{"gene":"PDZD8","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":2017,"finding":"PDZD8 is an ER-resident transmembrane protein whose SMP domain is functionally orthologous to the SMP domain of yeast Mmm1 (ERMES subunit). PDZD8 localizes to ER-mitochondria contact sites and is necessary for formation of these contacts in mammalian cells. In neurons, PDZD8 is required for Ca2+ uptake by mitochondria following synaptically induced Ca2+ release from ER, thereby regulating cytoplasmic Ca2+ dynamics.","method":"Live-cell imaging, subcellular fractionation, co-localization, siRNA knockdown with Ca2+ imaging readout, SMP domain functional complementation assay","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal localization, KD with defined physiological phenotype (Ca2+ dynamics), functional domain complementation; foundational paper replicated by multiple subsequent studies","pmids":["29097544"],"is_preprint":false},{"year":2020,"finding":"PDZD8 uses distinct domains to interact with GTP-bound Rab7 and the ER transmembrane protein Protrudin, localizing to ER-late endosome membrane contact sites. At these MCSs, mitochondria are also recruited, forming a three-way contact. PDZD8 thus serves as a shared component of both ER-mitochondria and ER-late endosome MCSs.","method":"Co-immunoprecipitation, proximity proteomics (BioID), live-cell imaging, CRISPR-Cas9 knockouts, domain mapping","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, proximity proteomics, domain mapping, replicated independently by multiple labs","pmids":["32686675"],"is_preprint":false},{"year":2019,"finding":"PDZD8, an intrinsic ER membrane protein with an SMP lipid transport domain, concentrates at ER-late endosome/lysosome contacts, where it interacts specifically with GTP-bound Rab7 (GTP-dependent interaction).","method":"Colocalization imaging, co-immunoprecipitation with GTP/GDP-loaded Rab7, knockdown","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 / Strong — GTP-dependency biochemically validated, replicated by multiple independent labs","pmids":["31636202"],"is_preprint":false},{"year":2020,"finding":"PDZD8 interacts with Protrudin at ER-endolysosome (LyLE) MCSs. The SMP domain of PDZD8 mediates lipid extraction from ER membranes. PDZD8 and Protrudin cooperatively promote endosome maturation by mediating ER-LyLE tethering and lipid extraction at MCSs, which is essential for neuronal polarity and integrity.","method":"Co-immunoprecipitation, overexpression and siRNA knockdown in HeLa cells and mouse primary neurons, live-cell imaging, endosomal morphology assays, lipid extraction assay","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, cell and primary neuron KD with defined phenotype (endosomal vacuolation, neuronal polarity), multiple orthogonal methods in one study","pmids":["32917905"],"is_preprint":false},{"year":2021,"finding":"The SMP domain of PDZD8 binds glycerophospholipids and ceramides both in vivo and in vitro, and can transport lipids between membranes in vitro. PDZD8 acts as a tether at ER-late endosome/lysosome MCSs and its lipid transfer activity is required for late endosome/lysosome positioning and neurite outgrowth.","method":"In vitro lipid binding assay, in vitro lipid transfer assay between liposomes, PDZD8 knockdown with neurite outgrowth and organelle positioning readouts, confocal imaging","journal":"Journal of Cell Science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstituted lipid transfer assay with domain mutagenesis, corroborated by cellular loss-of-function with defined phenotypes","pmids":["33912962"],"is_preprint":false},{"year":2021,"finding":"Crystal structure of the human PDZD8 C-terminal coiled-coil (CC) domain in complex with GTP-bound Rab7 was determined. The PDZD8 CC contains one short helix and two helices forming an antiparallel coiled-coil; two Rab7 molecules bind opposite sides of the CC in a 2:1 stoichiometry via their switch I/II and interswitch regions. Isothermal titration calorimetry confirmed the GTP-dependent 2:1 binding.","method":"X-ray crystallography, isothermal titration calorimetry (ITC)","journal":"Scientific Reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with biochemical validation (ITC), two orthogonal methods in one study","pmids":["34552186"],"is_preprint":false},{"year":2024,"finding":"PDZD8 is a substrate of AMPK; AMPK phosphorylates PDZD8 at threonine 527 (T527) under low-glucose conditions. This phosphorylation promotes the interaction of PDZD8 with glutaminase 1 (GLS1), activating GLS1 and promoting glutaminolysis. The PDZD8-T527A mutation abolishes this interaction and dampens glutaminolysis and pro-inflammatory cytokine secretion in macrophages.","method":"In vitro kinase assay, mass spectrometry phospho-mapping, co-immunoprecipitation, GLS1 activity assay, T527A phospho-dead mutant, in vivo skeletal muscle and macrophage assays","journal":"Cell Research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with phospho-site identification, phospho-dead mutant validation, and defined in vivo metabolic phenotype; multiple orthogonal methods","pmids":["38898113"],"is_preprint":false},{"year":2025,"finding":"FKBP8, an outer mitochondrial membrane (OMM) protein, is the direct tethering partner of ER-resident PDZD8 at ER-mitochondria contact sites (MERCS). Single-molecule tracking shows PDZD8 diffuses dynamically along the ER membrane with pauses and captures at MERCS. Overexpression of FKBP8 narrows the ER-OMM distance; combined deletion of PDZD8 and FKBP8 is interdependent for MERCS formation. PDZD8 enhances mitochondrial complexity (morphology) in a FKBP8-dependent manner.","method":"Unbiased proximity proteomics, CRISPR-Cas9 endogenous tagging, cryo-electron tomography, correlative light-electron microscopy (CLEM), single-molecule tracking, overexpression and double-KO epistasis","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural (cryo-ET), proximity proteomics, CLEM, single-molecule live imaging, and genetic epistasis in one study; multiple orthogonal methods","pmids":["40246839"],"is_preprint":false},{"year":2010,"finding":"PDZD8 interacts with HIV-1 Gag protein (identified by yeast two-hybrid, confirmed by co-immunoprecipitation). PDZD8 overexpression promotes initiation of reverse transcription and increases retroviral infection, while knockdown decreases HIV-1 infection. A PDZD8 mutant lacking its predicted coiled-coil domain fails to bind Gag and loses ability to promote HIV-1 infection, mapping the Gag-interacting region.","method":"Yeast two-hybrid, co-immunoprecipitation, overexpression and siRNA knockdown, reverse transcription efficiency assay, domain deletion mutagenesis","journal":"Journal of Virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP confirms Y2H result, domain mutagenesis maps binding region, functional knockdown assay; single lab","pmids":["20573829"],"is_preprint":false},{"year":2014,"finding":"PDZD8 is a critical component of a heat-labile, >100 kDa cytoplasmic factor that slows spontaneous disassembly of HIV-1 capsid-nucleocapsid (CA-NC) complexes in vitro. PDZD8 knockdown accelerates HIV-1 capsid disassembly (uncoating) in infected cells and decreases reverse transcription. The coiled-coil domain is sufficient for capsid binding, but the PDZ domain is additionally required for capsid stabilization and supporting HIV-1 infection.","method":"In vitro capsid disassembly assay, PDZD8 knockdown, immunoprecipitation, domain deletion mutagenesis, reverse transcription assay in cells","journal":"Journal of Virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro capsid assay, cellular KD with defined mechanistic readout, domain mutagenesis; single lab","pmids":["24554657"],"is_preprint":false},{"year":2015,"finding":"Stable CRISPR-Cas9 knockout of PDZD8 does not reduce HIV-1 or murine leukemia virus infection efficiency compared to parental cells, indicating PDZD8 is not absolutely required for retroviral infection.","method":"CRISPR-Cas9 knockout cell lines, HIV-1 and MLV infection assays","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean genetic KO with defined infection readout, single lab; negative result contradicting prior transient KD studies","pmids":["25771112"],"is_preprint":false},{"year":2011,"finding":"PDZD8 interacts with moesin (an ERM family protein) as identified by co-immunoprecipitation. Exogenous expression of PDZD8 or moesin reduces levels of stable (acetylated) microtubules, suggesting PDZD8 functions as part of a cytoskeletal regulatory complex. Overexpression or siRNA knockdown of PDZD8 affects HSV-1 infection levels.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, microtubule acetylation assay, HSV-1 infection assay","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP with functional follow-up (MT stability, viral infection); single lab","pmids":["21549406"],"is_preprint":false},{"year":2022,"finding":"In Drosophila, reducing pdzd8-mediated ER-mitochondria contacts (MERCs) in neurons slows age-associated locomotor decline and increases lifespan, correlating with increased mitophagy. Conversely, increasing MERCs via a synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation and reduces lifespan. Pdzd8 knockdown also rescues locomotor defects in a fly model of Alzheimer's disease expressing Aβ42, demonstrating that MERC reduction is protective in a neurodegenerative context.","method":"Drosophila neuron-specific RNAi knockdown, lifespan assay, locomotor assay, mitophagy reporter, synthetic ER-mitochondria tether overexpression, Aβ42 disease model","journal":"Life Science Alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KD and gain-of-function in vivo with defined phenotypes including mitophagy mechanistic link; single lab","pmids":["35831024"],"is_preprint":false},{"year":2022,"finding":"PDZD8-deficient mice accumulate cholesteryl esters (CEs) in the brain due to impaired lipophagy (lysosomal degradation of lipid droplets). PDZD8 functions as a Rab7 effector that transfers lipids (cholesterol and phosphatidylserine) between ER and Rab7-positive organelles to promote endolysosome maturation and fusion of CE-containing lipid droplets with lysosomes.","method":"PDZD8-KO mouse model, lipidomics (CE quantification), lipid droplet morphology assays, lysosomal fusion assays, Rab7 interaction studies","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with mechanistic phenotype (impaired lipophagy) and proposed lipid transport mechanism; single lab","pmids":["36465123"],"is_preprint":false},{"year":2023,"finding":"PDZD8 transports cholesterol to lipid droplets and promotes fusion of lipid droplets with lysosomes (lipophagy). PDZD8-KO mice exhibit impaired lipid droplet degradation and abnormal accumulation of cholesteryl esters in the brain, leading to behavioral abnormalities in emotion, cognition, and adaptation.","method":"PDZD8-KO mouse model, cholesterol transport assay, lipid droplet-lysosome fusion assay, behavioral battery (open field, fear conditioning, etc.)","journal":"Molecular Brain","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with mechanistic cellular assay (LD-lysosome fusion) and defined behavioral phenotype; single lab","pmids":["36658656"],"is_preprint":false},{"year":2025,"finding":"PDZD8 (LYVAC) is a general mediator of lysosomal vacuolation. Upon lysosomal osmotic stress, diverse vacuolation inducers trigger PDZD8 recruitment to lysosomes through multivalent interactions. Stress-induced lysosomal lipid signaling (phosphatidylserine and cholesterol) activates PDZD8's SMP lipid transfer domain, driving directional ER-to-lysosome lipid movement that causes osmotic membrane expansion of lysosomes.","method":"Human cell line imaging, PDZD8 KO and rescue, live-cell imaging, lipid sensing assays, domain mutagenesis of lipid-binding residues","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro lipid sensing, domain mutagenesis, KO rescue, mechanistic pathway reconstitution; multiple orthogonal methods in one study","pmids":["40839735"],"is_preprint":false},{"year":2025,"finding":"PDZD8 promotes autophagy at ER-late endosome/lysosome MCSs by promoting lysosome maturation and accelerating autophagic flux. PDZD8 is required for activity-dependent synaptic bouton formation in Drosophila neurons, and is sufficient to drive excess bouton formation through an autophagy-dependent mechanism. SMP domain mutational analysis suggests lipid transfer from ER to late endosomes/lysosomes is the mechanistic basis for lysosome maturation.","method":"In vivo CRISPR screen in Drosophila, genetic loss-of-function and gain-of-function, autophagic flux assays, lysosome maturation assays, SMP domain mutagenesis, synaptic bouton counting","journal":"Cell Reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo CRISPR KO with defined cellular (autophagy/lysosome) and organismal (synapse) phenotype, domain mutagenesis; single lab","pmids":["40156832"],"is_preprint":false},{"year":2025,"finding":"PDZD8 regulates endolysosomal maturation and acidification, which is required for proper translocation of TLR9 to endolysosomes and downstream NF-κB activation in kidney proximal tubular cells. PDZD8 KO mice show reduced cisplatin-induced AKI severity and reduced NF-κB pathway activation. PDZD8 knockdown did not alter mitochondrial morphology or cytosolic leakage of mitochondrial DNA.","method":"Pdzd8 KO mouse, in vitro PDZD8 knockdown in human proximal tubular cells, lysosomal acidification assay, TLR9 localization by immunofluorescence, NF-κB reporter, cisplatin AKI model","journal":"American Journal of Physiology - Renal Physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse plus cell-based KD with mechanistic pathway placement (TLR9-NF-κB) and defined negative result for mitochondrial involvement; single lab","pmids":["40897465"],"is_preprint":false},{"year":2024,"finding":"PDZD8 augments ER-mitochondria contact (MAM) formation in pancreatic β-cells. PDZD8 knockdown shortens MAM perimeter, suppresses MAM-related proteins (IP3R1, GRP75, VDAC1), inhibits VDAC1-IP3R1 interaction, alleviates mitochondrial Ca2+ overload, and decreases Cyclophilin D (CypD) expression, thereby reducing β-cell apoptosis. Cyclophilin D overexpression rescues β-cell death, placing PDZD8 upstream of CypD in the apoptotic pathway.","method":"PDZD8 knockdown in INS-1 cells and HFD mouse model, proximity ligation assay for VDAC1-IP3R1 interaction, Ca2+ imaging, mitochondrial membrane potential assay, Western blot, epistasis with CypD overexpression","journal":"Diabetes & Metabolism Journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway placement via epistasis (CypD rescue), PLA and Ca2+ imaging in cells and in vivo model; single lab","pmids":["39069376"],"is_preprint":false},{"year":2025,"finding":"PDZD8 dysregulation in RVLM neurons activates Ca2+-Calpain-2 (CAPN2) signaling: PDZD8 deficiency elevates cytoplasmic Ca2+ levels, which upregulates CAPN2, leading to ER stress, mitochondrial dysfunction, and neuronal apoptosis. CAPN2 inhibition rescues PDZD8-deficiency-induced ER stress and mitochondrial dysfunction. In vivo, PDZD8 upregulation in RVLM suppresses neuronal hyperexcitation and reduces blood pressure in stress-induced hypertension rats.","method":"PDZD8 siRNA knockdown in N2a cells, AAV2-mediated PDZD8 overexpression in rat RVLM, CAPN2 inhibitor treatment, Western blot, flow cytometry, immunofluorescence, RSNA/BP measurement","journal":"Molecular Neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic epistasis (CAPN2 inhibition rescuing PDZD8 KD phenotype), in vivo AAV rescue; single lab","pmids":["40418411"],"is_preprint":false},{"year":2025,"finding":"TMEM55B is identified as a lysosomal PDZD8-associated protein. TMEM55B depletion reduces lysosomal acidification, impairs lipid droplet turnover, attenuates lysosomal Ca2+ release and reuptake, and diminishes ER Ca2+ responses, consistent with impaired CICR at ER-lysosome MCSs. Mitochondrial Ca2+ dynamics were unaffected, suggesting specificity to the ER-lysosome axis.","method":"Co-immunoprecipitation/proximity assay for PDZD8-TMEM55B interaction, lysosomal pH assay, lipid droplet assay, Ca2+ imaging, siRNA knockdown","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, association demonstrated but mechanistic link to PDZD8 is indirect","pmids":["bio_10.1101_2025.10.21.683636"],"is_preprint":true},{"year":2025,"finding":"Live-cell imaging using a novel ddFP-based MERCS sensor (MERCdRED) demonstrated that large MERCS are more stable than smaller ones. Nutrient deprivation reduces MERCS area in a PDZD8-dependent manner, establishing PDZD8 as required for nutrient-regulated MERCS dynamics.","method":"Novel MERCS fluorescent sensor (ddFP-based), CLEM validation, live-cell imaging, PDZD8 KO under nutrient deprivation","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 2 / Weak — preprint, novel tool validated by CLEM, single lab, single method for the nutrient-PDZD8 finding","pmids":["bio_10.1101_2025.04.17.649323"],"is_preprint":true}],"current_model":"PDZD8 is an ER-anchored transmembrane protein with an SMP (lipid transfer) domain that functions as a tethering and lipid transport protein at multiple membrane contact sites: it bridges ER to mitochondria (via FKBP8 on the OMM) to regulate Ca2+ transfer and mitochondrial morphology, and bridges ER to late endosomes/lysosomes (via GTP-Rab7 and Protrudin) to mediate directional lipid transfer that drives endolysosomal maturation, lipophagy, lysosomal vacuolation, autophagic flux, and TLR9-NF-κB signaling; additionally, PDZD8 is phosphorylated by AMPK at T527 to promote glutaminolysis through GLS1 activation, and its coiled-coil domain interacts with the HIV-1 capsid to stabilize it during uncoating."},"narrative":{"mechanistic_narrative":"PDZD8 is an ER-anchored transmembrane protein that uses an SMP lipid-transfer domain to tether the endoplasmic reticulum to multiple partner organelles and to move lipids across the resulting membrane contact sites [PMID:29097544, PMID:33912962]. At ER–mitochondria contacts it is necessary for contact formation and for mitochondrial Ca2+ uptake following ER Ca2+ release, with the outer mitochondrial membrane protein FKBP8 serving as its direct tethering partner that sets ER–OMM distance and shapes mitochondrial morphology [PMID:29097544, PMID:40246839]. In parallel, PDZD8 localizes to ER–late endosome/lysosome contacts through a GTP-dependent interaction with Rab7 and an interaction with Protrudin, acting as a Rab7 effector whose SMP domain extracts and transfers glycerophospholipids, ceramides, cholesterol, and phosphatidylserine to drive endolysosomal maturation, lysosome positioning, lipophagy, and autophagic flux [PMID:31636202, PMID:32917905, PMID:33912962, PMID:34552186, PMID:36465123]. Stress-induced lysosomal lipid signaling activates this lipid-transfer activity to produce osmotic lysosomal vacuolation, and the same maturation function supports endolysosomal acidification required for TLR9 trafficking and downstream NF-κB activation [PMID:40839735, PMID:40897465]. Beyond its tethering roles, PDZD8 is a substrate of AMPK, which phosphorylates it at threonine 527 under low glucose to promote its interaction with and activation of glutaminase 1 (GLS1), linking PDZD8 to glutaminolysis and macrophage cytokine output [PMID:38898113]. Through these contact-site activities PDZD8 governs neuronal Ca2+ dynamics, neurite outgrowth, synaptic bouton formation, and brain lipid homeostasis [PMID:29097544, PMID:33912962, PMID:36658656, PMID:40156832].","teleology":[{"year":2017,"claim":"Established PDZD8 as the first mammalian ER protein required to build ER–mitochondria contacts and to couple ER Ca2+ release to mitochondrial Ca2+ uptake, defining its founding tethering function.","evidence":"Live-cell imaging, fractionation, siRNA knockdown with Ca2+ imaging, and SMP-domain complementation of yeast Mmm1 in mammalian cells","pmids":["29097544"],"confidence":"High","gaps":["Did not identify the mitochondrial tethering partner","Did not reconstitute lipid transfer by the SMP domain"]},{"year":2019,"claim":"Showed PDZD8 also concentrates at ER–late endosome/lysosome contacts via a GTP-dependent interaction with Rab7, extending its role beyond ER–mitochondria contacts.","evidence":"Colocalization imaging and Co-IP with GTP/GDP-loaded Rab7 plus knockdown","pmids":["31636202"],"confidence":"High","gaps":["Did not demonstrate lipid transfer at these contacts","Functional consequence of the Rab7 interaction unresolved"]},{"year":2020,"claim":"Identified Protrudin as a second ER-side partner and revealed PDZD8 as a shared component of ER–mitochondria and ER–late endosome contacts that can organize three-way contacts and drive endosome maturation.","evidence":"Co-IP, BioID proximity proteomics, CRISPR knockouts, domain mapping, and lipid-extraction assays in HeLa cells and primary neurons","pmids":["32686675","32917905"],"confidence":"High","gaps":["Direct lipid species transported not defined in these studies","How the three-way contact is coordinated mechanistically unclear"]},{"year":2021,"claim":"Demonstrated biochemically that the SMP domain binds and transfers glycerophospholipids and ceramides and that this lipid-transfer activity drives organelle positioning and neurite outgrowth, and solved the structural basis of the Rab7 interaction.","evidence":"In vitro lipid binding and liposome transfer assays with domain mutants, plus X-ray crystallography and ITC of the coiled-coil–Rab7 complex (2:1 GTP-dependent)","pmids":["33912962","34552186"],"confidence":"High","gaps":["Directionality of lipid flux in cells not established","Full-length protein structure and SMP domain orientation at the contact unresolved"]},{"year":2022,"claim":"Connected PDZD8-dependent contacts to organismal physiology and disease, showing in vivo that modulating ER–mitochondria contacts alters lifespan, mitophagy, and neurodegenerative phenotypes, and that PDZD8 loss causes brain cholesteryl-ester accumulation via impaired lipophagy.","evidence":"Drosophila neuron-specific RNAi with lifespan/locomotor/mitophagy and Aβ42 readouts; PDZD8-KO mouse lipidomics and lysosomal fusion assays","pmids":["35831024","36465123"],"confidence":"Medium","gaps":["Causal lipid species at the lysosome contact not fully isolated in vivo","Single-lab phenotypes not cross-validated"]},{"year":2023,"claim":"Linked PDZD8-mediated cholesterol transport and lipid-droplet–lysosome fusion to behavioral outcomes, establishing a brain lipid-homeostasis role.","evidence":"PDZD8-KO mice with cholesterol transport assays, lipid droplet–lysosome fusion assays, and behavioral battery","pmids":["36658656"],"confidence":"Medium","gaps":["Cell-type-specific contribution to behavior unresolved","Direct lipid-transfer step in vivo not reconstituted"]},{"year":2024,"claim":"Revealed an unexpected signaling-metabolic role: AMPK phosphorylates PDZD8 at T527 to activate GLS1 and glutaminolysis, coupling PDZD8 to nutrient stress and macrophage inflammation independent of its tethering function.","evidence":"In vitro kinase assay, MS phospho-mapping, Co-IP, GLS1 activity assay, T527A phospho-dead mutant, and in vivo muscle/macrophage assays","pmids":["38898113"],"confidence":"High","gaps":["Whether membrane contact-site localization is required for GLS1 activation is unclear","Structural basis of the PDZD8–GLS1 interaction undefined"]},{"year":2025,"claim":"Defined the direct ER–mitochondria tethering partner and the lipid-driven mechanism of lysosomal vacuolation, while extending PDZD8 function to endolysosomal acidification-dependent TLR9–NF-κB signaling and disease models.","evidence":"Cryo-ET, CLEM, proximity proteomics, single-molecule tracking and double-KO epistasis identifying FKBP8; SMP lipid-sensing/mutagenesis for lysosomal vacuolation; KO mouse and proximal tubular cell assays for TLR9–NF-κB; β-cell and RVLM neuron Ca2+ pathway studies","pmids":["40246839","40839735","40897465","40156832","39069376","40418411"],"confidence":"High","gaps":["How the same SMP domain achieves directional ER-to-lysosome versus ER-to-mitochondria transport is unresolved","Reconciliation of pro-apoptotic Ca2+ overload roles with protective neuronal roles across tissues incomplete"]},{"year":null,"claim":"It remains unresolved how PDZD8's lipid-transfer activity is directionally and selectively regulated across its distinct contact sites, and how its tethering, lipid-transport, and AMPK–GLS1 signaling functions are integrated within a single protein.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length structural model integrating SMP, coiled-coil, and PDZ domains","Regulation of partner choice (FKBP8 vs Rab7/Protrudin vs GLS1) not defined","Conflicting HIV-1 requirement (KD vs clean KO) unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[4,15]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[3,4,13]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,7,2]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,2,3]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,7]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[2,3,15]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,2,3]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[13,16]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[6,13,14]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[3,4]}],"complexes":[],"partners":["FKBP8","RAB7A","ZFYVE27","GLS","MSN","TMEM55B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NEN9","full_name":"PDZ domain-containing protein 8","aliases":["Sarcoma antigen NY-SAR-84/NY-SAR-104"],"length_aa":1154,"mass_kda":128.6,"function":"Molecular tethering protein that connects endoplasmic reticulum and mitochondria membranes (PubMed:29097544). PDZD8-dependent endoplasmic reticulum-mitochondria membrane tethering is essential for endoplasmic reticulum-mitochondria Ca(2+) transfer (PubMed:29097544). In neurons, involved in the regulation of dendritic Ca(2+) dynamics by regulating mitochondrial Ca(2+) uptake in neurons (PubMed:29097544). Plays an indirect role in the regulation of cell morphology and cytoskeletal organization (PubMed:21834987). May inhibit herpes simplex virus 1 infection at an early stage (PubMed:21549406)","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q8NEN9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PDZD8","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"COPA","stoichiometry":0.2},{"gene":"COPE","stoichiometry":0.2},{"gene":"RPL11","stoichiometry":0.2},{"gene":"RPL10A","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2},{"gene":"RPS18","stoichiometry":0.2},{"gene":"RPL19","stoichiometry":0.2},{"gene":"RPL27","stoichiometry":0.2},{"gene":"KIF11","stoichiometry":0.2},{"gene":"EIF4A1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PDZD8","total_profiled":1310},"omim":[{"mim_id":"620021","title":"INTELLECTUAL DEVELOPMENTAL DISORDER WITH AUTISM AND DYSMORPHIC FACIES; IDDADF","url":"https://www.omim.org/entry/620021"},{"mim_id":"619871","title":"CORNEAL DYSTROPHY, PUNCTIFORM AND POLYCHROMATIC PRE-DESCEMET; PPPCD","url":"https://www.omim.org/entry/619871"},{"mim_id":"614235","title":"PDZ DOMAIN-CONTAINING PROTEIN 8; PDZD8","url":"https://www.omim.org/entry/614235"},{"mim_id":"309845","title":"MOESIN; MSN","url":"https://www.omim.org/entry/309845"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli fibrillar center","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":68.9}],"url":"https://www.proteinatlas.org/search/PDZD8"},"hgnc":{"alias_symbol":["LYVAC","bA129M16.2","FLJ34427"],"prev_symbol":["PDZK8"]},"alphafold":{"accession":"Q8NEN9","domains":[{"cath_id":"-","chopping":"2-23_91-168_187-292","consensus_level":"high","plddt":79.8319,"start":2,"end":292},{"cath_id":"2.60.40.150","chopping":"319-361_676-797","consensus_level":"high","plddt":83.6,"start":319,"end":797},{"cath_id":"2.30.42.10","chopping":"363-449","consensus_level":"high","plddt":81.4053,"start":363,"end":449},{"cath_id":"3.30.60.20","chopping":"844-889","consensus_level":"high","plddt":86.9178,"start":844,"end":889},{"cath_id":"1.10.287","chopping":"1014-1115","consensus_level":"high","plddt":84.7541,"start":1014,"end":1115}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NEN9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NEN9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NEN9-F1-predicted_aligned_error_v6.png","plddt_mean":62.09},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PDZD8","jax_strain_url":"https://www.jax.org/strain/search?query=PDZD8"},"sequence":{"accession":"Q8NEN9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NEN9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NEN9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NEN9"}},"corpus_meta":[{"pmid":"29097544","id":"PMC_29097544","title":"ER-mitochondria 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PDZD8 localizes to ER-mitochondria contact sites and is necessary for formation of these contacts in mammalian cells. In neurons, PDZD8 is required for Ca2+ uptake by mitochondria following synaptically induced Ca2+ release from ER, thereby regulating cytoplasmic Ca2+ dynamics.\",\n      \"method\": \"Live-cell imaging, subcellular fractionation, co-localization, siRNA knockdown with Ca2+ imaging readout, SMP domain functional complementation assay\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal localization, KD with defined physiological phenotype (Ca2+ dynamics), functional domain complementation; foundational paper replicated by multiple subsequent studies\",\n      \"pmids\": [\"29097544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PDZD8 uses distinct domains to interact with GTP-bound Rab7 and the ER transmembrane protein Protrudin, localizing to ER-late endosome membrane contact sites. At these MCSs, mitochondria are also recruited, forming a three-way contact. PDZD8 thus serves as a shared component of both ER-mitochondria and ER-late endosome MCSs.\",\n      \"method\": \"Co-immunoprecipitation, proximity proteomics (BioID), live-cell imaging, CRISPR-Cas9 knockouts, domain mapping\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, proximity proteomics, domain mapping, replicated independently by multiple labs\",\n      \"pmids\": [\"32686675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PDZD8, an intrinsic ER membrane protein with an SMP lipid transport domain, concentrates at ER-late endosome/lysosome contacts, where it interacts specifically with GTP-bound Rab7 (GTP-dependent interaction).\",\n      \"method\": \"Colocalization imaging, co-immunoprecipitation with GTP/GDP-loaded Rab7, knockdown\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — GTP-dependency biochemically validated, replicated by multiple independent labs\",\n      \"pmids\": [\"31636202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PDZD8 interacts with Protrudin at ER-endolysosome (LyLE) MCSs. The SMP domain of PDZD8 mediates lipid extraction from ER membranes. PDZD8 and Protrudin cooperatively promote endosome maturation by mediating ER-LyLE tethering and lipid extraction at MCSs, which is essential for neuronal polarity and integrity.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and siRNA knockdown in HeLa cells and mouse primary neurons, live-cell imaging, endosomal morphology assays, lipid extraction assay\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, cell and primary neuron KD with defined phenotype (endosomal vacuolation, neuronal polarity), multiple orthogonal methods in one study\",\n      \"pmids\": [\"32917905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The SMP domain of PDZD8 binds glycerophospholipids and ceramides both in vivo and in vitro, and can transport lipids between membranes in vitro. PDZD8 acts as a tether at ER-late endosome/lysosome MCSs and its lipid transfer activity is required for late endosome/lysosome positioning and neurite outgrowth.\",\n      \"method\": \"In vitro lipid binding assay, in vitro lipid transfer assay between liposomes, PDZD8 knockdown with neurite outgrowth and organelle positioning readouts, confocal imaging\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstituted lipid transfer assay with domain mutagenesis, corroborated by cellular loss-of-function with defined phenotypes\",\n      \"pmids\": [\"33912962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structure of the human PDZD8 C-terminal coiled-coil (CC) domain in complex with GTP-bound Rab7 was determined. The PDZD8 CC contains one short helix and two helices forming an antiparallel coiled-coil; two Rab7 molecules bind opposite sides of the CC in a 2:1 stoichiometry via their switch I/II and interswitch regions. Isothermal titration calorimetry confirmed the GTP-dependent 2:1 binding.\",\n      \"method\": \"X-ray crystallography, isothermal titration calorimetry (ITC)\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with biochemical validation (ITC), two orthogonal methods in one study\",\n      \"pmids\": [\"34552186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PDZD8 is a substrate of AMPK; AMPK phosphorylates PDZD8 at threonine 527 (T527) under low-glucose conditions. This phosphorylation promotes the interaction of PDZD8 with glutaminase 1 (GLS1), activating GLS1 and promoting glutaminolysis. The PDZD8-T527A mutation abolishes this interaction and dampens glutaminolysis and pro-inflammatory cytokine secretion in macrophages.\",\n      \"method\": \"In vitro kinase assay, mass spectrometry phospho-mapping, co-immunoprecipitation, GLS1 activity assay, T527A phospho-dead mutant, in vivo skeletal muscle and macrophage assays\",\n      \"journal\": \"Cell Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with phospho-site identification, phospho-dead mutant validation, and defined in vivo metabolic phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"38898113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FKBP8, an outer mitochondrial membrane (OMM) protein, is the direct tethering partner of ER-resident PDZD8 at ER-mitochondria contact sites (MERCS). Single-molecule tracking shows PDZD8 diffuses dynamically along the ER membrane with pauses and captures at MERCS. Overexpression of FKBP8 narrows the ER-OMM distance; combined deletion of PDZD8 and FKBP8 is interdependent for MERCS formation. PDZD8 enhances mitochondrial complexity (morphology) in a FKBP8-dependent manner.\",\n      \"method\": \"Unbiased proximity proteomics, CRISPR-Cas9 endogenous tagging, cryo-electron tomography, correlative light-electron microscopy (CLEM), single-molecule tracking, overexpression and double-KO epistasis\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural (cryo-ET), proximity proteomics, CLEM, single-molecule live imaging, and genetic epistasis in one study; multiple orthogonal methods\",\n      \"pmids\": [\"40246839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PDZD8 interacts with HIV-1 Gag protein (identified by yeast two-hybrid, confirmed by co-immunoprecipitation). PDZD8 overexpression promotes initiation of reverse transcription and increases retroviral infection, while knockdown decreases HIV-1 infection. A PDZD8 mutant lacking its predicted coiled-coil domain fails to bind Gag and loses ability to promote HIV-1 infection, mapping the Gag-interacting region.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, overexpression and siRNA knockdown, reverse transcription efficiency assay, domain deletion mutagenesis\",\n      \"journal\": \"Journal of Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP confirms Y2H result, domain mutagenesis maps binding region, functional knockdown assay; single lab\",\n      \"pmids\": [\"20573829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PDZD8 is a critical component of a heat-labile, >100 kDa cytoplasmic factor that slows spontaneous disassembly of HIV-1 capsid-nucleocapsid (CA-NC) complexes in vitro. PDZD8 knockdown accelerates HIV-1 capsid disassembly (uncoating) in infected cells and decreases reverse transcription. The coiled-coil domain is sufficient for capsid binding, but the PDZ domain is additionally required for capsid stabilization and supporting HIV-1 infection.\",\n      \"method\": \"In vitro capsid disassembly assay, PDZD8 knockdown, immunoprecipitation, domain deletion mutagenesis, reverse transcription assay in cells\",\n      \"journal\": \"Journal of Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro capsid assay, cellular KD with defined mechanistic readout, domain mutagenesis; single lab\",\n      \"pmids\": [\"24554657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Stable CRISPR-Cas9 knockout of PDZD8 does not reduce HIV-1 or murine leukemia virus infection efficiency compared to parental cells, indicating PDZD8 is not absolutely required for retroviral infection.\",\n      \"method\": \"CRISPR-Cas9 knockout cell lines, HIV-1 and MLV infection assays\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean genetic KO with defined infection readout, single lab; negative result contradicting prior transient KD studies\",\n      \"pmids\": [\"25771112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PDZD8 interacts with moesin (an ERM family protein) as identified by co-immunoprecipitation. Exogenous expression of PDZD8 or moesin reduces levels of stable (acetylated) microtubules, suggesting PDZD8 functions as part of a cytoskeletal regulatory complex. Overexpression or siRNA knockdown of PDZD8 affects HSV-1 infection levels.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, microtubule acetylation assay, HSV-1 infection assay\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP with functional follow-up (MT stability, viral infection); single lab\",\n      \"pmids\": [\"21549406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Drosophila, reducing pdzd8-mediated ER-mitochondria contacts (MERCs) in neurons slows age-associated locomotor decline and increases lifespan, correlating with increased mitophagy. Conversely, increasing MERCs via a synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation and reduces lifespan. Pdzd8 knockdown also rescues locomotor defects in a fly model of Alzheimer's disease expressing Aβ42, demonstrating that MERC reduction is protective in a neurodegenerative context.\",\n      \"method\": \"Drosophila neuron-specific RNAi knockdown, lifespan assay, locomotor assay, mitophagy reporter, synthetic ER-mitochondria tether overexpression, Aβ42 disease model\",\n      \"journal\": \"Life Science Alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KD and gain-of-function in vivo with defined phenotypes including mitophagy mechanistic link; single lab\",\n      \"pmids\": [\"35831024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PDZD8-deficient mice accumulate cholesteryl esters (CEs) in the brain due to impaired lipophagy (lysosomal degradation of lipid droplets). PDZD8 functions as a Rab7 effector that transfers lipids (cholesterol and phosphatidylserine) between ER and Rab7-positive organelles to promote endolysosome maturation and fusion of CE-containing lipid droplets with lysosomes.\",\n      \"method\": \"PDZD8-KO mouse model, lipidomics (CE quantification), lipid droplet morphology assays, lysosomal fusion assays, Rab7 interaction studies\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with mechanistic phenotype (impaired lipophagy) and proposed lipid transport mechanism; single lab\",\n      \"pmids\": [\"36465123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PDZD8 transports cholesterol to lipid droplets and promotes fusion of lipid droplets with lysosomes (lipophagy). PDZD8-KO mice exhibit impaired lipid droplet degradation and abnormal accumulation of cholesteryl esters in the brain, leading to behavioral abnormalities in emotion, cognition, and adaptation.\",\n      \"method\": \"PDZD8-KO mouse model, cholesterol transport assay, lipid droplet-lysosome fusion assay, behavioral battery (open field, fear conditioning, etc.)\",\n      \"journal\": \"Molecular Brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with mechanistic cellular assay (LD-lysosome fusion) and defined behavioral phenotype; single lab\",\n      \"pmids\": [\"36658656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PDZD8 (LYVAC) is a general mediator of lysosomal vacuolation. Upon lysosomal osmotic stress, diverse vacuolation inducers trigger PDZD8 recruitment to lysosomes through multivalent interactions. Stress-induced lysosomal lipid signaling (phosphatidylserine and cholesterol) activates PDZD8's SMP lipid transfer domain, driving directional ER-to-lysosome lipid movement that causes osmotic membrane expansion of lysosomes.\",\n      \"method\": \"Human cell line imaging, PDZD8 KO and rescue, live-cell imaging, lipid sensing assays, domain mutagenesis of lipid-binding residues\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro lipid sensing, domain mutagenesis, KO rescue, mechanistic pathway reconstitution; multiple orthogonal methods in one study\",\n      \"pmids\": [\"40839735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PDZD8 promotes autophagy at ER-late endosome/lysosome MCSs by promoting lysosome maturation and accelerating autophagic flux. PDZD8 is required for activity-dependent synaptic bouton formation in Drosophila neurons, and is sufficient to drive excess bouton formation through an autophagy-dependent mechanism. SMP domain mutational analysis suggests lipid transfer from ER to late endosomes/lysosomes is the mechanistic basis for lysosome maturation.\",\n      \"method\": \"In vivo CRISPR screen in Drosophila, genetic loss-of-function and gain-of-function, autophagic flux assays, lysosome maturation assays, SMP domain mutagenesis, synaptic bouton counting\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo CRISPR KO with defined cellular (autophagy/lysosome) and organismal (synapse) phenotype, domain mutagenesis; single lab\",\n      \"pmids\": [\"40156832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PDZD8 regulates endolysosomal maturation and acidification, which is required for proper translocation of TLR9 to endolysosomes and downstream NF-κB activation in kidney proximal tubular cells. PDZD8 KO mice show reduced cisplatin-induced AKI severity and reduced NF-κB pathway activation. PDZD8 knockdown did not alter mitochondrial morphology or cytosolic leakage of mitochondrial DNA.\",\n      \"method\": \"Pdzd8 KO mouse, in vitro PDZD8 knockdown in human proximal tubular cells, lysosomal acidification assay, TLR9 localization by immunofluorescence, NF-κB reporter, cisplatin AKI model\",\n      \"journal\": \"American Journal of Physiology - Renal Physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse plus cell-based KD with mechanistic pathway placement (TLR9-NF-κB) and defined negative result for mitochondrial involvement; single lab\",\n      \"pmids\": [\"40897465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PDZD8 augments ER-mitochondria contact (MAM) formation in pancreatic β-cells. PDZD8 knockdown shortens MAM perimeter, suppresses MAM-related proteins (IP3R1, GRP75, VDAC1), inhibits VDAC1-IP3R1 interaction, alleviates mitochondrial Ca2+ overload, and decreases Cyclophilin D (CypD) expression, thereby reducing β-cell apoptosis. Cyclophilin D overexpression rescues β-cell death, placing PDZD8 upstream of CypD in the apoptotic pathway.\",\n      \"method\": \"PDZD8 knockdown in INS-1 cells and HFD mouse model, proximity ligation assay for VDAC1-IP3R1 interaction, Ca2+ imaging, mitochondrial membrane potential assay, Western blot, epistasis with CypD overexpression\",\n      \"journal\": \"Diabetes & Metabolism Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway placement via epistasis (CypD rescue), PLA and Ca2+ imaging in cells and in vivo model; single lab\",\n      \"pmids\": [\"39069376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PDZD8 dysregulation in RVLM neurons activates Ca2+-Calpain-2 (CAPN2) signaling: PDZD8 deficiency elevates cytoplasmic Ca2+ levels, which upregulates CAPN2, leading to ER stress, mitochondrial dysfunction, and neuronal apoptosis. CAPN2 inhibition rescues PDZD8-deficiency-induced ER stress and mitochondrial dysfunction. In vivo, PDZD8 upregulation in RVLM suppresses neuronal hyperexcitation and reduces blood pressure in stress-induced hypertension rats.\",\n      \"method\": \"PDZD8 siRNA knockdown in N2a cells, AAV2-mediated PDZD8 overexpression in rat RVLM, CAPN2 inhibitor treatment, Western blot, flow cytometry, immunofluorescence, RSNA/BP measurement\",\n      \"journal\": \"Molecular Neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic epistasis (CAPN2 inhibition rescuing PDZD8 KD phenotype), in vivo AAV rescue; single lab\",\n      \"pmids\": [\"40418411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM55B is identified as a lysosomal PDZD8-associated protein. TMEM55B depletion reduces lysosomal acidification, impairs lipid droplet turnover, attenuates lysosomal Ca2+ release and reuptake, and diminishes ER Ca2+ responses, consistent with impaired CICR at ER-lysosome MCSs. Mitochondrial Ca2+ dynamics were unaffected, suggesting specificity to the ER-lysosome axis.\",\n      \"method\": \"Co-immunoprecipitation/proximity assay for PDZD8-TMEM55B interaction, lysosomal pH assay, lipid droplet assay, Ca2+ imaging, siRNA knockdown\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, association demonstrated but mechanistic link to PDZD8 is indirect\",\n      \"pmids\": [\"bio_10.1101_2025.10.21.683636\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Live-cell imaging using a novel ddFP-based MERCS sensor (MERCdRED) demonstrated that large MERCS are more stable than smaller ones. Nutrient deprivation reduces MERCS area in a PDZD8-dependent manner, establishing PDZD8 as required for nutrient-regulated MERCS dynamics.\",\n      \"method\": \"Novel MERCS fluorescent sensor (ddFP-based), CLEM validation, live-cell imaging, PDZD8 KO under nutrient deprivation\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — preprint, novel tool validated by CLEM, single lab, single method for the nutrient-PDZD8 finding\",\n      \"pmids\": [\"bio_10.1101_2025.04.17.649323\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PDZD8 is an ER-anchored transmembrane protein with an SMP (lipid transfer) domain that functions as a tethering and lipid transport protein at multiple membrane contact sites: it bridges ER to mitochondria (via FKBP8 on the OMM) to regulate Ca2+ transfer and mitochondrial morphology, and bridges ER to late endosomes/lysosomes (via GTP-Rab7 and Protrudin) to mediate directional lipid transfer that drives endolysosomal maturation, lipophagy, lysosomal vacuolation, autophagic flux, and TLR9-NF-κB signaling; additionally, PDZD8 is phosphorylated by AMPK at T527 to promote glutaminolysis through GLS1 activation, and its coiled-coil domain interacts with the HIV-1 capsid to stabilize it during uncoating.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PDZD8 is an ER-anchored transmembrane protein that uses an SMP lipid-transfer domain to tether the endoplasmic reticulum to multiple partner organelles and to move lipids across the resulting membrane contact sites [#0, #4]. At ER–mitochondria contacts it is necessary for contact formation and for mitochondrial Ca2+ uptake following ER Ca2+ release, with the outer mitochondrial membrane protein FKBP8 serving as its direct tethering partner that sets ER–OMM distance and shapes mitochondrial morphology [#0, #7]. In parallel, PDZD8 localizes to ER–late endosome/lysosome contacts through a GTP-dependent interaction with Rab7 and an interaction with Protrudin, acting as a Rab7 effector whose SMP domain extracts and transfers glycerophospholipids, ceramides, cholesterol, and phosphatidylserine to drive endolysosomal maturation, lysosome positioning, lipophagy, and autophagic flux [#2, #3, #4, #5, #13]. Stress-induced lysosomal lipid signaling activates this lipid-transfer activity to produce osmotic lysosomal vacuolation, and the same maturation function supports endolysosomal acidification required for TLR9 trafficking and downstream NF-κB activation [#15, #17]. Beyond its tethering roles, PDZD8 is a substrate of AMPK, which phosphorylates it at threonine 527 under low glucose to promote its interaction with and activation of glutaminase 1 (GLS1), linking PDZD8 to glutaminolysis and macrophage cytokine output [#6]. Through these contact-site activities PDZD8 governs neuronal Ca2+ dynamics, neurite outgrowth, synaptic bouton formation, and brain lipid homeostasis [#0, #4, #14, #16].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established PDZD8 as the first mammalian ER protein required to build ER–mitochondria contacts and to couple ER Ca2+ release to mitochondrial Ca2+ uptake, defining its founding tethering function.\",\n      \"evidence\": \"Live-cell imaging, fractionation, siRNA knockdown with Ca2+ imaging, and SMP-domain complementation of yeast Mmm1 in mammalian cells\",\n      \"pmids\": [\"29097544\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the mitochondrial tethering partner\", \"Did not reconstitute lipid transfer by the SMP domain\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed PDZD8 also concentrates at ER–late endosome/lysosome contacts via a GTP-dependent interaction with Rab7, extending its role beyond ER–mitochondria contacts.\",\n      \"evidence\": \"Colocalization imaging and Co-IP with GTP/GDP-loaded Rab7 plus knockdown\",\n      \"pmids\": [\"31636202\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not demonstrate lipid transfer at these contacts\", \"Functional consequence of the Rab7 interaction unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified Protrudin as a second ER-side partner and revealed PDZD8 as a shared component of ER–mitochondria and ER–late endosome contacts that can organize three-way contacts and drive endosome maturation.\",\n      \"evidence\": \"Co-IP, BioID proximity proteomics, CRISPR knockouts, domain mapping, and lipid-extraction assays in HeLa cells and primary neurons\",\n      \"pmids\": [\"32686675\", \"32917905\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct lipid species transported not defined in these studies\", \"How the three-way contact is coordinated mechanistically unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated biochemically that the SMP domain binds and transfers glycerophospholipids and ceramides and that this lipid-transfer activity drives organelle positioning and neurite outgrowth, and solved the structural basis of the Rab7 interaction.\",\n      \"evidence\": \"In vitro lipid binding and liposome transfer assays with domain mutants, plus X-ray crystallography and ITC of the coiled-coil–Rab7 complex (2:1 GTP-dependent)\",\n      \"pmids\": [\"33912962\", \"34552186\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Directionality of lipid flux in cells not established\", \"Full-length protein structure and SMP domain orientation at the contact unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected PDZD8-dependent contacts to organismal physiology and disease, showing in vivo that modulating ER–mitochondria contacts alters lifespan, mitophagy, and neurodegenerative phenotypes, and that PDZD8 loss causes brain cholesteryl-ester accumulation via impaired lipophagy.\",\n      \"evidence\": \"Drosophila neuron-specific RNAi with lifespan/locomotor/mitophagy and Aβ42 readouts; PDZD8-KO mouse lipidomics and lysosomal fusion assays\",\n      \"pmids\": [\"35831024\", \"36465123\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal lipid species at the lysosome contact not fully isolated in vivo\", \"Single-lab phenotypes not cross-validated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked PDZD8-mediated cholesterol transport and lipid-droplet–lysosome fusion to behavioral outcomes, establishing a brain lipid-homeostasis role.\",\n      \"evidence\": \"PDZD8-KO mice with cholesterol transport assays, lipid droplet–lysosome fusion assays, and behavioral battery\",\n      \"pmids\": [\"36658656\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-type-specific contribution to behavior unresolved\", \"Direct lipid-transfer step in vivo not reconstituted\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed an unexpected signaling-metabolic role: AMPK phosphorylates PDZD8 at T527 to activate GLS1 and glutaminolysis, coupling PDZD8 to nutrient stress and macrophage inflammation independent of its tethering function.\",\n      \"evidence\": \"In vitro kinase assay, MS phospho-mapping, Co-IP, GLS1 activity assay, T527A phospho-dead mutant, and in vivo muscle/macrophage assays\",\n      \"pmids\": [\"38898113\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether membrane contact-site localization is required for GLS1 activation is unclear\", \"Structural basis of the PDZD8–GLS1 interaction undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined the direct ER–mitochondria tethering partner and the lipid-driven mechanism of lysosomal vacuolation, while extending PDZD8 function to endolysosomal acidification-dependent TLR9–NF-κB signaling and disease models.\",\n      \"evidence\": \"Cryo-ET, CLEM, proximity proteomics, single-molecule tracking and double-KO epistasis identifying FKBP8; SMP lipid-sensing/mutagenesis for lysosomal vacuolation; KO mouse and proximal tubular cell assays for TLR9–NF-κB; β-cell and RVLM neuron Ca2+ pathway studies\",\n      \"pmids\": [\"40246839\", \"40839735\", \"40897465\", \"40156832\", \"39069376\", \"40418411\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the same SMP domain achieves directional ER-to-lysosome versus ER-to-mitochondria transport is unresolved\", \"Reconciliation of pro-apoptotic Ca2+ overload roles with protective neuronal roles across tissues incomplete\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how PDZD8's lipid-transfer activity is directionally and selectively regulated across its distinct contact sites, and how its tethering, lipid-transport, and AMPK–GLS1 signaling functions are integrated within a single protein.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length structural model integrating SMP, coiled-coil, and PDZ domains\", \"Regulation of partner choice (FKBP8 vs Rab7/Protrudin vs GLS1) not defined\", \"Conflicting HIV-1 requirement (KD vs clean KO) unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [4, 15]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [3, 4, 13]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 7, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [2, 3, 15]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [13, 16]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [6, 13, 14]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"FKBP8\", \"RAB7A\", \"ZFYVE27\", \"GLS\", \"MSN\", \"TMEM55B\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}