth [#2, #3, #5]. Through hydrophobic intramembrane hairpin domains and interactions with ER-shaping atlastins and reticulons, it regulates the ER sheet-to-tubule balance and stabilizes the tubular ER network where it predominantly localizes [#2, #3]. As a multidomain scaffold bearing a non-canonical FYVE domain that binds phosphatidylinositol 3-phosphate, an FFAT motif, a Rab-binding domain, and a KIF5-interacting domain, protrudin acts as a tethering factor at ER\\u2013endosome/lysosome contact sites, inactivating the small GTPase Rab11 and coupling to the kinesin motor KIF5; it partners with the SMP-domain lipid-transfer protein PDZD8 at ER\\u2013late endosome/lysosome contacts to position endolysosomes and promote neurite outgrowth [#5, #7]. Oligomerization via its third hydrophobic region (HR3) is required for protrusion formation, and a neuron-specific microexon included by SRRM4-dependent alternative splicing yields a long isoform that more potently drives neurite outgrowth [#1, #4]. Protrudin was identified as a spastin-binding protein, and an HSP-associated mutation impairs spastin binding while increasing protein stability and rendering cells susceptible to ER stress; knockout mice lack HSP signs, indicating the disease mutations act by a gain-of-toxic-function mechanism [#0, #3, #6]. Beyond the neural system, protrudin supports endothelial FAK and mTOR/S6K signaling required for cell migration and retinal angiogenesis [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing protrudin's first molecular partner answered how a disease-linked protein connects to HSP biology by showing it binds the microtubule-severing protein spastin and that a disease mutation disrupts this interaction.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-immunoprecipitation, and colocalization in mammalian cells with a disease-associated mutant\",\n      \"pmids\": [\"16826525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the spastin interaction not resolved\", \"Mechanism linking mutation to the aberrant tubular pattern unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defining protrudin as a PI3P-binding peripheral membrane protein that self-oligomerizes via HR3 answered how it acts mechanistically, showing oligomerization is functionally required for protrusion formation.\",\n      \"evidence\": \"Sucrose gradient centrifugation, Triton X-114 phase separation, lipid-binding assays, and dominant-negative \\u0394HR3 overexpression\",\n      \"pmids\": [\"22216323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of HR3-mediated oligomerization not determined\", \"Stoichiometry in vivo unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapping protrudin's domain architecture and ER-shaping partners answered what cellular structure it builds, placing it as a regulator of tubular ER morphogenesis.\",\n      \"evidence\": \"Co-immunoprecipitation with atlastins/ER-shaping proteins, domain analysis, and ER morphology assays with live-cell imaging\",\n      \"pmids\": [\"23969831\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each domain to morphology not separated\", \"Direct membrane-bending activity not reconstituted\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"An in vivo interactome and topology study answered which HSP-network proteins protrudin engages and how a disease mutation alters protein behavior, revealing increased mutant stability and ER stress susceptibility.\",\n      \"evidence\": \"Proteomics of purified protrudin complexes from transgenic mouse brain, membrane topology mapping, and ER stress assays\",\n      \"pmids\": [\"24668814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal link between increased stability and toxicity not established\", \"ER stress mechanism not molecularly defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identifying SRRM4-dependent microexon inclusion answered how neurons tune protrudin function, generating a long isoform with enhanced neurite-promoting activity.\",\n      \"evidence\": \"Splicing assays, SRRM4 knockdown, CRISPR exon deletion, motif mutation, and neurite outgrowth assays in Neuro2A and embryonic stem cells\",\n      \"pmids\": [\"28106138\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for enhanced activity of the long isoform unknown\", \"In vivo relevance of the microexon not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Synthesizing prior data framed protrudin as a membrane contact site tether, integrating Rab11 inactivation, ER membrane bending, and KIF5/VAP interactions into one mechanism.\",\n      \"evidence\": \"Review consolidating organelle fractionation, domain-function, and MCS tethering experiments\",\n      \"pmids\": [\"31406056\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Review presents no primary data\", \"Direct ordering of tethering events not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A knockout mouse answered whether HSP mutations act by loss or gain of function, showing behavioral abnormalities but no HSP phenotype and pointing to a gain-of-toxic-function disease mechanism.\",\n      \"evidence\": \"Protrudin knockout mice subjected to a behavioral test battery\",\n      \"pmids\": [\"33172474\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular pathway underlying behavioral deficits not resolved\", \"Toxic gain-of-function mechanism not directly demonstrated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying PDZD8 as a partner answered how lipids are delivered at protrudin tethers, showing SMP-domain lipid transfer is required for endolysosome positioning and neurite outgrowth.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro lipid-binding and lipid-transfer assays, organelle positioning, and knockdown neurite outgrowth readouts\",\n      \"pmids\": [\"33912962\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direction and selectivity of in vivo lipid flux unknown\", \"Whether protrudin regulates PDZD8 transfer activity unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extending protrudin beyond neurons answered whether it has vascular roles, linking it to FAK and mTOR/S6K signaling required for endothelial migration and retinal angiogenesis.\",\n      \"evidence\": \"siRNA knockdown with phospho-FAK imaging and mTOR/S6K immunoblotting, tube formation assays, and retinal vascular analysis in knockout mice\",\n      \"pmids\": [\"35368213\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between protrudin and FAK activation not defined\", \"Whether the contact-site/trafficking function underlies the signaling phenotype unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How protrudin's gain-of-toxic-function HSP mutations mechanistically cause neurodegeneration, and how its membrane-bending and lipid-transfer tethering activities are coordinated in vivo, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the protrudin tethering complex\", \"Causal chain from mutant stability/ER stress to neurodegeneration unestablished\", \"Physiological substrates of lipid transfer at protrudin MCSs undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 5, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005789\", \"supporting_discovery_ids\": []}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [5, 7]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SPAST\", \"KIF5A\", \"ATL1\", \"REEP1\", \"VAPA\", \"PDZD8\", \"RAB11A\", \"PLP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}