{"gene":"VPS35","run_date":"2026-04-28T23:00:23","timeline":{"discoveries":[{"year":1992,"finding":"VPS35 (Vps35p) peripherally associates with a membranous particulate cell fraction in yeast and is required for sorting of carboxypeptidase Y (CPY) to the vacuole; null mutants cause selective missorting and secretion of CPY but not other vacuolar hydrolases, indicating alternative sorting pathways exist.","method":"Gene disruption (null allele), subcellular fractionation, vacuolar protein sorting assays in S. cerevisiae","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 — foundational genetic and biochemical characterization with multiple phenotypic readouts in yeast ortholog","pmids":["1498362"],"is_preprint":false},{"year":1997,"finding":"Vps35p is required for retrograde retrieval of the vacuolar sorting receptor Vps10p from the prevacuolar endosome back to the Golgi; loss of VPS35 shifts Vps10p to the vacuolar membrane via a Pep12p (endosomal t-SNARE)-dependent route; Vps35p co-fractionates with Vps10p even in vps29 mutants, suggesting direct interaction.","method":"Temperature-conditional VPS35 allele, subcellular fractionation, genetic epistasis with sec1 and pep12 mutants, co-fractionation assays in S. cerevisiae","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (conditional allele, fractionation, epistasis) in foundational yeast study","pmids":["9105038"],"is_preprint":false},{"year":2006,"finding":"Crystal structure of human Vps26A at 2.1-Å resolution reveals an arrestin-fold; the Vps35-binding site maps to a mobile loop (residues 235–246) near the tip of the C-terminal domain; hydrophobic residues and a glycine in this loop are required for Vps26 integration into the retromer complex and endosomal localization.","method":"X-ray crystallography (2.1-Å), site-directed mutagenesis of Vps26 loop residues, endosomal localization assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis validation","pmids":["16732284"],"is_preprint":false},{"year":2007,"finding":"A conserved PRLYL motif in the N-terminal domain of Vps35 is essential for retromer subcomplex assembly: the R107W mutation in human VPS35 disrupts co-precipitation with Vps26 and causes dominant-negative trafficking defects when expressed in yeast, shifting VPS35 from endosomes to a juxtanuclear compartment and affecting mannose phosphate receptor and insulin trafficking.","method":"Dominant-negative expression in yeast, co-immunoprecipitation, subcellular fractionation, site-directed mutagenesis","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis plus Co-IP plus cross-species functional rescue experiments","pmids":["17916227"],"is_preprint":false},{"year":2007,"finding":"A dominant-negative N-terminal deletion mutant of VPS35 reduces canonical Wnt signaling in HEK-293 cells; GST pulldown identified a possible indirect interaction between the LRP6 intracellular domain and VPS35, linking retromer to Wnt/LRP6 endosomal signaling.","method":"Dominant-negative overexpression, GST-fusion pulldown, Wnt reporter assay (Tcf promoter)","journal":"Neurobiology of disease","confidence":"Low","confidence_rationale":"Tier 3 — single pulldown and overexpression without direct mechanistic dissection","pmids":["17239604"],"is_preprint":false},{"year":2007,"finding":"Drosophila Vps35 loss impairs scavenger receptor-ligand endocytosis, causes mislocalisation of receptors and endocytic proteins, negatively regulates actin polymerisation, and leads to overproliferation of larval blood cells and upregulation of TGFβ/BMP signaling at the neuromuscular junction.","method":"RNAi knockdown in S2 cells and Drosophila, endocytosis assay, genetic interaction analysis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — clean RNAi phenotype with multiple cellular readouts in Drosophila ortholog","pmids":["18057029"],"is_preprint":false},{"year":2010,"finding":"VPS35 (retromer) mediates transport of the mitochondrial SUMO E3 ligase MAPL from mitochondria to peroxisomes via mitochondria-derived vesicles (MDVs); Vps35 and Vps26 are found in complex with MAPL by unbiased screen, and Vps35 is recruited to mitochondrial vesicles; silencing Vps35 or Vps26A significantly reduces MAPL delivery to peroxisomes.","method":"Unbiased protein interaction screen, Co-immunoprecipitation, confocal imaging of Vps35 on MDVs, siRNA silencing with peroxisomal MAPL quantification","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, imaging, and functional knockdown with defined cargo readout","pmids":["20619655"],"is_preprint":false},{"year":2010,"finding":"Loss of Vps26b in mice destabilizes the Vps26b-Vps29-Vps35 retromer complex and leads to ~20% increase in sortilin levels, while SorLA is unaffected, implicating this complex in sortilin retrieval from endosomes to the TGN.","method":"Vps26b knockout mice, Co-immunoprecipitation, Western blot","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — knockout mouse with defined biochemical phenotype","pmids":["21040701"],"is_preprint":false},{"year":2011,"finding":"VPS35 interacts with BACE1 and is required for BACE1 endosome-to-Golgi retrieval; VPS35 haploinsufficiency in Tg2576 AD mice increases BACE1 activity in endosomes, elevates Aβ levels, and causes earlier-onset AD-like phenotypes including cognitive deficits and impaired LTP.","method":"Co-immunoprecipitation (VPS35-BACE1), hemizygous Vps35 deletion in Tg2576 mice, BACE1 activity assay, immunofluorescence localization, behavioral testing","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus in vivo loss-of-function with multiple orthogonal functional readouts","pmids":["22105352"],"is_preprint":false},{"year":2012,"finding":"The WASH complex is recruited to endosomes through direct binding of its FAM21 subunit's unstructured tail domain to VPS35; this interaction is necessary and sufficient for WASH complex endosomal targeting; elevated FAM21-tail expression inhibits WASH-retromer association and causes cell-spreading defects.","method":"Biochemical binding assays, overexpression of FAM21 tail fragments, endosomal localization assays, cell spreading phenotype","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — domain mapping plus functional consequence of disrupting interaction","pmids":["22070227"],"is_preprint":false},{"year":2012,"finding":"VPS35 is required for BACE1 retrograde trafficking in developing hippocampal neurons; Vps35 depletion impairs retrograde BACE1 transport and alters BACE1 distribution, leading to shortened apical dendrites, reduced dendritic spines, and swollen commissural axons; BACE1 suppression partially rescues these deficits.","method":"In utero electroporation of microRNA against Vps35, immunofluorescence, genetic epistasis with BACE1 knockdown","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KD with defined cargo (BACE1) and epistasis rescue","pmids":["23259059"],"is_preprint":false},{"year":2013,"finding":"VPS35 D620N mutant redistributes retromer-positive endosomes to a perinuclear localization and enlarges endosomes; D620N is correctly folded and binds Vps29 and Vps26A with normal affinity; D620N interacts with CI-M6PR but disrupts cathepsin D trafficking, which is responsible for α-synuclein degradation.","method":"Subcellular localization imaging, binding affinity measurements, cathepsin D trafficking assay, patient fibroblast analysis","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal assays including patient cells and structural integrity assessment","pmids":["24152121"],"is_preprint":false},{"year":2013,"finding":"VPS35 loss of function alters RANK distribution upon RANKL stimulation, enhances RANKL sensitivity, sustains RANKL signaling, and increases hyperresorptive osteoclast formation; hemizygous Vps35 deletion in mice causes osteoporosis with decreased trabecular bone volume.","method":"VPS35 siRNA knockdown, hemizygous Vps35 knockout mice, RANKL signaling assays, bone histomorphometry","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — in vivo mouse model plus cellular mechanistic assays with defined signaling readout","pmids":["23509071"],"is_preprint":false},{"year":2014,"finding":"VPS35 D620N mutation impairs WASH complex association with retromer and reduces WASH recruitment to endosomes; autophagy is impaired in D620N-expressing cells or WASH-depleted cells due to abnormal trafficking of ATG9A; retromer is required for endosomal WASH complex recruitment.","method":"Co-immunoprecipitation, endosomal recruitment assay, ATG9A trafficking assay, autophagy flux measurement","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods establishing WASH recruitment mechanism and functional autophagy consequence","pmids":["24819384"],"is_preprint":false},{"year":2014,"finding":"The primary molecular defect of VPS35 D620N mutation is a 2.2-fold decrease in binding affinity for the WASH complex component FAM21, measured by isothermal calorimetry; SILAC-based interactome comparison confirms reduced WASH complex association; D620N perturbs endosome-to-TGN transport but not endosome-to-plasma membrane recycling.","method":"SILAC-based quantitative proteomics, isothermal calorimetry, retromer cargo trafficking assays, patient-derived cells","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1–2 — biophysical affinity measurement plus quantitative proteomics plus functional cargo assay","pmids":["24980502"],"is_preprint":false},{"year":2014,"finding":"Rab7 recruits retromer to late endosomes via direct interactions with N-terminal conserved regions in Vps35; association of Vps26 with Vps35 allosterically enhances high-affinity Rab7 binding to the Vps sub-complex; disrupting the Vps35-Vps26 interaction perturbs Rab7-mediated retromer recruitment.","method":"FRET-based interaction assay in HeLa cells, biophysical binding measurements, site-directed mutagenesis of Vps35-Vps26 interface","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1–2 — FRET in vivo plus biophysical measurements plus mutagenesis","pmids":["25367362"],"is_preprint":false},{"year":2014,"finding":"VPS35 D620N acts as a loss-of-function mutation for synaptic function: it localizes to dendritic spines and normally regulates AMPA receptor (AMPAR) trafficking; D620N expression impairs excitatory synaptic transmission and AMPAR surface expression and synaptic recycling in mouse cortical neurons and iPSC-derived dopamine neurons from human D620N carriers.","method":"Electrophysiology, AMPAR surface expression assay, synaptic recycling assay, iPSC-derived neurons from patients","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — human patient-derived neurons plus electrophysiology plus receptor trafficking assays","pmids":["25416282"],"is_preprint":false},{"year":2014,"finding":"VPS35 is required for endosome-to-Golgi retrieval of Lamp2a; VPS35-deficient or D620N-mutant DA neurons exhibit accelerated Lamp2a degradation, impairing chaperone-mediated autophagy and leading to α-synuclein accumulation; Lamp2a overexpression in VPS35-deficient neurons reduces α-synuclein.","method":"Conditional VPS35 knockout in dopamine neurons, Lamp2a trafficking assay, α-synuclein immunostaining, epistasis rescue with Lamp2a overexpression","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with defined cargo (Lamp2a) plus epistasis rescue","pmids":["26203154"],"is_preprint":false},{"year":2014,"finding":"VPS35 dysfunction impairs retromer-mediated retrieval of the mannose 6-phosphate receptor, reducing lysosomal turnover of M6PR and affecting cathepsin D (CTSD) maturation and trafficking; VPS35 knockdown in Drosophila accumulates detergent-insoluble α-synuclein and exacerbates locomotor impairment.","method":"VPS35 RNAi knockdown in Drosophila, CTSD maturation assay, α-synuclein fractionation, behavioral assay","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2 — Drosophila in vivo KD with defined biochemical and behavioral readouts","pmids":["25107340"],"is_preprint":false},{"year":2015,"finding":"PD-associated VPS35 mutations (especially D620N) cause mitochondrial fragmentation and cell death through enhanced interaction with dynamin-like protein DLP1, which increases turnover of mitochondrial DLP1 complexes via mitochondria-derived vesicle trafficking to lysosomes; inhibition of mitochondrial fission prevents VPS35-induced mitochondrial deficits; oxidative stress increases the VPS35-DLP1 interaction.","method":"Co-immunoprecipitation (VPS35-DLP1), mitochondrial fission inhibition rescue, MDV tracking, patient fibroblast and in vivo mouse substantia nigra analysis","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus rescue experiments plus patient cells plus in vivo validation","pmids":["26618722"],"is_preprint":false},{"year":2015,"finding":"VPS35 deficiency or D620N mutation increases mitochondrial E3 ubiquitin ligase MUL1 levels, leading to MFN2 (mitofusin 2) ubiquitin-dependent degradation and mitochondrial fragmentation; MUL1 suppression rescues MFN2 reduction and DA neuron loss but not α-synuclein accumulation.","method":"Conditional VPS35 KO in DA neurons, MUL1/MFN2 Western blot, MUL1 knockdown epistasis rescue, TH+ neuron counting","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — in vivo conditional KO plus epistasis with mechanistic pathway placement","pmids":["26321632"],"is_preprint":false},{"year":2015,"finding":"VPS35 interacts with AMPA receptor subunits GluA1 and GluA2; VPS35 deficiency impairs dendritic spine maturation and decreases surface AMPA receptor levels; GluA2 overexpression partially rescues spine maturation deficits in VPS35-deficient neurons.","method":"Co-immunoprecipitation (VPS35-GluA1/GluA2), surface receptor biotinylation, GluA2 rescue epistasis, spine morphology imaging","journal":"Molecular brain","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional rescue, single lab","pmids":["26521016"],"is_preprint":false},{"year":2015,"finding":"VPS29-VPS35 form a biologically stable intermediate sub-complex in vivo; deficiency of VPS35 or VPS29 causes degradation of other retromer subunits, while VPS26 deficiency does not affect VPS29/VPS35 levels; VPS26-VPS35 is more susceptible to ubiquitin-proteasome degradation than VPS29-VPS35.","method":"siRNA knockdown of individual subunits, in vitro sub-complex reconstitution, ubiquitin-proteasome pathway inhibitor treatment","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro reconstitution and KD with biochemical readouts, single lab","pmids":["25937119"],"is_preprint":false},{"year":2016,"finding":"Parkinson disease-associated Vps35 variant R524W, but not P316S, is a loss-of-function mutation: R524W reduces association with the retromer regulatory network and dysregulates endosomal receptor (CI-M6PR) sorting; R524W expression causes accumulation of intracellular α-synuclein-positive aggregates; this phenotype is partially rescued by retromer-stabilizing compound R55.","method":"Co-immunoprecipitation, CI-M6PR trafficking assay, α-synuclein aggregation imaging, pharmacological rescue with R55","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal assays plus pharmacological rescue","pmids":["27385586"],"is_preprint":false},{"year":2016,"finding":"VPS35 interacts with and recycles dopamine receptor D1 (DRD1) from endosomes to the cell surface; VPS35 overexpression increases surface DRD1 and CREB/ERK phosphorylation downstream of dopamine; D620N mutant fails to recycle DRD1 or restore CREB/ERK signaling.","method":"Co-immunoprecipitation, surface receptor recycling assay, Western blot of CREB/ERK phosphorylation, VPS35 knockdown/overexpression","journal":"Neurobiology of aging","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional recycling assay plus signaling readout, single lab","pmids":["27460146"],"is_preprint":false},{"year":2016,"finding":"VPS35 binds farnesylated (but not palmitoylated or GTP-loaded) N-Ras in the cytosol within a high-molecular-weight complex; silencing VPS35 increases N-Ras association with cytoplasmic vesicles, diminishes Ras GTP loading, and inhibits MAPK signaling and growth of N-Ras-dependent melanoma cells.","method":"Affinity purification of cytosolic Ras + mass spectrometry, Co-IP with farnesyl-dependence test, VPS35 siRNA + Ras GTP loading assay + MAPK signaling","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — mass spectrometry ID plus biochemical validation of farnesyl-dependence plus functional signaling readout","pmids":["27502489"],"is_preprint":false},{"year":2017,"finding":"VPS35 D620N mutation causes mitochondrial fragmentation through the VPS35-DLP1 interaction via a conserved FLV motif in the C-terminus of DLP1; a decoy peptide based on this FLV motif blocks VPS35-DLP1 interaction and rescues D620N-induced mitochondrial fragmentation and respiratory deficits.","method":"Site-directed mutagenesis of DLP1 FLV motif, decoy peptide inhibitor, Co-IP, mitochondrial morphology and respiration assays in M17 cells and patient fibroblasts","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1–2 — motif mutagenesis plus peptide inhibitor rescue plus functional respiratory assay","pmids":["28040727"],"is_preprint":false},{"year":2017,"finding":"VPS35 regulates lysosomal degradation of parkin substrate AIMP2; VPS35 co-immunoprecipitates with AIMP2 and Lamp2a; D620N mutation disrupts this association; VPS35 overexpression prevents AIMP2-induced PARP1-dependent cell death; VPS35 knockdown causes AIMP2-dependent PARP1 activation and cell death.","method":"Co-immunoprecipitation (VPS35-AIMP2-Lamp2a), VPS35 overexpression/knockdown, PARP1 activation assay, epistasis with AIMP2 knockdown","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus epistasis, single lab","pmids":["28383562"],"is_preprint":false},{"year":2017,"finding":"Drosophila Vps35 loss affects synaptic vesicle recycling and dopaminergic synaptic release; dLRRK together with Rab5 and Rab11 is also involved; manipulation of LRRK/Rab5/Rab11 activities improves Vps35 synaptic phenotypes, placing Vps35 and LRRK2 in the same synaptic vesicle recycling pathway.","method":"Drosophila vps35 null mutants, genetic interaction with LRRK/Rab5/Rab11 transgenes, dopamine release measurement, behavioral assay","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis in Drosophila ortholog with defined neurotransmission readout","pmids":["28482024"],"is_preprint":false},{"year":2018,"finding":"VPS35 D620N knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts and mouse tissues; VPS35 D620N increases LRRK2 kinase activity; knockout or knockdown of VPS35 suppresses LRRK2-mediated Rab phosphorylation, indicating VPS35 controls LRRK2 activity; D620N mutation is a gain-of-function for LRRK2 hyperactivation.","method":"VPS35 D620N knock-in mice, phospho-Rab8A/10/12 Western blot, VPS35 KO/KD in multiple cell types, patient neutrophil/monocyte analysis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — knock-in mouse model plus KO/KD plus patient-derived cells, multiple orthogonal readouts","pmids":["29743203"],"is_preprint":false},{"year":2018,"finding":"Parkin interacts with VPS35 and mediates its poly-ubiquitination at three C-terminal lysine residues via an atypical poly-ubiquitin chain that does not promote proteasomal degradation; parkin KO mice show markedly decreased WASH complex components in brain; parkin silencing in neurons disrupts ATG9A vesicular sorting (a WASH-dependent retromer cargo).","method":"Co-immunoprecipitation, ubiquitination assay with lysine mutants, parkin KO mouse brain analysis, ATG9A trafficking assay","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus ubiquitin mapping plus KO mouse plus cargo trafficking assay","pmids":["29893854"],"is_preprint":false},{"year":2016,"finding":"Trem2 undergoes clathrin-dependent endocytosis and is recycled to the plasma membrane via VPS35/retromer; VPS35 knockdown causes Trem2 accumulation in lysosomes; VPS35 deficiency leads to excessive LPS-induced iNOS and IL-6 production in microglia that is reversed by Trem2 overexpression; AD-associated R47H Trem2 mutant fails to interact with Vps35 and becomes unstable.","method":"Co-immunoprecipitation (VPS35-Trem2), VPS35 knockdown, lysosome trafficking assay, inflammatory cytokine measurement, rescue with Trem2 overexpression","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional rescue, single lab","pmids":["27717139"],"is_preprint":false},{"year":2019,"finding":"VPS35 regulates tau phosphorylation through cathepsin D availability: VPS35 overexpression reduces pathological tau, while VPS35 silencing increases tau accumulation; cathepsin D mediates this effect; VPS35 downregulation in a tauopathy mouse model exacerbates motor and learning impairments and tau accumulation.","method":"VPS35 overexpression/silencing in neuronal cells, cathepsin D activity assay, tauopathy transgenic mouse model with VPS35 downregulation","journal":"Molecular psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro plus in vivo with mechanistic cathepsin D link, single lab","pmids":["31289348"],"is_preprint":false},{"year":2020,"finding":"VPS35 deficiency impairs Cdk5/p35 lysosomal degradation; loss of VPS35 increases p35 levels and Cdk5 activity, leading to tau hyperphosphorylation and retinal ganglion cell degeneration; Cdk5 inhibitor roscovitine reduces hyperphosphorylated tau caused by VPS35 deficiency; p35 is identified as a VPS35 cargo.","method":"VPS35 downregulation/overexpression in RGCs, Co-immunoprecipitation (Vps35-p35), Cdk5 activity assay, roscovitine rescue, in vivo retinal glutamate excitotoxicity model","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus pharmacological rescue plus in vivo model, single lab","pmids":["31995153"],"is_preprint":false},{"year":2021,"finding":"VPS35 D620N mutation reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy: D620N cells are desensitized to CCCP-induced mitochondrial potential collapse, show altered fragmentation, cannot accumulate PINK1 at the mitochondrial surface, and consequently fail to recruit Parkin for mitophagy initiation.","method":"CRISPR-Cas9 heterozygous D620N knock-in in SH-SY5Y cells, mitochondrial membrane potential assay, PINK1/Parkin recruitment imaging, CCCP treatment","journal":"Translational neurodegeneration","confidence":"High","confidence_rationale":"Tier 2 — endogenous knock-in model with multiple functional mitophagy readouts","pmids":["34127073"],"is_preprint":false},{"year":2022,"finding":"Upon mtDNA damage, VPS35 mediates maturation of early endosomes to late autophagy vesicles for selective mtDNA degradation; SAMM50 acts as a gatekeeper controlling nucleoid release and transfer to endosomes; the ATAD3-SAMM50 axis is disrupted by mtDNA damage, facilitating endosomal recruitment near nucleoid sub-compartments.","method":"Proximity labeling (Twinkle as nucleoid marker), VPS35 knockdown, lysosomal function assays, ATG5 KO, mouse model of mtDNA alterations treated with rapamycin","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — proximity labeling plus KD plus mouse model with multiple orthogonal readouts","pmids":["36344526"],"is_preprint":false},{"year":2021,"finding":"VPS35 D620N mutation promotes LRRK2-mediated lysosomal recruitment of RILPL1 and its binding to the lysosomal membrane protein TMEM55B; VPS35 D620N reduces RILPL1 levels in mouse tissues in a manner reversed by LRRK2 inhibition and proteasome inhibitors; RILPL1 KO enhances Rab substrate phosphorylation; TMEM55B KO increases RILPL1 levels.","method":"Quantitative lysosomal proteomics (~220 proteins altered), Co-IP of RILPL1-TMEM55B, VPS35 D620N knock-in mouse tissues, LRRK2 inhibitor treatment, RILPL1/TMEM55B knockout cells","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 — quantitative proteomics plus Co-IP plus KO plus pharmacological rescue in multiple model systems","pmids":["38091401"],"is_preprint":false},{"year":2023,"finding":"VPS35 selectively binds endocytosed EGFR in early endosomes and recycles it to the cell surface, activating downstream ERK1/2 signaling; high VPS35 expression increases sensitivity to EGFR inhibitors in gastric cancer xenograft and organoid models.","method":"Co-immunoprecipitation (VPS35-EGFR), biotin surface assay, patient-derived xenografts and organoids, EGFR inhibitor response assay","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional recycling assay plus PDX/organoid validation, single lab","pmids":["36738481"],"is_preprint":false},{"year":2021,"finding":"VPS35 promotes hepatoma cell proliferation via the PI3K/AKT signaling pathway; VPS35 knockout reduces membrane FGFR3 distribution, demonstrating VPS35's role in sorting and trafficking of transmembrane receptor FGFR3 in hepatoma cells.","method":"VPS35 knockout in hepatoma cells, FGFR3 membrane localization assay, PI3K/AKT signaling assay, in vivo xenograft","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — KO plus receptor trafficking assay, single lab","pmids":["32071398"],"is_preprint":false},{"year":2023,"finding":"VPS35 D620N significantly elevates LRRK2-mediated phosphorylation of Rab10, Rab12, and Rab29 in knock-in mice; LRRK2 kinase inhibitor MLi-2 normalizes striatal dopamine transporter (DAT) expression and function and abolishes increased amphetamine-induced hyperlocomotion in VKI mice; VPS35 haploinsufficiency reduces Rab12 phosphorylation and impairs DAT similarly but is not reversed by MLi-2, confirming D620N is a gain-of-function for LRRK2 kinase.","method":"VPS35 D620N knock-in and haploinsufficient mice, phospho-Rab Western blot, DAT expression/function assay, LRRK2 inhibitor MLi-2 treatment, behavioral testing","journal":"NPJ Parkinson's disease","confidence":"High","confidence_rationale":"Tier 2 — knock-in + haploinsufficient mouse comparison plus pharmacological rescue with defined biochemical and behavioral readouts","pmids":["38110354"],"is_preprint":false}],"current_model":"VPS35 is the central cargo-recognition subunit of the retromer complex that mediates endosome-to-TGN/plasma membrane retrograde trafficking of diverse transmembrane cargoes (including CI-M6PR, BACE1, LAMP2a, Trem2, DRD1, AMPA receptors, EGFR, and RANK); it recruits the WASH complex to endosomes via FAM21 binding to nucleate actin for tubule formation, is targeted to endosomes through Rab7 interaction with its N-terminal domain, is ubiquitinated by parkin in a non-degradative manner that modulates sorting, and the PD-linked D620N mutation primarily disrupts FAM21/WASH complex binding—causing impaired cargo retrieval, ATG9A mis-sorting, LRRK2 hyperactivation (with elevated Rab8A/10/12 phosphorylation and lysosomal recruitment of RILPL1-TMEM55B), enhanced VPS35-DLP1 interaction leading to mitochondrial fragmentation via MUL1-dependent MFN2 degradation, and impaired PINK1/Parkin-mediated mitophagy."},"narrative":{"teleology":[{"year":1992,"claim":"Establishing that VPS35 is a membrane-associated protein required for vacuolar protein sorting resolved a key component of the endosomal sorting machinery, though its precise molecular role was unknown.","evidence":"Gene disruption, subcellular fractionation, and CPY secretion assays in S. cerevisiae","pmids":["1498362"],"confidence":"High","gaps":["Mechanism of VPS35 action on sorting unknown","No direct cargo interaction demonstrated","Mammalian ortholog function not addressed"]},{"year":1997,"claim":"Demonstrating that VPS35 is specifically required for retrograde retrieval of the sorting receptor Vps10p from endosomes to the Golgi established the retromer concept and positioned VPS35 as a cargo-recognition factor rather than a general sorting component.","evidence":"Temperature-conditional VPS35 allele, co-fractionation with Vps10p, genetic epistasis with pep12 in S. cerevisiae","pmids":["9105038"],"confidence":"High","gaps":["Direct physical interaction with cargo not yet proven by binding assay","Structural basis unknown","Mammalian cargo repertoire undefined"]},{"year":2006,"claim":"Structural determination of Vps26A and mapping of its VPS35-binding interface provided the first atomic-level understanding of retromer subcomplex assembly, revealing that a mobile loop in Vps26 is critical for integration into the complex.","evidence":"X-ray crystallography at 2.1 Å, site-directed mutagenesis, endosomal localization assays","pmids":["16732284"],"confidence":"High","gaps":["Full VPS35 structure not solved","VPS35-Vps29 interface not mapped","Cargo-binding site on VPS35 not identified"]},{"year":2007,"claim":"Identification of the conserved PRLYL motif in VPS35's N-terminus as essential for Vps26 binding and retromer integrity, together with evidence for dominant-negative trafficking defects, established that VPS35 serves as a scaffold nucleating retromer assembly.","evidence":"R107W mutagenesis, Co-IP, cross-species dominant-negative expression in yeast and mammalian cells","pmids":["17916227"],"confidence":"High","gaps":["VPS35-Vps29 binding determinants not mapped","Full cargo-binding surface unresolved"]},{"year":2010,"claim":"Discovery that VPS35 is recruited to mitochondria-derived vesicles and mediates MAPL delivery from mitochondria to peroxisomes expanded retromer function beyond canonical endosome-to-Golgi trafficking to inter-organelle transport.","evidence":"Unbiased protein interaction screen, Co-IP, confocal imaging of VPS35 on MDVs, siRNA knockdown with peroxisomal MAPL quantification","pmids":["20619655"],"confidence":"High","gaps":["Mechanism of VPS35 recruitment to mitochondrial membranes unknown","Whether MDV cargo selectivity requires VPS35 directly or through associated factors unclear"]},{"year":2011,"claim":"Showing that VPS35 interacts with BACE1 and that haploinsufficiency increases endosomal BACE1 activity and Aβ production in AD mice linked retromer dysfunction to Alzheimer disease pathogenesis through a specific cargo-sorting defect.","evidence":"Co-IP of VPS35-BACE1, hemizygous Vps35 deletion in Tg2576 mice, BACE1 activity assay, behavioral testing","pmids":["22105352"],"confidence":"High","gaps":["Direct BACE1 sorting signal on VPS35 not mapped","Whether VPS35 loss affects amyloidogenesis independent of BACE1 not determined"]},{"year":2012,"claim":"Identification of FAM21 as the direct bridge between VPS35 and the WASH actin-nucleation complex on endosomes resolved how retromer coordinates membrane remodeling with cargo sorting.","evidence":"Biochemical binding assays, FAM21 tail fragment overexpression, endosomal localization assays","pmids":["22070227"],"confidence":"High","gaps":["Binding site on VPS35 for FAM21 not mapped at residue level","Whether WASH recruitment is required for all retromer cargoes unknown"]},{"year":2014,"claim":"Biophysical and proteomic characterization of VPS35 D620N revealed that the primary molecular defect is a ~2-fold reduction in FAM21/WASH binding affinity while retromer trimer assembly is unaffected, establishing D620N as a selective WASH-recruitment defect that impairs endosome-to-TGN transport and ATG9A sorting for autophagy.","evidence":"Isothermal calorimetry, SILAC-based quantitative proteomics, ATG9A trafficking assay, patient fibroblasts","pmids":["24980502","24819384","24152121"],"confidence":"High","gaps":["Structural basis of D620N-FAM21 affinity loss not resolved","Whether all D620N phenotypes are WASH-dependent not tested"]},{"year":2014,"claim":"Demonstration that Rab7 recruits retromer to late endosomes via VPS35's N-terminal domain, with allosteric enhancement by Vps26 binding, established the molecular logic of retromer membrane targeting.","evidence":"FRET-based interaction assay in HeLa cells, biophysical binding measurements, mutagenesis of Vps35-Vps26 interface","pmids":["25367362"],"confidence":"High","gaps":["Whether Rab7 GTP/GDP state regulates the interaction quantitatively not fully resolved","Role of SNX-BAR dimers in coordinating with Rab7-VPS35 binding unclear"]},{"year":2014,"claim":"Showing that VPS35 retrieves Lamp2a from endosomes and that D620N causes accelerated Lamp2a degradation, impairing chaperone-mediated autophagy and leading to α-synuclein accumulation, established a mechanistic link between retromer dysfunction and Parkinson-relevant protein aggregation.","evidence":"Conditional VPS35 KO in DA neurons, Lamp2a trafficking, α-synuclein immunostaining, Lamp2a overexpression rescue","pmids":["26203154"],"confidence":"High","gaps":["Sorting signal on Lamp2a recognized by VPS35 not identified","Whether CMA impairment is the dominant pathway for α-synuclein accumulation vs. macroautophagy defects not resolved"]},{"year":2015,"claim":"Discovery that D620N enhances VPS35–DLP1 interaction, increasing mitochondrial DLP1 turnover and causing mitochondrial fragmentation, and that VPS35 deficiency elevates MUL1 leading to MFN2 degradation, established a mitochondrial fragmentation axis in PD pathogenesis separable from α-synuclein accumulation.","evidence":"Co-IP of VPS35-DLP1, mitochondrial fission inhibitor rescue, conditional KO with MUL1 epistasis, patient fibroblasts","pmids":["26618722","26321632"],"confidence":"High","gaps":["Whether enhanced VPS35-DLP1 binding is a direct consequence of WASH loss or an independent D620N effect unclear","Structural basis of VPS35-DLP1 interaction resolved only at motif level (FLV)"]},{"year":2016,"claim":"Expanding VPS35's cargo repertoire to include Trem2, DRD1, N-Ras, and RANK demonstrated that retromer-mediated receptor recycling controls inflammatory signaling in microglia, dopaminergic signaling, Ras-MAPK signaling, and osteoclast formation.","evidence":"Co-IP, surface recycling assays, inflammatory cytokine measurement, RANK signaling in osteoclasts, farnesyl-dependent N-Ras binding and MAPK readout, hemizygous KO mouse bone phenotype","pmids":["27717139","27460146","27502489","23509071"],"confidence":"High","gaps":["Sorting signals on these diverse cargoes not systematically identified","Whether all cargoes require WASH-dependent sorting not tested"]},{"year":2018,"claim":"Demonstrating that D620N is a gain-of-function for LRRK2 kinase activity—elevating Rab8A/10/12 phosphorylation in knock-in mice and patient cells—while VPS35 loss suppresses LRRK2-mediated Rab phosphorylation, established VPS35 as an upstream activator of LRRK2 and linked the two major familial PD genes in a common pathway.","evidence":"VPS35 D620N knock-in mice, phospho-Rab Western blot, VPS35 KO/KD, patient neutrophils/monocytes","pmids":["29743203"],"confidence":"High","gaps":["Mechanism by which VPS35 activates LRRK2 kinase not identified (direct binding vs. membrane platform)","Whether wild-type VPS35 normally stimulates LRRK2 at physiological levels unclear"]},{"year":2018,"claim":"Parkin was shown to ubiquitinate VPS35 at C-terminal lysines via non-degradative poly-ubiquitin chains, and parkin loss reduced WASH complex components and disrupted ATG9A sorting, positioning VPS35 as a Parkin substrate that integrates retromer and mitophagy pathways.","evidence":"Co-IP, ubiquitin chain type and lysine mapping, parkin KO mouse brain analysis, ATG9A trafficking","pmids":["29893854"],"confidence":"High","gaps":["Functional consequence of VPS35 ubiquitination on WASH recruitment not directly tested","Whether parkin-mediated ubiquitination regulates VPS35-LRRK2 interplay unknown"]},{"year":2021,"claim":"D620N was shown to impair PINK1/Parkin-mediated mitophagy by preventing PINK1 accumulation on depolarized mitochondria, connecting retromer dysfunction to a second mitochondrial quality control pathway beyond fission.","evidence":"CRISPR heterozygous D620N knock-in in SH-SY5Y cells, mitochondrial membrane potential, PINK1/Parkin recruitment imaging","pmids":["34127073"],"confidence":"High","gaps":["Whether the mitophagy defect is secondary to altered mitochondrial membrane potential or a direct VPS35 role in PINK1 stabilization not resolved"]},{"year":2021,"claim":"Quantitative lysosomal proteomics in D620N knock-in mice revealed LRRK2-mediated lysosomal recruitment of RILPL1 via TMEM55B binding, identifying a downstream effector pathway of the VPS35-LRRK2 axis that alters lysosomal composition.","evidence":"Lysosomal proteomics (~220 altered proteins), Co-IP of RILPL1-TMEM55B, LRRK2 inhibitor reversal, RILPL1/TMEM55B KO cells","pmids":["38091401"],"confidence":"High","gaps":["Functional consequence of RILPL1 lysosomal recruitment on lysosomal degradation or autophagy not fully defined","Whether this pathway operates in human dopaminergic neurons not confirmed"]},{"year":2022,"claim":"VPS35 was found to mediate endosomal maturation for selective mtDNA degradation upon mitochondrial DNA damage, with SAMM50 acting as a gatekeeper for nucleoid release, extending VPS35's mitochondrial roles to genome maintenance.","evidence":"Proximity labeling with Twinkle, VPS35 knockdown, ATG5 KO, mouse model with mtDNA alterations","pmids":["36344526"],"confidence":"High","gaps":["Whether this pathway is retromer-dependent or uses VPS35 in a retromer-independent capacity not resolved","Mechanism of VPS35 recruitment to damaged nucleoid compartments unknown"]},{"year":2023,"claim":"Comparison of D620N knock-in versus haploinsufficient mice confirmed D620N as a gain-of-function for LRRK2 kinase (reversed by inhibitor MLi-2) while haploinsufficiency impairs DAT independently of LRRK2, demonstrating that loss-of-function and gain-of-toxic-function coexist in D620N pathogenesis.","evidence":"VPS35 D620N KI and haploinsufficient mice, phospho-Rab blots, DAT expression/function, LRRK2 inhibitor MLi-2, behavioral testing","pmids":["38110354"],"confidence":"High","gaps":["How D620N simultaneously causes loss of WASH binding and gain of LRRK2 activation structurally unresolved","Cell-type-specific contributions of these dual mechanisms in dopaminergic neurons not separated"]},{"year":null,"claim":"A high-resolution structure of full-length VPS35 in complex with FAM21, Rab7, and cargo peptides is needed to explain how D620N simultaneously impairs WASH recruitment and hyperactivates LRRK2, and whether these are structurally linked or independent surfaces.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length VPS35 structure with bound FAM21 or LRRK2","Direct VPS35-LRRK2 binding interface not mapped","Cargo-binding specificity determinants on VPS35 for its >10 known cargoes not structurally resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,9,15,17]},{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[1,8,11,17,37]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1,9,11,13,15]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[6,19,20,34,35]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[6,11,35]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[25]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,8,9,11,14,17,24,37]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[13,34,35]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[6,19,20,34]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11,29,39]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[25,37,38]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[16,21,28]}],"complexes":["Retromer (VPS35-VPS29-VPS26)"],"partners":["VPS26A","VPS29","FAM21","RAB7A","DLP1","LRRK2","PRKN","BACE1"],"other_free_text":[]},"mechanistic_narrative":"VPS35 is the central cargo-recognition subunit of the retromer complex, mediating retrograde retrieval of diverse transmembrane cargoes—including sorting receptors (Vps10p, CI-M6PR), BACE1, Lamp2a, EGFR, DRD1, AMPA receptors, RANK, and Trem2—from endosomes to the trans-Golgi network or plasma membrane [PMID:9105038, PMID:22105352, PMID:26203154, PMID:36738481, PMID:27460146, PMID:25416282, PMID:23509071, PMID:27717139]. It is recruited to late endosomes through direct interaction with Rab7 at its N-terminal domain and recruits the WASH actin-nucleation complex to endosomes via binding of the FAM21 subunit, thereby promoting actin-dependent membrane tubulation required for cargo sorting; the Parkinson disease-linked D620N mutation selectively reduces FAM21/WASH binding affinity, impairing ATG9A trafficking and autophagy [PMID:25367362, PMID:22070227, PMID:24980502, PMID:24819384]. The D620N mutation additionally causes LRRK2 kinase hyperactivation with elevated phosphorylation of Rab8A, Rab10, and Rab12, enhanced VPS35–DLP1 interaction leading to MUL1-dependent MFN2 degradation and mitochondrial fragmentation, and impaired PINK1/Parkin-mediated mitophagy, establishing convergent pathogenic mechanisms in familial Parkinson disease [PMID:29743203, PMID:26618722, PMID:26321632, PMID:34127073]. VPS35 also participates in mitochondria-derived vesicle trafficking, mediating transport of MAPL from mitochondria to peroxisomes and selective degradation of damaged mtDNA through endosomal maturation [PMID:20619655, PMID:36344526]."},"prefetch_data":{"uniprot":{"accession":"Q96QK1","full_name":"Vacuolar protein sorting-associated protein 35","aliases":["Maternal-embryonic 3","Vesicle protein sorting 35"],"length_aa":796,"mass_kda":91.7,"function":"Acts as a component of the retromer cargo-selective complex (CSC). The CSC is believed to be the core functional component of retromer or respective retromer complex variants acting to prevent missorting of selected transmembrane cargo proteins into the lysosomal degradation pathway. The recruitment of the CSC to the endosomal membrane involves RAB7A and SNX3. The CSC seems to associate with the cytoplasmic domain of cargo proteins predominantly via VPS35; however, these interactions seem to be of low affinity and retromer SNX proteins may also contribute to cargo selectivity thus questioning the classical function of the CSC. The SNX-BAR retromer mediates retrograde transport of cargo proteins from endosomes to the trans-Golgi network (TGN) and is involved in endosome-to-plasma membrane transport for cargo protein recycling. The SNX3-retromer mediates the retrograde endosome-to-TGN transport of WLS distinct from the SNX-BAR retromer pathway (PubMed:30213940). The SNX27-retromer is believed to be involved in endosome-to-plasma membrane trafficking and recycling of a broad spectrum of cargo proteins. The CSC seems to act as recruitment hub for other proteins, such as the WASH complex and TBC1D5 (Probable). Required for retrograde transport of lysosomal enzyme receptor IGF2R and SLC11A2. Required to regulate transcytosis of the polymeric immunoglobulin receptor (pIgR-pIgA) (PubMed:15078903, PubMed:15247922, PubMed:20164305). Required for endosomal localization of WASHC2C (PubMed:22070227, PubMed:28892079). Mediates the association of the CSC with the WASH complex via WASHC2 (PubMed:22070227, PubMed:24819384, PubMed:24980502). Required for the endosomal localization of TBC1D5 (PubMed:20923837) (Microbial infection) The heterotrimeric retromer cargo-selective complex (CSC) mediates the exit of human papillomavirus from the early endosome and the delivery to the Golgi apparatus","subcellular_location":"Cytoplasm; Membrane; Endosome; Early endosome; Late endosome","url":"https://www.uniprot.org/uniprotkb/Q96QK1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/VPS35","classification":"Common Essential","n_dependent_lines":864,"n_total_lines":1208,"dependency_fraction":0.7152317880794702},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000069329","cell_line_id":"CID001488","localizations":[{"compartment":"vesicles","grade":3}],"interactors":[{"gene":"CAPZB","stoichiometry":10.0},{"gene":"VPS29","stoichiometry":10.0},{"gene":"CAPZA1","stoichiometry":10.0},{"gene":"CAPZA2","stoichiometry":10.0},{"gene":"WASH3P;WASH2P;WASH6P;WASH1;WASH4P","stoichiometry":10.0},{"gene":"KIAA1033","stoichiometry":10.0},{"gene":"VPS26A","stoichiometry":10.0},{"gene":"VPS26B","stoichiometry":10.0},{"gene":"FKBP15","stoichiometry":10.0},{"gene":"RAB7A","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/target/CID001488","total_profiled":1310},"omim":[{"mim_id":"619856","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 50; ANKRD50","url":"https://www.omim.org/entry/619856"},{"mim_id":"619135","title":"RITSCHER-SCHINZEL SYNDROME 3; RTSC3","url":"https://www.omim.org/entry/619135"},{"mim_id":"618981","title":"VPS35 ENDOSOMAL PROTEIN-SORTING FACTOR-LIKE; VPS35L","url":"https://www.omim.org/entry/618981"},{"mim_id":"615740","title":"TBC1 DOMAIN FAMILY, MEMBER 5; TBC1D5","url":"https://www.omim.org/entry/615740"},{"mim_id":"614905","title":"SORTING NEXIN 8; SNX8","url":"https://www.omim.org/entry/614905"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Endosomes","reliability":"Approved"},{"location":"Lysosomes","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/VPS35"},"hgnc":{"alias_symbol":["FLJ10752","MEM3","PARK17"],"prev_symbol":[]},"alphafold":{"accession":"Q96QK1","domains":[{"cath_id":"1.25.40","chopping":"9-160","consensus_level":"medium","plddt":92.4794,"start":9,"end":160}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96QK1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96QK1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96QK1-F1-predicted_aligned_error_v6.png","plddt_mean":91.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VPS35","jax_strain_url":"https://www.jax.org/strain/search?query=VPS35"},"sequence":{"accession":"Q96QK1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96QK1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96QK1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96QK1"}},"corpus_meta":[{"pmid":"21763483","id":"PMC_21763483","title":"A mutation in VPS35, 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cerevisiae\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — foundational genetic and biochemical characterization with multiple phenotypic readouts in yeast ortholog\",\n      \"pmids\": [\"1498362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Vps35p is required for retrograde retrieval of the vacuolar sorting receptor Vps10p from the prevacuolar endosome back to the Golgi; loss of VPS35 shifts Vps10p to the vacuolar membrane via a Pep12p (endosomal t-SNARE)-dependent route; Vps35p co-fractionates with Vps10p even in vps29 mutants, suggesting direct interaction.\",\n      \"method\": \"Temperature-conditional VPS35 allele, subcellular fractionation, genetic epistasis with sec1 and pep12 mutants, co-fractionation assays in S. cerevisiae\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (conditional allele, fractionation, epistasis) in foundational yeast study\",\n      \"pmids\": [\"9105038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structure of human Vps26A at 2.1-Å resolution reveals an arrestin-fold; the Vps35-binding site maps to a mobile loop (residues 235–246) near the tip of the C-terminal domain; hydrophobic residues and a glycine in this loop are required for Vps26 integration into the retromer complex and endosomal localization.\",\n      \"method\": \"X-ray crystallography (2.1-Å), site-directed mutagenesis of Vps26 loop residues, endosomal localization assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis validation\",\n      \"pmids\": [\"16732284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A conserved PRLYL motif in the N-terminal domain of Vps35 is essential for retromer subcomplex assembly: the R107W mutation in human VPS35 disrupts co-precipitation with Vps26 and causes dominant-negative trafficking defects when expressed in yeast, shifting VPS35 from endosomes to a juxtanuclear compartment and affecting mannose phosphate receptor and insulin trafficking.\",\n      \"method\": \"Dominant-negative expression in yeast, co-immunoprecipitation, subcellular fractionation, site-directed mutagenesis\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis plus Co-IP plus cross-species functional rescue experiments\",\n      \"pmids\": [\"17916227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"A dominant-negative N-terminal deletion mutant of VPS35 reduces canonical Wnt signaling in HEK-293 cells; GST pulldown identified a possible indirect interaction between the LRP6 intracellular domain and VPS35, linking retromer to Wnt/LRP6 endosomal signaling.\",\n      \"method\": \"Dominant-negative overexpression, GST-fusion pulldown, Wnt reporter assay (Tcf promoter)\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single pulldown and overexpression without direct mechanistic dissection\",\n      \"pmids\": [\"17239604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Drosophila Vps35 loss impairs scavenger receptor-ligand endocytosis, causes mislocalisation of receptors and endocytic proteins, negatively regulates actin polymerisation, and leads to overproliferation of larval blood cells and upregulation of TGFβ/BMP signaling at the neuromuscular junction.\",\n      \"method\": \"RNAi knockdown in S2 cells and Drosophila, endocytosis assay, genetic interaction analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean RNAi phenotype with multiple cellular readouts in Drosophila ortholog\",\n      \"pmids\": [\"18057029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"VPS35 (retromer) mediates transport of the mitochondrial SUMO E3 ligase MAPL from mitochondria to peroxisomes via mitochondria-derived vesicles (MDVs); Vps35 and Vps26 are found in complex with MAPL by unbiased screen, and Vps35 is recruited to mitochondrial vesicles; silencing Vps35 or Vps26A significantly reduces MAPL delivery to peroxisomes.\",\n      \"method\": \"Unbiased protein interaction screen, Co-immunoprecipitation, confocal imaging of Vps35 on MDVs, siRNA silencing with peroxisomal MAPL quantification\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, imaging, and functional knockdown with defined cargo readout\",\n      \"pmids\": [\"20619655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Loss of Vps26b in mice destabilizes the Vps26b-Vps29-Vps35 retromer complex and leads to ~20% increase in sortilin levels, while SorLA is unaffected, implicating this complex in sortilin retrieval from endosomes to the TGN.\",\n      \"method\": \"Vps26b knockout mice, Co-immunoprecipitation, Western blot\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knockout mouse with defined biochemical phenotype\",\n      \"pmids\": [\"21040701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"VPS35 interacts with BACE1 and is required for BACE1 endosome-to-Golgi retrieval; VPS35 haploinsufficiency in Tg2576 AD mice increases BACE1 activity in endosomes, elevates Aβ levels, and causes earlier-onset AD-like phenotypes including cognitive deficits and impaired LTP.\",\n      \"method\": \"Co-immunoprecipitation (VPS35-BACE1), hemizygous Vps35 deletion in Tg2576 mice, BACE1 activity assay, immunofluorescence localization, behavioral testing\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus in vivo loss-of-function with multiple orthogonal functional readouts\",\n      \"pmids\": [\"22105352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The WASH complex is recruited to endosomes through direct binding of its FAM21 subunit's unstructured tail domain to VPS35; this interaction is necessary and sufficient for WASH complex endosomal targeting; elevated FAM21-tail expression inhibits WASH-retromer association and causes cell-spreading defects.\",\n      \"method\": \"Biochemical binding assays, overexpression of FAM21 tail fragments, endosomal localization assays, cell spreading phenotype\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping plus functional consequence of disrupting interaction\",\n      \"pmids\": [\"22070227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VPS35 is required for BACE1 retrograde trafficking in developing hippocampal neurons; Vps35 depletion impairs retrograde BACE1 transport and alters BACE1 distribution, leading to shortened apical dendrites, reduced dendritic spines, and swollen commissural axons; BACE1 suppression partially rescues these deficits.\",\n      \"method\": \"In utero electroporation of microRNA against Vps35, immunofluorescence, genetic epistasis with BACE1 knockdown\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KD with defined cargo (BACE1) and epistasis rescue\",\n      \"pmids\": [\"23259059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"VPS35 D620N mutant redistributes retromer-positive endosomes to a perinuclear localization and enlarges endosomes; D620N is correctly folded and binds Vps29 and Vps26A with normal affinity; D620N interacts with CI-M6PR but disrupts cathepsin D trafficking, which is responsible for α-synuclein degradation.\",\n      \"method\": \"Subcellular localization imaging, binding affinity measurements, cathepsin D trafficking assay, patient fibroblast analysis\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays including patient cells and structural integrity assessment\",\n      \"pmids\": [\"24152121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"VPS35 loss of function alters RANK distribution upon RANKL stimulation, enhances RANKL sensitivity, sustains RANKL signaling, and increases hyperresorptive osteoclast formation; hemizygous Vps35 deletion in mice causes osteoporosis with decreased trabecular bone volume.\",\n      \"method\": \"VPS35 siRNA knockdown, hemizygous Vps35 knockout mice, RANKL signaling assays, bone histomorphometry\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo mouse model plus cellular mechanistic assays with defined signaling readout\",\n      \"pmids\": [\"23509071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"VPS35 D620N mutation impairs WASH complex association with retromer and reduces WASH recruitment to endosomes; autophagy is impaired in D620N-expressing cells or WASH-depleted cells due to abnormal trafficking of ATG9A; retromer is required for endosomal WASH complex recruitment.\",\n      \"method\": \"Co-immunoprecipitation, endosomal recruitment assay, ATG9A trafficking assay, autophagy flux measurement\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods establishing WASH recruitment mechanism and functional autophagy consequence\",\n      \"pmids\": [\"24819384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The primary molecular defect of VPS35 D620N mutation is a 2.2-fold decrease in binding affinity for the WASH complex component FAM21, measured by isothermal calorimetry; SILAC-based interactome comparison confirms reduced WASH complex association; D620N perturbs endosome-to-TGN transport but not endosome-to-plasma membrane recycling.\",\n      \"method\": \"SILAC-based quantitative proteomics, isothermal calorimetry, retromer cargo trafficking assays, patient-derived cells\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biophysical affinity measurement plus quantitative proteomics plus functional cargo assay\",\n      \"pmids\": [\"24980502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Rab7 recruits retromer to late endosomes via direct interactions with N-terminal conserved regions in Vps35; association of Vps26 with Vps35 allosterically enhances high-affinity Rab7 binding to the Vps sub-complex; disrupting the Vps35-Vps26 interaction perturbs Rab7-mediated retromer recruitment.\",\n      \"method\": \"FRET-based interaction assay in HeLa cells, biophysical binding measurements, site-directed mutagenesis of Vps35-Vps26 interface\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — FRET in vivo plus biophysical measurements plus mutagenesis\",\n      \"pmids\": [\"25367362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"VPS35 D620N acts as a loss-of-function mutation for synaptic function: it localizes to dendritic spines and normally regulates AMPA receptor (AMPAR) trafficking; D620N expression impairs excitatory synaptic transmission and AMPAR surface expression and synaptic recycling in mouse cortical neurons and iPSC-derived dopamine neurons from human D620N carriers.\",\n      \"method\": \"Electrophysiology, AMPAR surface expression assay, synaptic recycling assay, iPSC-derived neurons from patients\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human patient-derived neurons plus electrophysiology plus receptor trafficking assays\",\n      \"pmids\": [\"25416282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"VPS35 is required for endosome-to-Golgi retrieval of Lamp2a; VPS35-deficient or D620N-mutant DA neurons exhibit accelerated Lamp2a degradation, impairing chaperone-mediated autophagy and leading to α-synuclein accumulation; Lamp2a overexpression in VPS35-deficient neurons reduces α-synuclein.\",\n      \"method\": \"Conditional VPS35 knockout in dopamine neurons, Lamp2a trafficking assay, α-synuclein immunostaining, epistasis rescue with Lamp2a overexpression\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with defined cargo (Lamp2a) plus epistasis rescue\",\n      \"pmids\": [\"26203154\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"VPS35 dysfunction impairs retromer-mediated retrieval of the mannose 6-phosphate receptor, reducing lysosomal turnover of M6PR and affecting cathepsin D (CTSD) maturation and trafficking; VPS35 knockdown in Drosophila accumulates detergent-insoluble α-synuclein and exacerbates locomotor impairment.\",\n      \"method\": \"VPS35 RNAi knockdown in Drosophila, CTSD maturation assay, α-synuclein fractionation, behavioral assay\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Drosophila in vivo KD with defined biochemical and behavioral readouts\",\n      \"pmids\": [\"25107340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PD-associated VPS35 mutations (especially D620N) cause mitochondrial fragmentation and cell death through enhanced interaction with dynamin-like protein DLP1, which increases turnover of mitochondrial DLP1 complexes via mitochondria-derived vesicle trafficking to lysosomes; inhibition of mitochondrial fission prevents VPS35-induced mitochondrial deficits; oxidative stress increases the VPS35-DLP1 interaction.\",\n      \"method\": \"Co-immunoprecipitation (VPS35-DLP1), mitochondrial fission inhibition rescue, MDV tracking, patient fibroblast and in vivo mouse substantia nigra analysis\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus rescue experiments plus patient cells plus in vivo validation\",\n      \"pmids\": [\"26618722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"VPS35 deficiency or D620N mutation increases mitochondrial E3 ubiquitin ligase MUL1 levels, leading to MFN2 (mitofusin 2) ubiquitin-dependent degradation and mitochondrial fragmentation; MUL1 suppression rescues MFN2 reduction and DA neuron loss but not α-synuclein accumulation.\",\n      \"method\": \"Conditional VPS35 KO in DA neurons, MUL1/MFN2 Western blot, MUL1 knockdown epistasis rescue, TH+ neuron counting\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo conditional KO plus epistasis with mechanistic pathway placement\",\n      \"pmids\": [\"26321632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"VPS35 interacts with AMPA receptor subunits GluA1 and GluA2; VPS35 deficiency impairs dendritic spine maturation and decreases surface AMPA receptor levels; GluA2 overexpression partially rescues spine maturation deficits in VPS35-deficient neurons.\",\n      \"method\": \"Co-immunoprecipitation (VPS35-GluA1/GluA2), surface receptor biotinylation, GluA2 rescue epistasis, spine morphology imaging\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional rescue, single lab\",\n      \"pmids\": [\"26521016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"VPS29-VPS35 form a biologically stable intermediate sub-complex in vivo; deficiency of VPS35 or VPS29 causes degradation of other retromer subunits, while VPS26 deficiency does not affect VPS29/VPS35 levels; VPS26-VPS35 is more susceptible to ubiquitin-proteasome degradation than VPS29-VPS35.\",\n      \"method\": \"siRNA knockdown of individual subunits, in vitro sub-complex reconstitution, ubiquitin-proteasome pathway inhibitor treatment\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro reconstitution and KD with biochemical readouts, single lab\",\n      \"pmids\": [\"25937119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Parkinson disease-associated Vps35 variant R524W, but not P316S, is a loss-of-function mutation: R524W reduces association with the retromer regulatory network and dysregulates endosomal receptor (CI-M6PR) sorting; R524W expression causes accumulation of intracellular α-synuclein-positive aggregates; this phenotype is partially rescued by retromer-stabilizing compound R55.\",\n      \"method\": \"Co-immunoprecipitation, CI-M6PR trafficking assay, α-synuclein aggregation imaging, pharmacological rescue with R55\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays plus pharmacological rescue\",\n      \"pmids\": [\"27385586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"VPS35 interacts with and recycles dopamine receptor D1 (DRD1) from endosomes to the cell surface; VPS35 overexpression increases surface DRD1 and CREB/ERK phosphorylation downstream of dopamine; D620N mutant fails to recycle DRD1 or restore CREB/ERK signaling.\",\n      \"method\": \"Co-immunoprecipitation, surface receptor recycling assay, Western blot of CREB/ERK phosphorylation, VPS35 knockdown/overexpression\",\n      \"journal\": \"Neurobiology of aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional recycling assay plus signaling readout, single lab\",\n      \"pmids\": [\"27460146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"VPS35 binds farnesylated (but not palmitoylated or GTP-loaded) N-Ras in the cytosol within a high-molecular-weight complex; silencing VPS35 increases N-Ras association with cytoplasmic vesicles, diminishes Ras GTP loading, and inhibits MAPK signaling and growth of N-Ras-dependent melanoma cells.\",\n      \"method\": \"Affinity purification of cytosolic Ras + mass spectrometry, Co-IP with farnesyl-dependence test, VPS35 siRNA + Ras GTP loading assay + MAPK signaling\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mass spectrometry ID plus biochemical validation of farnesyl-dependence plus functional signaling readout\",\n      \"pmids\": [\"27502489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"VPS35 D620N mutation causes mitochondrial fragmentation through the VPS35-DLP1 interaction via a conserved FLV motif in the C-terminus of DLP1; a decoy peptide based on this FLV motif blocks VPS35-DLP1 interaction and rescues D620N-induced mitochondrial fragmentation and respiratory deficits.\",\n      \"method\": \"Site-directed mutagenesis of DLP1 FLV motif, decoy peptide inhibitor, Co-IP, mitochondrial morphology and respiration assays in M17 cells and patient fibroblasts\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — motif mutagenesis plus peptide inhibitor rescue plus functional respiratory assay\",\n      \"pmids\": [\"28040727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"VPS35 regulates lysosomal degradation of parkin substrate AIMP2; VPS35 co-immunoprecipitates with AIMP2 and Lamp2a; D620N mutation disrupts this association; VPS35 overexpression prevents AIMP2-induced PARP1-dependent cell death; VPS35 knockdown causes AIMP2-dependent PARP1 activation and cell death.\",\n      \"method\": \"Co-immunoprecipitation (VPS35-AIMP2-Lamp2a), VPS35 overexpression/knockdown, PARP1 activation assay, epistasis with AIMP2 knockdown\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus epistasis, single lab\",\n      \"pmids\": [\"28383562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Drosophila Vps35 loss affects synaptic vesicle recycling and dopaminergic synaptic release; dLRRK together with Rab5 and Rab11 is also involved; manipulation of LRRK/Rab5/Rab11 activities improves Vps35 synaptic phenotypes, placing Vps35 and LRRK2 in the same synaptic vesicle recycling pathway.\",\n      \"method\": \"Drosophila vps35 null mutants, genetic interaction with LRRK/Rab5/Rab11 transgenes, dopamine release measurement, behavioral assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in Drosophila ortholog with defined neurotransmission readout\",\n      \"pmids\": [\"28482024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"VPS35 D620N knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts and mouse tissues; VPS35 D620N increases LRRK2 kinase activity; knockout or knockdown of VPS35 suppresses LRRK2-mediated Rab phosphorylation, indicating VPS35 controls LRRK2 activity; D620N mutation is a gain-of-function for LRRK2 hyperactivation.\",\n      \"method\": \"VPS35 D620N knock-in mice, phospho-Rab8A/10/12 Western blot, VPS35 KO/KD in multiple cell types, patient neutrophil/monocyte analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knock-in mouse model plus KO/KD plus patient-derived cells, multiple orthogonal readouts\",\n      \"pmids\": [\"29743203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Parkin interacts with VPS35 and mediates its poly-ubiquitination at three C-terminal lysine residues via an atypical poly-ubiquitin chain that does not promote proteasomal degradation; parkin KO mice show markedly decreased WASH complex components in brain; parkin silencing in neurons disrupts ATG9A vesicular sorting (a WASH-dependent retromer cargo).\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with lysine mutants, parkin KO mouse brain analysis, ATG9A trafficking assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus ubiquitin mapping plus KO mouse plus cargo trafficking assay\",\n      \"pmids\": [\"29893854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Trem2 undergoes clathrin-dependent endocytosis and is recycled to the plasma membrane via VPS35/retromer; VPS35 knockdown causes Trem2 accumulation in lysosomes; VPS35 deficiency leads to excessive LPS-induced iNOS and IL-6 production in microglia that is reversed by Trem2 overexpression; AD-associated R47H Trem2 mutant fails to interact with Vps35 and becomes unstable.\",\n      \"method\": \"Co-immunoprecipitation (VPS35-Trem2), VPS35 knockdown, lysosome trafficking assay, inflammatory cytokine measurement, rescue with Trem2 overexpression\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional rescue, single lab\",\n      \"pmids\": [\"27717139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"VPS35 regulates tau phosphorylation through cathepsin D availability: VPS35 overexpression reduces pathological tau, while VPS35 silencing increases tau accumulation; cathepsin D mediates this effect; VPS35 downregulation in a tauopathy mouse model exacerbates motor and learning impairments and tau accumulation.\",\n      \"method\": \"VPS35 overexpression/silencing in neuronal cells, cathepsin D activity assay, tauopathy transgenic mouse model with VPS35 downregulation\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro plus in vivo with mechanistic cathepsin D link, single lab\",\n      \"pmids\": [\"31289348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"VPS35 deficiency impairs Cdk5/p35 lysosomal degradation; loss of VPS35 increases p35 levels and Cdk5 activity, leading to tau hyperphosphorylation and retinal ganglion cell degeneration; Cdk5 inhibitor roscovitine reduces hyperphosphorylated tau caused by VPS35 deficiency; p35 is identified as a VPS35 cargo.\",\n      \"method\": \"VPS35 downregulation/overexpression in RGCs, Co-immunoprecipitation (Vps35-p35), Cdk5 activity assay, roscovitine rescue, in vivo retinal glutamate excitotoxicity model\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus pharmacological rescue plus in vivo model, single lab\",\n      \"pmids\": [\"31995153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"VPS35 D620N mutation reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy: D620N cells are desensitized to CCCP-induced mitochondrial potential collapse, show altered fragmentation, cannot accumulate PINK1 at the mitochondrial surface, and consequently fail to recruit Parkin for mitophagy initiation.\",\n      \"method\": \"CRISPR-Cas9 heterozygous D620N knock-in in SH-SY5Y cells, mitochondrial membrane potential assay, PINK1/Parkin recruitment imaging, CCCP treatment\",\n      \"journal\": \"Translational neurodegeneration\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — endogenous knock-in model with multiple functional mitophagy readouts\",\n      \"pmids\": [\"34127073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Upon mtDNA damage, VPS35 mediates maturation of early endosomes to late autophagy vesicles for selective mtDNA degradation; SAMM50 acts as a gatekeeper controlling nucleoid release and transfer to endosomes; the ATAD3-SAMM50 axis is disrupted by mtDNA damage, facilitating endosomal recruitment near nucleoid sub-compartments.\",\n      \"method\": \"Proximity labeling (Twinkle as nucleoid marker), VPS35 knockdown, lysosomal function assays, ATG5 KO, mouse model of mtDNA alterations treated with rapamycin\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — proximity labeling plus KD plus mouse model with multiple orthogonal readouts\",\n      \"pmids\": [\"36344526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"VPS35 D620N mutation promotes LRRK2-mediated lysosomal recruitment of RILPL1 and its binding to the lysosomal membrane protein TMEM55B; VPS35 D620N reduces RILPL1 levels in mouse tissues in a manner reversed by LRRK2 inhibition and proteasome inhibitors; RILPL1 KO enhances Rab substrate phosphorylation; TMEM55B KO increases RILPL1 levels.\",\n      \"method\": \"Quantitative lysosomal proteomics (~220 proteins altered), Co-IP of RILPL1-TMEM55B, VPS35 D620N knock-in mouse tissues, LRRK2 inhibitor treatment, RILPL1/TMEM55B knockout cells\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — quantitative proteomics plus Co-IP plus KO plus pharmacological rescue in multiple model systems\",\n      \"pmids\": [\"38091401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"VPS35 selectively binds endocytosed EGFR in early endosomes and recycles it to the cell surface, activating downstream ERK1/2 signaling; high VPS35 expression increases sensitivity to EGFR inhibitors in gastric cancer xenograft and organoid models.\",\n      \"method\": \"Co-immunoprecipitation (VPS35-EGFR), biotin surface assay, patient-derived xenografts and organoids, EGFR inhibitor response assay\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional recycling assay plus PDX/organoid validation, single lab\",\n      \"pmids\": [\"36738481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"VPS35 promotes hepatoma cell proliferation via the PI3K/AKT signaling pathway; VPS35 knockout reduces membrane FGFR3 distribution, demonstrating VPS35's role in sorting and trafficking of transmembrane receptor FGFR3 in hepatoma cells.\",\n      \"method\": \"VPS35 knockout in hepatoma cells, FGFR3 membrane localization assay, PI3K/AKT signaling assay, in vivo xenograft\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO plus receptor trafficking assay, single lab\",\n      \"pmids\": [\"32071398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"VPS35 D620N significantly elevates LRRK2-mediated phosphorylation of Rab10, Rab12, and Rab29 in knock-in mice; LRRK2 kinase inhibitor MLi-2 normalizes striatal dopamine transporter (DAT) expression and function and abolishes increased amphetamine-induced hyperlocomotion in VKI mice; VPS35 haploinsufficiency reduces Rab12 phosphorylation and impairs DAT similarly but is not reversed by MLi-2, confirming D620N is a gain-of-function for LRRK2 kinase.\",\n      \"method\": \"VPS35 D620N knock-in and haploinsufficient mice, phospho-Rab Western blot, DAT expression/function assay, LRRK2 inhibitor MLi-2 treatment, behavioral testing\",\n      \"journal\": \"NPJ Parkinson's disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knock-in + haploinsufficient mouse comparison plus pharmacological rescue with defined biochemical and behavioral readouts\",\n      \"pmids\": [\"38110354\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"VPS35 is the central cargo-recognition subunit of the retromer complex that mediates endosome-to-TGN/plasma membrane retrograde trafficking of diverse transmembrane cargoes (including CI-M6PR, BACE1, LAMP2a, Trem2, DRD1, AMPA receptors, EGFR, and RANK); it recruits the WASH complex to endosomes via FAM21 binding to nucleate actin for tubule formation, is targeted to endosomes through Rab7 interaction with its N-terminal domain, is ubiquitinated by parkin in a non-degradative manner that modulates sorting, and the PD-linked D620N mutation primarily disrupts FAM21/WASH complex binding—causing impaired cargo retrieval, ATG9A mis-sorting, LRRK2 hyperactivation (with elevated Rab8A/10/12 phosphorylation and lysosomal recruitment of RILPL1-TMEM55B), enhanced VPS35-DLP1 interaction leading to mitochondrial fragmentation via MUL1-dependent MFN2 degradation, and impaired PINK1/Parkin-mediated mitophagy.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"VPS35 is the central cargo-recognition subunit of the retromer complex, mediating retrograde retrieval of diverse transmembrane cargoes—including sorting receptors (Vps10p, CI-M6PR), BACE1, Lamp2a, EGFR, DRD1, AMPA receptors, RANK, and Trem2—from endosomes to the trans-Golgi network or plasma membrane [PMID:9105038, PMID:22105352, PMID:26203154, PMID:36738481, PMID:27460146, PMID:25416282, PMID:23509071, PMID:27717139]. It is recruited to late endosomes through direct interaction with Rab7 at its N-terminal domain and recruits the WASH actin-nucleation complex to endosomes via binding of the FAM21 subunit, thereby promoting actin-dependent membrane tubulation required for cargo sorting; the Parkinson disease-linked D620N mutation selectively reduces FAM21/WASH binding affinity, impairing ATG9A trafficking and autophagy [PMID:25367362, PMID:22070227, PMID:24980502, PMID:24819384]. The D620N mutation additionally causes LRRK2 kinase hyperactivation with elevated phosphorylation of Rab8A, Rab10, and Rab12, enhanced VPS35–DLP1 interaction leading to MUL1-dependent MFN2 degradation and mitochondrial fragmentation, and impaired PINK1/Parkin-mediated mitophagy, establishing convergent pathogenic mechanisms in familial Parkinson disease [PMID:29743203, PMID:26618722, PMID:26321632, PMID:34127073]. VPS35 also participates in mitochondria-derived vesicle trafficking, mediating transport of MAPL from mitochondria to peroxisomes and selective degradation of damaged mtDNA through endosomal maturation [PMID:20619655, PMID:36344526].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Establishing that VPS35 is a membrane-associated protein required for vacuolar protein sorting resolved a key component of the endosomal sorting machinery, though its precise molecular role was unknown.\",\n      \"evidence\": \"Gene disruption, subcellular fractionation, and CPY secretion assays in S. cerevisiae\",\n      \"pmids\": [\"1498362\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of VPS35 action on sorting unknown\", \"No direct cargo interaction demonstrated\", \"Mammalian ortholog function not addressed\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Demonstrating that VPS35 is specifically required for retrograde retrieval of the sorting receptor Vps10p from endosomes to the Golgi established the retromer concept and positioned VPS35 as a cargo-recognition factor rather than a general sorting component.\",\n      \"evidence\": \"Temperature-conditional VPS35 allele, co-fractionation with Vps10p, genetic epistasis with pep12 in S. cerevisiae\",\n      \"pmids\": [\"9105038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical interaction with cargo not yet proven by binding assay\", \"Structural basis unknown\", \"Mammalian cargo repertoire undefined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Structural determination of Vps26A and mapping of its VPS35-binding interface provided the first atomic-level understanding of retromer subcomplex assembly, revealing that a mobile loop in Vps26 is critical for integration into the complex.\",\n      \"evidence\": \"X-ray crystallography at 2.1 Å, site-directed mutagenesis, endosomal localization assays\",\n      \"pmids\": [\"16732284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full VPS35 structure not solved\", \"VPS35-Vps29 interface not mapped\", \"Cargo-binding site on VPS35 not identified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of the conserved PRLYL motif in VPS35's N-terminus as essential for Vps26 binding and retromer integrity, together with evidence for dominant-negative trafficking defects, established that VPS35 serves as a scaffold nucleating retromer assembly.\",\n      \"evidence\": \"R107W mutagenesis, Co-IP, cross-species dominant-negative expression in yeast and mammalian cells\",\n      \"pmids\": [\"17916227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"VPS35-Vps29 binding determinants not mapped\", \"Full cargo-binding surface unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovery that VPS35 is recruited to mitochondria-derived vesicles and mediates MAPL delivery from mitochondria to peroxisomes expanded retromer function beyond canonical endosome-to-Golgi trafficking to inter-organelle transport.\",\n      \"evidence\": \"Unbiased protein interaction screen, Co-IP, confocal imaging of VPS35 on MDVs, siRNA knockdown with peroxisomal MAPL quantification\",\n      \"pmids\": [\"20619655\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of VPS35 recruitment to mitochondrial membranes unknown\", \"Whether MDV cargo selectivity requires VPS35 directly or through associated factors unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showing that VPS35 interacts with BACE1 and that haploinsufficiency increases endosomal BACE1 activity and Aβ production in AD mice linked retromer dysfunction to Alzheimer disease pathogenesis through a specific cargo-sorting defect.\",\n      \"evidence\": \"Co-IP of VPS35-BACE1, hemizygous Vps35 deletion in Tg2576 mice, BACE1 activity assay, behavioral testing\",\n      \"pmids\": [\"22105352\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct BACE1 sorting signal on VPS35 not mapped\", \"Whether VPS35 loss affects amyloidogenesis independent of BACE1 not determined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identification of FAM21 as the direct bridge between VPS35 and the WASH actin-nucleation complex on endosomes resolved how retromer coordinates membrane remodeling with cargo sorting.\",\n      \"evidence\": \"Biochemical binding assays, FAM21 tail fragment overexpression, endosomal localization assays\",\n      \"pmids\": [\"22070227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding site on VPS35 for FAM21 not mapped at residue level\", \"Whether WASH recruitment is required for all retromer cargoes unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Biophysical and proteomic characterization of VPS35 D620N revealed that the primary molecular defect is a ~2-fold reduction in FAM21/WASH binding affinity while retromer trimer assembly is unaffected, establishing D620N as a selective WASH-recruitment defect that impairs endosome-to-TGN transport and ATG9A sorting for autophagy.\",\n      \"evidence\": \"Isothermal calorimetry, SILAC-based quantitative proteomics, ATG9A trafficking assay, patient fibroblasts\",\n      \"pmids\": [\"24980502\", \"24819384\", \"24152121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of D620N-FAM21 affinity loss not resolved\", \"Whether all D620N phenotypes are WASH-dependent not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstration that Rab7 recruits retromer to late endosomes via VPS35's N-terminal domain, with allosteric enhancement by Vps26 binding, established the molecular logic of retromer membrane targeting.\",\n      \"evidence\": \"FRET-based interaction assay in HeLa cells, biophysical binding measurements, mutagenesis of Vps35-Vps26 interface\",\n      \"pmids\": [\"25367362\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Rab7 GTP/GDP state regulates the interaction quantitatively not fully resolved\", \"Role of SNX-BAR dimers in coordinating with Rab7-VPS35 binding unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showing that VPS35 retrieves Lamp2a from endosomes and that D620N causes accelerated Lamp2a degradation, impairing chaperone-mediated autophagy and leading to α-synuclein accumulation, established a mechanistic link between retromer dysfunction and Parkinson-relevant protein aggregation.\",\n      \"evidence\": \"Conditional VPS35 KO in DA neurons, Lamp2a trafficking, α-synuclein immunostaining, Lamp2a overexpression rescue\",\n      \"pmids\": [\"26203154\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sorting signal on Lamp2a recognized by VPS35 not identified\", \"Whether CMA impairment is the dominant pathway for α-synuclein accumulation vs. macroautophagy defects not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovery that D620N enhances VPS35–DLP1 interaction, increasing mitochondrial DLP1 turnover and causing mitochondrial fragmentation, and that VPS35 deficiency elevates MUL1 leading to MFN2 degradation, established a mitochondrial fragmentation axis in PD pathogenesis separable from α-synuclein accumulation.\",\n      \"evidence\": \"Co-IP of VPS35-DLP1, mitochondrial fission inhibitor rescue, conditional KO with MUL1 epistasis, patient fibroblasts\",\n      \"pmids\": [\"26618722\", \"26321632\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether enhanced VPS35-DLP1 binding is a direct consequence of WASH loss or an independent D620N effect unclear\", \"Structural basis of VPS35-DLP1 interaction resolved only at motif level (FLV)\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Expanding VPS35's cargo repertoire to include Trem2, DRD1, N-Ras, and RANK demonstrated that retromer-mediated receptor recycling controls inflammatory signaling in microglia, dopaminergic signaling, Ras-MAPK signaling, and osteoclast formation.\",\n      \"evidence\": \"Co-IP, surface recycling assays, inflammatory cytokine measurement, RANK signaling in osteoclasts, farnesyl-dependent N-Ras binding and MAPK readout, hemizygous KO mouse bone phenotype\",\n      \"pmids\": [\"27717139\", \"27460146\", \"27502489\", \"23509071\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sorting signals on these diverse cargoes not systematically identified\", \"Whether all cargoes require WASH-dependent sorting not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that D620N is a gain-of-function for LRRK2 kinase activity—elevating Rab8A/10/12 phosphorylation in knock-in mice and patient cells—while VPS35 loss suppresses LRRK2-mediated Rab phosphorylation, established VPS35 as an upstream activator of LRRK2 and linked the two major familial PD genes in a common pathway.\",\n      \"evidence\": \"VPS35 D620N knock-in mice, phospho-Rab Western blot, VPS35 KO/KD, patient neutrophils/monocytes\",\n      \"pmids\": [\"29743203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which VPS35 activates LRRK2 kinase not identified (direct binding vs. membrane platform)\", \"Whether wild-type VPS35 normally stimulates LRRK2 at physiological levels unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Parkin was shown to ubiquitinate VPS35 at C-terminal lysines via non-degradative poly-ubiquitin chains, and parkin loss reduced WASH complex components and disrupted ATG9A sorting, positioning VPS35 as a Parkin substrate that integrates retromer and mitophagy pathways.\",\n      \"evidence\": \"Co-IP, ubiquitin chain type and lysine mapping, parkin KO mouse brain analysis, ATG9A trafficking\",\n      \"pmids\": [\"29893854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of VPS35 ubiquitination on WASH recruitment not directly tested\", \"Whether parkin-mediated ubiquitination regulates VPS35-LRRK2 interplay unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"D620N was shown to impair PINK1/Parkin-mediated mitophagy by preventing PINK1 accumulation on depolarized mitochondria, connecting retromer dysfunction to a second mitochondrial quality control pathway beyond fission.\",\n      \"evidence\": \"CRISPR heterozygous D620N knock-in in SH-SY5Y cells, mitochondrial membrane potential, PINK1/Parkin recruitment imaging\",\n      \"pmids\": [\"34127073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the mitophagy defect is secondary to altered mitochondrial membrane potential or a direct VPS35 role in PINK1 stabilization not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Quantitative lysosomal proteomics in D620N knock-in mice revealed LRRK2-mediated lysosomal recruitment of RILPL1 via TMEM55B binding, identifying a downstream effector pathway of the VPS35-LRRK2 axis that alters lysosomal composition.\",\n      \"evidence\": \"Lysosomal proteomics (~220 altered proteins), Co-IP of RILPL1-TMEM55B, LRRK2 inhibitor reversal, RILPL1/TMEM55B KO cells\",\n      \"pmids\": [\"38091401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of RILPL1 lysosomal recruitment on lysosomal degradation or autophagy not fully defined\", \"Whether this pathway operates in human dopaminergic neurons not confirmed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"VPS35 was found to mediate endosomal maturation for selective mtDNA degradation upon mitochondrial DNA damage, with SAMM50 acting as a gatekeeper for nucleoid release, extending VPS35's mitochondrial roles to genome maintenance.\",\n      \"evidence\": \"Proximity labeling with Twinkle, VPS35 knockdown, ATG5 KO, mouse model with mtDNA alterations\",\n      \"pmids\": [\"36344526\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this pathway is retromer-dependent or uses VPS35 in a retromer-independent capacity not resolved\", \"Mechanism of VPS35 recruitment to damaged nucleoid compartments unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Comparison of D620N knock-in versus haploinsufficient mice confirmed D620N as a gain-of-function for LRRK2 kinase (reversed by inhibitor MLi-2) while haploinsufficiency impairs DAT independently of LRRK2, demonstrating that loss-of-function and gain-of-toxic-function coexist in D620N pathogenesis.\",\n      \"evidence\": \"VPS35 D620N KI and haploinsufficient mice, phospho-Rab blots, DAT expression/function, LRRK2 inhibitor MLi-2, behavioral testing\",\n      \"pmids\": [\"38110354\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How D620N simultaneously causes loss of WASH binding and gain of LRRK2 activation structurally unresolved\", \"Cell-type-specific contributions of these dual mechanisms in dopaminergic neurons not separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of full-length VPS35 in complex with FAM21, Rab7, and cargo peptides is needed to explain how D620N simultaneously impairs WASH recruitment and hyperactivates LRRK2, and whether these are structurally linked or independent surfaces.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length VPS35 structure with bound FAM21 or LRRK2\", \"Direct VPS35-LRRK2 binding interface not mapped\", \"Cargo-binding specificity determinants on VPS35 for its >10 known cargoes not structurally resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 9, 15, 17]},\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [1, 8, 11, 17, 37]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1, 9, 11, 13, 15]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [6, 19, 20, 34, 35]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [6, 11, 35]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 8, 9, 11, 14, 17, 24, 37]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [13, 34, 35]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [6, 19, 20, 34]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11, 29, 39]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [25, 37, 38]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [16, 21, 28]}\n    ],\n    \"complexes\": [\n      \"Retromer (VPS35-VPS29-VPS26)\"\n    ],\n    \"partners\": [\n      \"VPS26A\",\n      \"VPS29\",\n      \"FAM21\",\n      \"RAB7A\",\n      \"DLP1\",\n      \"LRRK2\",\n      \"PRKN\",\n      \"BACE1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}