{"gene":"SEC22B","run_date":"2026-06-10T07:46:30","timeline":{"discoveries":[{"year":2001,"finding":"Crystal structure of the 130-amino acid N-terminal (longin) domain of mouse Sec22b was solved at 2.4 Å resolution, revealing a mixed α/β fold resembling a circular permutation of profilin and GAF/PAS modules. This domain is structurally distinct from syntaxin 1A's N-terminal domain and, unlike syntaxin 1A, does not affect the rate of SNARE assembly in vitro.","method":"X-ray crystallography (2.4 Å), in vitro SNARE assembly kinetics assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution crystal structure combined with in vitro functional assay in the same study","pmids":["11309394"],"is_preprint":false},{"year":1999,"finding":"Endogenous Sec22b/ERS-24 localizes to the pre-Golgi intermediate compartment (IC), and antibodies against Sec22b inhibit ER-to-Golgi transport of VSVG prior to the EGTA-sensitive docking/fusion step, causing VSVG accumulation in pre-Golgi vesicular intermediates.","method":"Immunofluorescence co-labeling, semi-intact cell ER-Golgi transport reconstitution assay with inhibitory antibodies, EGTA block-release","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstituted transport assay with antibody inhibition combined with morphological localization and multiple pharmacological conditions","pmids":["9950687"],"is_preprint":false},{"year":2004,"finding":"Sec22b localizes on ER-derived vesicles that are recruited to the Legionella-containing vacuole (LCV); Sec22b is delivered to the LCV membrane and is functionally required for biogenesis of the replicative organelle supporting Legionella intracellular growth. Rab1 recruits ER-derived vesicles to the LCV upstream of Sec22b-dependent fusion.","method":"Immunofluorescence/electron microscopy localization, genetic inhibition (dominant-negative constructs), intracellular growth assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (morphological, genetic loss-of-function, bacterial growth readout); replicated in subsequent studies","pmids":["15117975"],"is_preprint":false},{"year":2008,"finding":"Sec22b (R-SNARE) associates with ER-localized syntaxin 18 (Qa-SNARE), and this binary interaction induces increased α-helicity in both SNARE motifs, creating high-affinity binding sites for BNIP1 (Qb) and p31/Use1 (Qc), thereby driving sequential Q-SNARE assembly. This R-SNARE-dependent Q-SNARE assembly mechanism is distinct from that of non-ER SNAREs.","method":"Pulldown assays, CD spectroscopy measuring α-helicity changes upon SNARE motif association","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro biophysical assay (CD spectroscopy) plus pulldown; single lab, two complementary methods","pmids":["17979832"],"is_preprint":false},{"year":2009,"finding":"Overexpression of Sec22b in J774 macrophages nearly abolishes phagocytosis without affecting Fc receptor surface expression, whereas suppression of endogenous Sec22b increases phagocytic capacity. Domain analysis shows the R-SNARE motif (responsible for SNARE complex formation with syntaxin 18 and/or D12) mediates this inhibition, identifying Sec22b as a negative regulator of phagocytosis likely by titrating free syntaxin 18/D12.","method":"Stable overexpression, shRNA knockdown, domain deletion/mutagenesis analysis, phagocytosis assay, flow cytometry","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with domain mapping; single lab, multiple orthogonal approaches","pmids":["19710423"],"is_preprint":false},{"year":2010,"finding":"During virulent L. pneumophila infection, Sec22b (ER v-SNARE) undergoes non-canonical pairing with plasma membrane syntaxins (Stx2, Stx3, Stx4) and SNAP23 on the LCV. Depletion of plasma membrane syntaxins delays calnexin acquisition and retains Rab1 on phagosomes. Addition of α-SNAP and NSF dissociates these non-canonical SNARE complexes, demonstrating they are functional.","method":"RNAi depletion, co-immunoprecipitation, NSF/α-SNAP disassembly assay, immunofluorescence","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional co-IP validated by NSF/α-SNAP disassembly assay, multiple syntaxins tested, replicated conceptually by later studies","pmids":["20163564"],"is_preprint":false},{"year":2011,"finding":"Sec22b localizes to the ERGIC and pairs with plasma membrane SNARE syntaxin 4 present on phagosomes. Depletion of Sec22b in dendritic cells inhibits recruitment of ER-resident proteins to phagosomes (and to T. gondii-containing vacuoles), impairs antigen export to the cytosol, accelerates lysosomal recruitment, and blocks MHC class I cross-presentation after phagocytosis or endocytosis of antigen.","method":"siRNA knockdown, immunofluorescence, co-immunoprecipitation (Sec22b–syntaxin 4 pairing), antigen cross-presentation assay, phagosome maturation assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, loss-of-function, trafficking, functional T cell assay) in a high-profile journal; widely cited and replicated","pmids":["22153078"],"is_preprint":false},{"year":2017,"finding":"DC-specific conditional knockout of Sec22b in mice impairs cross-presentation ex vivo and cross-priming of CD8+ T cells in vivo, and abolishes antitumor immune responses and response to anti-PD-1 therapy, supporting Sec22b-dependent ER-phagosome traffic as required for cross-presentation.","method":"Conditional Cre-lox DC-specific knockout mice, ex vivo cross-presentation assay, in vivo tumor challenge, anti-PD-1 treatment","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean conditional KO with defined cellular and in vivo phenotypes; contradicted by another conditional KO study (PMID:28658614), reducing confidence","pmids":["28663435"],"is_preprint":false},{"year":2017,"finding":"A separate DC-specific Sec22b knockout mouse (CD11c-Cre Sec22b fl/fl) shows that SEC22B-deficient DCs can efficiently cross-present antigen both in vivo and in vitro. shRNA-mediated Sec22b silencing reduces cross-presentation even in SEC22B-knockout BMDCs, indicating the shRNA effect is due to off-target activity rather than Sec22b loss.","method":"Conditional Cre-lox DC-specific knockout mice, in vitro/in vivo cross-presentation assays, shRNA in KO cells, RNA-seq of shRNA-treated KO BMDCs","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rigorous genetic controls (shRNA in KO background) with RNA-seq; directly contradicts PMID:28663435, lowering overall field confidence","pmids":["28658614"],"is_preprint":false},{"year":2020,"finding":"SEC22B interacts with NBEAL2 (gray platelet syndrome protein) via a region spanning NBEAL2 amino acids 1798–1903; GPS-associated missense variants E1833K and R1839C in NBEAL2 abolish this interaction. NBEAL2 can simultaneously bind SEC22B and P-selectin. CRISPR/Cas9 knockout of SEC22B in imMKCL cells decreases NBEAL2 protein and blocks α-granule biogenesis, demonstrating SEC22B is required for megakaryocyte α-granule production.","method":"Co-immunoprecipitation (tagged and endogenous proteins), CRISPR/Cas9 knockout, immunofluorescence microscopy, α-granule content assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with endogenous proteins, domain mapping with disease-variant validation, and CRISPR KO phenotype; multiple orthogonal methods in one study","pmids":["32384141"],"is_preprint":false},{"year":2020,"finding":"Sec22b interacts with members of the extended synaptotagmin (E-Syt) family via the longin domain of Sec22b, stabilizing Sec22b–syntaxin 1 (Stx1) non-fusogenic SNARE complexes at ER-PM contacts. Overexpression of wild-type E-Syt2 (but not lipid-transfer-deficient or ER-anchoring-deficient mutants) increases axonal filopodia formation and neurite ramification; this effect is blocked by clostridial neurotoxin cleaving Stx1, longin domain expression, or a Sec22b mutant with an extended SNARE-TM linker.","method":"Co-immunoprecipitation, overexpression/dominant-negative constructs, neurite morphology assay, clostridial neurotoxin inhibition","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping plus functional rescue/inhibition experiments; single lab, multiple orthogonal approaches","pmids":["32843578"],"is_preprint":false},{"year":2021,"finding":"Sec22b depletion in endothelial cells causes loss of elongated Weibel-Palade body morphology, disintegration of Golgi and dilation of rough ER, reduced proteolytic processing of VWF, accumulation of VWF in dilated rER, and reduced VWF secretion, demonstrating that Sec22b-containing SNARE complexes governing ER-to-Golgi anterograde transport determine WPB length and VWF hemostatic activity.","method":"shRNA-mediated knockdown, immunofluorescence, electron microscopy, VWF secretion assay","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean loss-of-function with multiple morphological and biochemical readouts; single lab","pmids":["32336681"],"is_preprint":false},{"year":2021,"finding":"Sec22b co-localizes with inducible NO synthase (iNOS) at ERGIC/Golgi compartments and phagosomes. siRNA silencing of Sec22b in bone marrow-derived dendritic cells abrogates NO and cytokine production at both protein and mRNA levels and reduces nuclear translocation of NF-κB. Sec22b was found to co-occur with NF-κB in both cytoplasm and nucleus, implicating Sec22b in NF-κB shuttling.","method":"Immunofluorescence co-localization, siRNA knockdown, NO/cytokine measurement, NF-κB nuclear translocation assay, pharmacological secretory pathway blockade","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — loss-of-function with multiple functional readouts and co-localization data; single lab, mechanistic link to NF-κB shuttling is correlative","pmids":["34580108"],"is_preprint":false},{"year":2022,"finding":"Sec22b and Ykt6 act as opposing regulators of autophagosome axonal retrograde transport in neurons. Ischemia-reperfusion increases Sec22b and decreases Ykt6 in neurons. Sec22b knockdown and Ykt6 overexpression rescue axonal autophagosome retrograde transport, restore autophagic flux, and reduce infarct size in a murine MCAO/R model in an autophagy-dependent manner.","method":"Knockdown/overexpression in primary neurons and in vivo murine MCAO/R model, live autophagosome tracking, autophagic flux assays, Atg7 conditional KO epistasis","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo and in vitro loss/gain-of-function with epistasis (Atg7 KO); single lab","pmids":["35654605"],"is_preprint":false},{"year":2022,"finding":"In liver cancer cells, cytosolic proteins associate with E-Syt1 on the ER, then localize inside SEC22B+ vesicles. SEC22B on these vesicles tethers to the plasma membrane via Q-SNAREs (SNAP23, SNX3, SNX4) for secretion of cytosolic cargo, identifying SEC22B as a mediator of unconventional cytosolic protein secretion at ER-PM contact sites.","method":"Co-immunoprecipitation, subcellular fractionation, immunofluorescence, proximity ligation, inhibition of PKCδ–E-Syt1 interaction","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple Co-IP and localization methods; single lab, functional link established by inhibition of cargo–E-Syt1 interaction","pmids":["36044553"],"is_preprint":false},{"year":2023,"finding":"Sec22b tethers ER-phagosome membrane contact sites (MCS) independently of STIM1. Sec22b knockdown increases phagosomal calcium signaling, accelerates phagolysosome fusion and antigen degradation, and alters phagosomal phospholipids (PI(3)P, PS, PI(4)P). Sec22b co-precipitates with the PS/PI(4)P exchange protein ORP8; wild-type but not catalytic-mutant ORP8 rescues phagosomal PI(4)P levels and reduces antigen degradation. The MCS-disrupting Sec22b-P33 mutant fails to rescue, identifying tethering as the mechanistic basis.","method":"siRNA knockdown, co-immunoprecipitation (Sec22b–ORP8), artificial tether (MAPPER) rescue, Sec22b-P33 MCS-disrupting mutant, phagosomal lipid quantification, calcium imaging, phagolysosome fusion assay","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, mutant rescue, artificial tether, lipid profiling, calcium imaging) establishing mechanistic pathway; single lab but comprehensive","pmids":["37794132"],"is_preprint":false},{"year":2023,"finding":"The L. pneumophila effector Lug15 is a novel E3 ubiquitin ligase that ubiquitinates host Sec22b, mediates Sec22b recruitment to the LCV, and promotes non-canonical SNARE pairing of Sec22b with plasma membrane syntaxin 3.","method":"In vitro ubiquitination assay, co-immunoprecipitation, intracellular bacterial growth assay","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzymatic assay plus Co-IP and functional readout; single lab","pmids":["37882795"],"is_preprint":false},{"year":2023,"finding":"Sec22b is a critical non-redundant regulator of plasma cell maintenance: Sec22b-deficient mice show near-absence of plasma cells and dramatically reduced serum antibody titers. Mechanistically, Sec22b contributes to efficient antibody secretion and regulates transcriptional identity of plasma cells as well as morphology of the ER and mitochondria.","method":"Conditional knockout mice, flow cytometry, ELISA for antibody titers, immunofluorescence of organelle morphology, transcriptional profiling","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with multiple cellular and functional readouts; single lab","pmids":["36595686"],"is_preprint":false},{"year":2024,"finding":"Glucagon signaling phosphorylates SEC22B at serine 137 in the liver; hepatocyte-specific loss- and gain-of-function experiments show SEC22B is a key regulator of hepatic glycogen, lipid, and amino acid metabolism. SEC22B-S137 phosphorylation affects several protein binding partners and mediates glucagon's metabolic actions.","method":"In situ time-resolved liver phosphoproteomics, hepatocyte-specific KO and overexpression mouse models, co-immunoprecipitation of SEC22B binding partners with phospho-site variants","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics discovery combined with conditional KO/OE and Co-IP binding partner analysis; single lab, multiple orthogonal approaches","pmids":["39333498"],"is_preprint":false},{"year":2025,"finding":"The L. pneumophila effector SdeA catalyzes conjugation of phosphoribosyl-linked ubiquitin specifically to serine 137 of Sec22b; subsequently the canonical ubiquitin system adds polyubiquitin chains. This multimodal ubiquitination of Sec22b facilitates non-canonical SNARE pairing (Sec22b with Stx3) during early infection. The Legionella deubiquitinase LotB cleaves the polyubiquitin chains from Sec22b, causing Sec22b dissociation from syntaxin 3.","method":"In vitro ubiquitination assay, site-directed mutagenesis (Sec22b-S137), co-immunoprecipitation, deubiquitinase assay (LotB)","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzymatic assay with site-directed mutagenesis and Co-IP; single lab, multiple complementary experiments","pmids":["41669755"],"is_preprint":false},{"year":2025,"finding":"SEC22B specifically interacts with wild-type ClC-5 chloride/proton antiporter but not with pathogenic ClC-5 mutants in renal proximal tubule cells. SEC22B deletion impairs ClC-5 trafficking, causing its retention at the Golgi and endosomes, identifying SEC22B as a trafficking factor for ClC-5 in the secretory pathway.","method":"Interactome analysis (Co-IP), CRISPR/Cas9 or siRNA knockdown, immunofluorescence of ClC-5 localization","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, Co-IP and localization without in vitro reconstitution","pmids":["bio_10.1101_2025.11.03.686312"],"is_preprint":true},{"year":2025,"finding":"SEC22B (R-SNARE), together with Q-SNAREs STX4 and SNAP23, mediates fusion of UBB+1-containing autophagosomes with the plasma membrane during secretory autophagy. Disruption of SEC22B impairs autophagosome-PM fusion and reduces UBB+1 secretion without affecting its intracellular turnover.","method":"siRNA knockdown, secretion assay, autophagosome-PM fusion assay","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, knockdown with secretion readout; no in vitro reconstitution","pmids":["bio_10.1101_2024.12.31.630908"],"is_preprint":true},{"year":2025,"finding":"In axons, the NRZ–SEC22B tethering complex links axonal ER exit (ERES-dependent, Golgi-independent) to plasma membrane delivery of locally translated transmembrane proteins, dependent on ER-PM contacts. This coupling supports axon growth and bouton assembly.","method":"Live imaging in neurons, overexpression/dominant-negative constructs, ERES marker colocalization, axon growth and bouton assays","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab; mechanism inferred from colocalization and dominant-negative without reconstitution","pmids":["bio_10.1101_2025.09.09.674816"],"is_preprint":true}],"current_model":"SEC22B is an R-SNARE resident in the ERGIC/ER that mediates anterograde ER-to-Golgi membrane fusion via canonical complexes with syntaxin 18, BNIP1, and p31/Use1; its longin domain (structurally a profilin/GAF-like fold) enables non-fusogenic pairing with plasma membrane syntaxins (e.g., Stx1, Stx4) at ER-PM contact sites to support neurite growth and unconventional secretion; in phagocytes it tethers ER-phagosome contact sites through ORP8 to control phagosomal phospholipid composition and maturation; it is ubiquitinated on S137 by bacterial effectors (Lug15/SdeA) to drive non-canonical SNARE pairing during Legionella infection; and its S137 phosphorylation by glucagon signaling in hepatocytes regulates glycogen, lipid, and amino acid metabolism."},"narrative":{"mechanistic_narrative":"SEC22B is an R-SNARE of the ER/ERGIC that mediates anterograde ER-to-Golgi membrane transport and, through non-fusogenic SNARE pairings, participates in membrane contact-site biology, unconventional secretion, and immune membrane traffic [PMID:9950687, PMID:22153078]. Its N-terminal longin domain adopts a profilin/GAF-like α/β fold structurally distinct from syntaxin 1A and dispensable for SNARE assembly kinetics in vitro [PMID:11309394]. In canonical ER fusion, SEC22B binding to the Qa-SNARE syntaxin 18 increases α-helicity in both SNARE motifs to nucleate sequential recruitment of the Qb/Qc-SNAREs BNIP1 and p31/Use1 [PMID:17979832], and loss of SEC22B-dependent ER-to-Golgi transport disorganizes the Golgi, dilates the rough ER, and impairs VWF processing and secretion [PMID:32336681]. Beyond classical fusion, the longin domain pairs SEC22B with plasma-membrane syntaxins (Stx1, Stx4) at ER-PM contact sites, stabilized by extended synaptotagmins, to drive neurite outgrowth [PMID:32843578] and to support unconventional secretion of cytosolic cargo loaded into SEC22B+ vesicles that tether to the PM via SNAP23-containing Q-SNARE complexes [PMID:36044553]. In phagocytes, SEC22B pairs with phagosomal syntaxin 4 to deliver ER components to maturing phagosomes, sustain MHC class I cross-presentation, and tether ER-phagosome contact sites independently of STIM1; this tethering recruits the lipid-exchange protein ORP8 to set phagosomal PI(4)P, PS and PI(3)P content and restrain phagolysosome fusion and antigen degradation [PMID:22153078, PMID:37794132]. SEC22B is also required for megakaryocyte α-granule biogenesis through its interaction with NBEAL2 [PMID:32384141] and for plasma cell maintenance and efficient antibody secretion [PMID:36595686]. Intracellular Legionella exploits SEC22B by recruiting ER-derived vesicles to the Legionella-containing vacuole and driving non-canonical SEC22B pairing with plasma-membrane syntaxins; the bacterial effectors Lug15 and SdeA ubiquitinate SEC22B—SdeA installing phosphoribosyl-ubiquitin at serine 137 followed by canonical polyubiquitination—to promote these non-canonical complexes [PMID:15117975, PMID:20163564, PMID:37882795, PMID:41669755]. The same residue is targeted physiologically: glucagon signaling phosphorylates SEC22B at S137 in hepatocytes to control glycogen, lipid, and amino acid metabolism [PMID:39333498].","teleology":[{"year":1999,"claim":"Established SEC22B as a functional component of early secretory traffic by showing it acts at the pre-Golgi intermediate compartment before the docking/fusion step of ER-to-Golgi transport.","evidence":"Immunofluorescence localization and antibody-inhibited semi-intact cell ER-Golgi transport reconstitution","pmids":["9950687"],"confidence":"High","gaps":["Did not define the cognate Q-SNARE partners","Mechanism of vesicle docking versus fusion not resolved"]},{"year":2001,"claim":"Resolved the architecture of the SEC22B N-terminal domain, revealing a longin (profilin/GAF-like) fold that is structurally distinct from syntaxin 1A and does not govern SNARE assembly rate, reframing the domain's role away from kinetic regulation of fusion.","evidence":"X-ray crystallography at 2.4 Å plus in vitro SNARE assembly kinetics","pmids":["11309394"],"confidence":"High","gaps":["Functional role of the longin fold left undefined","No partner identified for the domain at this stage"]},{"year":2008,"claim":"Defined the molecular logic of canonical ER SNARE assembly, showing SEC22B binding to syntaxin 18 induces α-helicity that creates high-affinity sites for BNIP1 and p31/Use1.","evidence":"Pulldown assays and CD spectroscopy of SNARE motif α-helicity changes","pmids":["17979832"],"confidence":"Medium","gaps":["In vitro biophysics not validated by reconstituted fusion","Single-lab biophysical analysis"]},{"year":2009,"claim":"Showed SEC22B abundance negatively tunes phagocytosis through its R-SNARE motif, implicating it in regulating Q-SNARE availability in phagocytes.","evidence":"Overexpression, shRNA knockdown, and domain mapping with phagocytosis assays in macrophages","pmids":["19710423"],"confidence":"Medium","gaps":["Titration model of free syntaxin 18/D12 not directly demonstrated","Single cell-line context"]},{"year":2011,"claim":"Connected SEC22B to immune membrane traffic by showing it pairs with phagosomal syntaxin 4 to deliver ER components to phagosomes and enable MHC class I cross-presentation.","evidence":"siRNA knockdown, Co-IP of SEC22B-syntaxin 4, phagosome maturation and cross-presentation assays in dendritic cells","pmids":["22153078"],"confidence":"High","gaps":["Whether SEC22B mediates fusion versus non-fusogenic tethering at phagosomes unresolved here","Did not address contact-site lipid transfer"]},{"year":2017,"claim":"Tested the genetic requirement for SEC22B in cross-presentation in vivo, yielding conflicting conclusions on whether SEC22B is essential for DC cross-priming.","evidence":"Two independent DC-specific conditional knockout mouse lines with ex vivo/in vivo cross-presentation assays; one used shRNA-in-KO controls","pmids":["28663435","28658614"],"confidence":"Medium","gaps":["The two conditional KO studies reach opposite conclusions","shRNA off-target effects confound earlier knockdown data"]},{"year":2020,"claim":"Extended SEC22B function beyond bulk traffic, establishing it as a longin-domain-mediated organizer at ER-PM contacts (with E-Syts and Stx1 for neurite growth) and as an essential factor for megakaryocyte α-granule biogenesis via NBEAL2.","evidence":"Co-IP with domain and disease-variant mapping, CRISPR knockout, and neurite/α-granule morphology assays","pmids":["32843578","32384141"],"confidence":"High","gaps":["Whether the NBEAL2 and E-Syt pathways intersect mechanistically is unknown","Stoichiometry of non-fusogenic SEC22B-Stx1 complexes not defined"]},{"year":2021,"claim":"Linked SEC22B-dependent anterograde transport to specialized secretory organelle biology (Weibel-Palade body morphology and VWF processing) and broadened its immune signaling role to NO/cytokine production and NF-κB shuttling.","evidence":"shRNA knockdown with EM, VWF secretion assays in endothelial cells, and siRNA with NO/cytokine and NF-κB translocation readouts in dendritic cells","pmids":["32336681","34580108"],"confidence":"Medium","gaps":["NF-κB shuttling link is correlative","Direct SNARE partners at WPBs not mapped"]},{"year":2022,"claim":"Identified SEC22B as a regulator of autophagosome retrograde transport and as a mediator of unconventional cytosolic protein secretion through SEC22B+ vesicles tethering to the PM at ER-PM contacts.","evidence":"Knockdown/overexpression with Atg7 epistasis in an MCAO/R model; Co-IP, fractionation, proximity ligation and cargo-E-Syt1 inhibition in liver cancer cells","pmids":["35654605","36044553"],"confidence":"Medium","gaps":["Opposing SEC22B/Ykt6 regulation mechanism not biochemically defined","Cargo selection into SEC22B+ vesicles unresolved"]},{"year":2023,"claim":"Defined the mechanistic basis of SEC22B at ER-phagosome contacts as a tether that recruits ORP8 to set phagosomal phospholipid composition, and established non-redundant requirements in plasma cell maintenance and antibody secretion.","evidence":"siRNA, Co-IP with ORP8, MAPPER artificial-tether and P33 MCS-disrupting mutant rescue, lipid profiling and calcium imaging; conditional KO mice with ELISA and organelle morphology","pmids":["37794132","36595686"],"confidence":"High","gaps":["How tethering is coordinated with SNARE pairing at the same membrane is unclear","Transcriptional identity link in plasma cells is correlative"]},{"year":2023,"claim":"Revealed that Legionella effectors hijack SEC22B by ubiquitinating it (via the E3 ligase Lug15) to drive recruitment to the LCV and non-canonical pairing with plasma-membrane syntaxin 3.","evidence":"In vitro ubiquitination, Co-IP, and intracellular bacterial growth assays","pmids":["37882795"],"confidence":"Medium","gaps":["Ubiquitination site not defined in this study","Host enzymatic background not excluded"]},{"year":2024,"claim":"Identified SEC22B serine 137 as a physiological glucagon-regulated phosphosite controlling hepatic glycogen, lipid, and amino acid metabolism, expanding SEC22B's role into metabolic signaling.","evidence":"Time-resolved liver phosphoproteomics, hepatocyte-specific KO/overexpression mice, and Co-IP with phospho-site variants","pmids":["39333498"],"confidence":"Medium","gaps":["Phospho-dependent binding partners not fully resolved","Link between SNARE/trafficking activity and metabolic output not mechanistically closed"]},{"year":2025,"claim":"Resolved the chemistry of bacterial SEC22B ubiquitination, showing SdeA installs phosphoribosyl-ubiquitin at S137 followed by canonical polyubiquitination, with the deubiquitinase LotB reversing the syntaxin 3 pairing.","evidence":"In vitro ubiquitination, S137 mutagenesis, Co-IP, and LotB deubiquitinase assay","pmids":["41669755"],"confidence":"Medium","gaps":["Temporal interplay of Lug15 and SdeA modifications not defined","Single-lab in vitro reconstitution"]},{"year":2025,"claim":"Preprint-stage evidence extends SEC22B to ClC-5 secretory trafficking, secretory autophagy of UBB+1, and NRZ-coupled axonal ER-to-PM delivery of locally translated proteins.","evidence":"Co-IP and localization (ClC-5), siRNA secretion/fusion assays (UBB+1), live imaging and dominant-negative constructs (axonal NRZ-SEC22B)","pmids":["bio_10.1101_2025.11.03.686312","bio_10.1101_2024.12.31.630908","bio_10.1101_2025.09.09.674816"],"confidence":"Low","gaps":["All three are preprints lacking in vitro reconstitution","Single-lab observations not independently confirmed"]},{"year":null,"claim":"How SEC22B switches between fusogenic ER-to-Golgi transport and non-fusogenic tethering at ER-PM/ER-phagosome contacts, and how S137 modification states integrate metabolic, infection, and trafficking outputs, remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model linking longin-domain tethering to SNARE-zippering decisions","Regulatory hierarchy of S137 phosphorylation versus ubiquitination unknown","Contradictory cross-presentation KO data still unreconciled"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,3,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,15]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[15]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,11,15]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[10,14]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[2,14]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,11]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,14]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,17]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[13]}],"complexes":["SEC22B-syntaxin18-BNIP1-Use1 ER SNARE complex","NRZ tethering complex"],"partners":["STX18","BNIP1","USE1","STX4","STX1","ORP8","NBEAL2","SNAP23"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75396","full_name":"Vesicle-trafficking protein SEC22b","aliases":["ER-Golgi SNARE of 24 kDa","ERS-24","ERS24","SEC22 vesicle-trafficking protein homolog B","SEC22 vesicle-trafficking protein-like 1"],"length_aa":215,"mass_kda":24.7,"function":"SNARE involved in targeting and fusion of ER-derived transport vesicles with the Golgi complex as well as Golgi-derived retrograde transport vesicles with the ER","subcellular_location":"Endoplasmic reticulum membrane; Endoplasmic reticulum-Golgi intermediate compartment membrane; Golgi apparatus, cis-Golgi network membrane; Golgi apparatus, trans-Golgi network membrane; Melanosome","url":"https://www.uniprot.org/uniprotkb/O75396/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/SEC22B","classification":"Common Essential","n_dependent_lines":72,"n_total_lines":74,"dependency_fraction":0.972972972972973},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000265808","cell_line_id":"CID000748","localizations":[{"compartment":"vesicles","grade":3},{"compartment":"er","grade":2}],"interactors":[{"gene":"BNIP1","stoichiometry":10.0},{"gene":"GOSR2","stoichiometry":10.0},{"gene":"STX18","stoichiometry":10.0},{"gene":"STX5","stoichiometry":10.0},{"gene":"SCFD1","stoichiometry":4.0},{"gene":"NSF","stoichiometry":4.0},{"gene":"GORASP2","stoichiometry":0.2},{"gene":"MIF","stoichiometry":0.2},{"gene":"NAPA","stoichiometry":0.2},{"gene":"PIN4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000748","total_profiled":1310},"omim":[{"mim_id":"619558","title":"RAB39A, MEMBER RAS ONCOGENE FAMILY; RAB39A","url":"https://www.omim.org/entry/619558"},{"mim_id":"610675","title":"UNCONVENTIONAL SNARE IN THE ER 1; USE1","url":"https://www.omim.org/entry/610675"},{"mim_id":"605456","title":"BET1 GOLGI VESICULAR MEMBRANE-TRAFFICKING PROTEIN; BET1","url":"https://www.omim.org/entry/605456"},{"mim_id":"604029","title":"SEC22 HOMOLOG B, VESICLE TRAFFICKING PROTEIN; SEC22B","url":"https://www.omim.org/entry/604029"},{"mim_id":"604028","title":"SEC22 HOMOLOG C, VESICLE TRAFFICKING PROTEIN; SEC22C","url":"https://www.omim.org/entry/604028"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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Injury.","date":"2022","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/35654605","citation_count":14,"is_preprint":false},{"pmid":"36044553","id":"PMC_36044553","title":"Extended-synaptotagmin 1 engages in unconventional protein secretion mediated via SEC22B+ vesicle pathway in liver cancer.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/36044553","citation_count":13,"is_preprint":false},{"pmid":"31391476","id":"PMC_31391476","title":"SNARE protein SEC22B regulates early embryonic development.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31391476","citation_count":9,"is_preprint":false},{"pmid":"37794132","id":"PMC_37794132","title":"Sec22b regulates phagosome maturation by promoting ORP8-mediated lipid exchange at endoplasmic reticulum-phagosome contact 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/36789450","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.03.686312","title":"TMEM9 and SEC22B interact with ClC-5 to shape renal proximal tubule function and Dent’s Disease type I pathogenesis","date":"2025-11-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.03.686312","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.31.630908","title":"Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant, UBB  <sup>+1</sup>","date":"2025-01-02","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.31.630908","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.09.09.674816","title":"The axonal ER couples translation and secretion machineries for local delivery of axonal transmembrane proteins to promote axonal development","date":"2025-09-09","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.09.674816","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17034,"output_tokens":6248,"usd":0.072411,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15061,"output_tokens":5175,"usd":0.10234,"stage2_stop_reason":"end_turn"},"total_usd":0.174751,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"Crystal structure of the 130-amino acid N-terminal (longin) domain of mouse Sec22b was solved at 2.4 Å resolution, revealing a mixed α/β fold resembling a circular permutation of profilin and GAF/PAS modules. This domain is structurally distinct from syntaxin 1A's N-terminal domain and, unlike syntaxin 1A, does not affect the rate of SNARE assembly in vitro.\",\n      \"method\": \"X-ray crystallography (2.4 Å), in vitro SNARE assembly kinetics assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution crystal structure combined with in vitro functional assay in the same study\",\n      \"pmids\": [\"11309394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Endogenous Sec22b/ERS-24 localizes to the pre-Golgi intermediate compartment (IC), and antibodies against Sec22b inhibit ER-to-Golgi transport of VSVG prior to the EGTA-sensitive docking/fusion step, causing VSVG accumulation in pre-Golgi vesicular intermediates.\",\n      \"method\": \"Immunofluorescence co-labeling, semi-intact cell ER-Golgi transport reconstitution assay with inhibitory antibodies, EGTA block-release\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstituted transport assay with antibody inhibition combined with morphological localization and multiple pharmacological conditions\",\n      \"pmids\": [\"9950687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Sec22b localizes on ER-derived vesicles that are recruited to the Legionella-containing vacuole (LCV); Sec22b is delivered to the LCV membrane and is functionally required for biogenesis of the replicative organelle supporting Legionella intracellular growth. Rab1 recruits ER-derived vesicles to the LCV upstream of Sec22b-dependent fusion.\",\n      \"method\": \"Immunofluorescence/electron microscopy localization, genetic inhibition (dominant-negative constructs), intracellular growth assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (morphological, genetic loss-of-function, bacterial growth readout); replicated in subsequent studies\",\n      \"pmids\": [\"15117975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Sec22b (R-SNARE) associates with ER-localized syntaxin 18 (Qa-SNARE), and this binary interaction induces increased α-helicity in both SNARE motifs, creating high-affinity binding sites for BNIP1 (Qb) and p31/Use1 (Qc), thereby driving sequential Q-SNARE assembly. This R-SNARE-dependent Q-SNARE assembly mechanism is distinct from that of non-ER SNAREs.\",\n      \"method\": \"Pulldown assays, CD spectroscopy measuring α-helicity changes upon SNARE motif association\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro biophysical assay (CD spectroscopy) plus pulldown; single lab, two complementary methods\",\n      \"pmids\": [\"17979832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Overexpression of Sec22b in J774 macrophages nearly abolishes phagocytosis without affecting Fc receptor surface expression, whereas suppression of endogenous Sec22b increases phagocytic capacity. Domain analysis shows the R-SNARE motif (responsible for SNARE complex formation with syntaxin 18 and/or D12) mediates this inhibition, identifying Sec22b as a negative regulator of phagocytosis likely by titrating free syntaxin 18/D12.\",\n      \"method\": \"Stable overexpression, shRNA knockdown, domain deletion/mutagenesis analysis, phagocytosis assay, flow cytometry\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with domain mapping; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"19710423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"During virulent L. pneumophila infection, Sec22b (ER v-SNARE) undergoes non-canonical pairing with plasma membrane syntaxins (Stx2, Stx3, Stx4) and SNAP23 on the LCV. Depletion of plasma membrane syntaxins delays calnexin acquisition and retains Rab1 on phagosomes. Addition of α-SNAP and NSF dissociates these non-canonical SNARE complexes, demonstrating they are functional.\",\n      \"method\": \"RNAi depletion, co-immunoprecipitation, NSF/α-SNAP disassembly assay, immunofluorescence\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional co-IP validated by NSF/α-SNAP disassembly assay, multiple syntaxins tested, replicated conceptually by later studies\",\n      \"pmids\": [\"20163564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Sec22b localizes to the ERGIC and pairs with plasma membrane SNARE syntaxin 4 present on phagosomes. Depletion of Sec22b in dendritic cells inhibits recruitment of ER-resident proteins to phagosomes (and to T. gondii-containing vacuoles), impairs antigen export to the cytosol, accelerates lysosomal recruitment, and blocks MHC class I cross-presentation after phagocytosis or endocytosis of antigen.\",\n      \"method\": \"siRNA knockdown, immunofluorescence, co-immunoprecipitation (Sec22b–syntaxin 4 pairing), antigen cross-presentation assay, phagosome maturation assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, loss-of-function, trafficking, functional T cell assay) in a high-profile journal; widely cited and replicated\",\n      \"pmids\": [\"22153078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DC-specific conditional knockout of Sec22b in mice impairs cross-presentation ex vivo and cross-priming of CD8+ T cells in vivo, and abolishes antitumor immune responses and response to anti-PD-1 therapy, supporting Sec22b-dependent ER-phagosome traffic as required for cross-presentation.\",\n      \"method\": \"Conditional Cre-lox DC-specific knockout mice, ex vivo cross-presentation assay, in vivo tumor challenge, anti-PD-1 treatment\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional KO with defined cellular and in vivo phenotypes; contradicted by another conditional KO study (PMID:28658614), reducing confidence\",\n      \"pmids\": [\"28663435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A separate DC-specific Sec22b knockout mouse (CD11c-Cre Sec22b fl/fl) shows that SEC22B-deficient DCs can efficiently cross-present antigen both in vivo and in vitro. shRNA-mediated Sec22b silencing reduces cross-presentation even in SEC22B-knockout BMDCs, indicating the shRNA effect is due to off-target activity rather than Sec22b loss.\",\n      \"method\": \"Conditional Cre-lox DC-specific knockout mice, in vitro/in vivo cross-presentation assays, shRNA in KO cells, RNA-seq of shRNA-treated KO BMDCs\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rigorous genetic controls (shRNA in KO background) with RNA-seq; directly contradicts PMID:28663435, lowering overall field confidence\",\n      \"pmids\": [\"28658614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SEC22B interacts with NBEAL2 (gray platelet syndrome protein) via a region spanning NBEAL2 amino acids 1798–1903; GPS-associated missense variants E1833K and R1839C in NBEAL2 abolish this interaction. NBEAL2 can simultaneously bind SEC22B and P-selectin. CRISPR/Cas9 knockout of SEC22B in imMKCL cells decreases NBEAL2 protein and blocks α-granule biogenesis, demonstrating SEC22B is required for megakaryocyte α-granule production.\",\n      \"method\": \"Co-immunoprecipitation (tagged and endogenous proteins), CRISPR/Cas9 knockout, immunofluorescence microscopy, α-granule content assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with endogenous proteins, domain mapping with disease-variant validation, and CRISPR KO phenotype; multiple orthogonal methods in one study\",\n      \"pmids\": [\"32384141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Sec22b interacts with members of the extended synaptotagmin (E-Syt) family via the longin domain of Sec22b, stabilizing Sec22b–syntaxin 1 (Stx1) non-fusogenic SNARE complexes at ER-PM contacts. Overexpression of wild-type E-Syt2 (but not lipid-transfer-deficient or ER-anchoring-deficient mutants) increases axonal filopodia formation and neurite ramification; this effect is blocked by clostridial neurotoxin cleaving Stx1, longin domain expression, or a Sec22b mutant with an extended SNARE-TM linker.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/dominant-negative constructs, neurite morphology assay, clostridial neurotoxin inhibition\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping plus functional rescue/inhibition experiments; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"32843578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Sec22b depletion in endothelial cells causes loss of elongated Weibel-Palade body morphology, disintegration of Golgi and dilation of rough ER, reduced proteolytic processing of VWF, accumulation of VWF in dilated rER, and reduced VWF secretion, demonstrating that Sec22b-containing SNARE complexes governing ER-to-Golgi anterograde transport determine WPB length and VWF hemostatic activity.\",\n      \"method\": \"shRNA-mediated knockdown, immunofluorescence, electron microscopy, VWF secretion assay\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean loss-of-function with multiple morphological and biochemical readouts; single lab\",\n      \"pmids\": [\"32336681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Sec22b co-localizes with inducible NO synthase (iNOS) at ERGIC/Golgi compartments and phagosomes. siRNA silencing of Sec22b in bone marrow-derived dendritic cells abrogates NO and cytokine production at both protein and mRNA levels and reduces nuclear translocation of NF-κB. Sec22b was found to co-occur with NF-κB in both cytoplasm and nucleus, implicating Sec22b in NF-κB shuttling.\",\n      \"method\": \"Immunofluorescence co-localization, siRNA knockdown, NO/cytokine measurement, NF-κB nuclear translocation assay, pharmacological secretory pathway blockade\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — loss-of-function with multiple functional readouts and co-localization data; single lab, mechanistic link to NF-κB shuttling is correlative\",\n      \"pmids\": [\"34580108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Sec22b and Ykt6 act as opposing regulators of autophagosome axonal retrograde transport in neurons. Ischemia-reperfusion increases Sec22b and decreases Ykt6 in neurons. Sec22b knockdown and Ykt6 overexpression rescue axonal autophagosome retrograde transport, restore autophagic flux, and reduce infarct size in a murine MCAO/R model in an autophagy-dependent manner.\",\n      \"method\": \"Knockdown/overexpression in primary neurons and in vivo murine MCAO/R model, live autophagosome tracking, autophagic flux assays, Atg7 conditional KO epistasis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo and in vitro loss/gain-of-function with epistasis (Atg7 KO); single lab\",\n      \"pmids\": [\"35654605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In liver cancer cells, cytosolic proteins associate with E-Syt1 on the ER, then localize inside SEC22B+ vesicles. SEC22B on these vesicles tethers to the plasma membrane via Q-SNAREs (SNAP23, SNX3, SNX4) for secretion of cytosolic cargo, identifying SEC22B as a mediator of unconventional cytosolic protein secretion at ER-PM contact sites.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, immunofluorescence, proximity ligation, inhibition of PKCδ–E-Syt1 interaction\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple Co-IP and localization methods; single lab, functional link established by inhibition of cargo–E-Syt1 interaction\",\n      \"pmids\": [\"36044553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Sec22b tethers ER-phagosome membrane contact sites (MCS) independently of STIM1. Sec22b knockdown increases phagosomal calcium signaling, accelerates phagolysosome fusion and antigen degradation, and alters phagosomal phospholipids (PI(3)P, PS, PI(4)P). Sec22b co-precipitates with the PS/PI(4)P exchange protein ORP8; wild-type but not catalytic-mutant ORP8 rescues phagosomal PI(4)P levels and reduces antigen degradation. The MCS-disrupting Sec22b-P33 mutant fails to rescue, identifying tethering as the mechanistic basis.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation (Sec22b–ORP8), artificial tether (MAPPER) rescue, Sec22b-P33 MCS-disrupting mutant, phagosomal lipid quantification, calcium imaging, phagolysosome fusion assay\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, mutant rescue, artificial tether, lipid profiling, calcium imaging) establishing mechanistic pathway; single lab but comprehensive\",\n      \"pmids\": [\"37794132\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The L. pneumophila effector Lug15 is a novel E3 ubiquitin ligase that ubiquitinates host Sec22b, mediates Sec22b recruitment to the LCV, and promotes non-canonical SNARE pairing of Sec22b with plasma membrane syntaxin 3.\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation, intracellular bacterial growth assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzymatic assay plus Co-IP and functional readout; single lab\",\n      \"pmids\": [\"37882795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Sec22b is a critical non-redundant regulator of plasma cell maintenance: Sec22b-deficient mice show near-absence of plasma cells and dramatically reduced serum antibody titers. Mechanistically, Sec22b contributes to efficient antibody secretion and regulates transcriptional identity of plasma cells as well as morphology of the ER and mitochondria.\",\n      \"method\": \"Conditional knockout mice, flow cytometry, ELISA for antibody titers, immunofluorescence of organelle morphology, transcriptional profiling\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with multiple cellular and functional readouts; single lab\",\n      \"pmids\": [\"36595686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Glucagon signaling phosphorylates SEC22B at serine 137 in the liver; hepatocyte-specific loss- and gain-of-function experiments show SEC22B is a key regulator of hepatic glycogen, lipid, and amino acid metabolism. SEC22B-S137 phosphorylation affects several protein binding partners and mediates glucagon's metabolic actions.\",\n      \"method\": \"In situ time-resolved liver phosphoproteomics, hepatocyte-specific KO and overexpression mouse models, co-immunoprecipitation of SEC22B binding partners with phospho-site variants\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics discovery combined with conditional KO/OE and Co-IP binding partner analysis; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"39333498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The L. pneumophila effector SdeA catalyzes conjugation of phosphoribosyl-linked ubiquitin specifically to serine 137 of Sec22b; subsequently the canonical ubiquitin system adds polyubiquitin chains. This multimodal ubiquitination of Sec22b facilitates non-canonical SNARE pairing (Sec22b with Stx3) during early infection. The Legionella deubiquitinase LotB cleaves the polyubiquitin chains from Sec22b, causing Sec22b dissociation from syntaxin 3.\",\n      \"method\": \"In vitro ubiquitination assay, site-directed mutagenesis (Sec22b-S137), co-immunoprecipitation, deubiquitinase assay (LotB)\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzymatic assay with site-directed mutagenesis and Co-IP; single lab, multiple complementary experiments\",\n      \"pmids\": [\"41669755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SEC22B specifically interacts with wild-type ClC-5 chloride/proton antiporter but not with pathogenic ClC-5 mutants in renal proximal tubule cells. SEC22B deletion impairs ClC-5 trafficking, causing its retention at the Golgi and endosomes, identifying SEC22B as a trafficking factor for ClC-5 in the secretory pathway.\",\n      \"method\": \"Interactome analysis (Co-IP), CRISPR/Cas9 or siRNA knockdown, immunofluorescence of ClC-5 localization\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, Co-IP and localization without in vitro reconstitution\",\n      \"pmids\": [\"bio_10.1101_2025.11.03.686312\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SEC22B (R-SNARE), together with Q-SNAREs STX4 and SNAP23, mediates fusion of UBB+1-containing autophagosomes with the plasma membrane during secretory autophagy. Disruption of SEC22B impairs autophagosome-PM fusion and reduces UBB+1 secretion without affecting its intracellular turnover.\",\n      \"method\": \"siRNA knockdown, secretion assay, autophagosome-PM fusion assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, knockdown with secretion readout; no in vitro reconstitution\",\n      \"pmids\": [\"bio_10.1101_2024.12.31.630908\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In axons, the NRZ–SEC22B tethering complex links axonal ER exit (ERES-dependent, Golgi-independent) to plasma membrane delivery of locally translated transmembrane proteins, dependent on ER-PM contacts. This coupling supports axon growth and bouton assembly.\",\n      \"method\": \"Live imaging in neurons, overexpression/dominant-negative constructs, ERES marker colocalization, axon growth and bouton assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab; mechanism inferred from colocalization and dominant-negative without reconstitution\",\n      \"pmids\": [\"bio_10.1101_2025.09.09.674816\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SEC22B is an R-SNARE resident in the ERGIC/ER that mediates anterograde ER-to-Golgi membrane fusion via canonical complexes with syntaxin 18, BNIP1, and p31/Use1; its longin domain (structurally a profilin/GAF-like fold) enables non-fusogenic pairing with plasma membrane syntaxins (e.g., Stx1, Stx4) at ER-PM contact sites to support neurite growth and unconventional secretion; in phagocytes it tethers ER-phagosome contact sites through ORP8 to control phagosomal phospholipid composition and maturation; it is ubiquitinated on S137 by bacterial effectors (Lug15/SdeA) to drive non-canonical SNARE pairing during Legionella infection; and its S137 phosphorylation by glucagon signaling in hepatocytes regulates glycogen, lipid, and amino acid metabolism.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SEC22B is an R-SNARE of the ER/ERGIC that mediates anterograde ER-to-Golgi membrane transport and, through non-fusogenic SNARE pairings, participates in membrane contact-site biology, unconventional secretion, and immune membrane traffic [#1, #6]. Its N-terminal longin domain adopts a profilin/GAF-like α/β fold structurally distinct from syntaxin 1A and dispensable for SNARE assembly kinetics in vitro [#0]. In canonical ER fusion, SEC22B binding to the Qa-SNARE syntaxin 18 increases α-helicity in both SNARE motifs to nucleate sequential recruitment of the Qb/Qc-SNAREs BNIP1 and p31/Use1 [#3], and loss of SEC22B-dependent ER-to-Golgi transport disorganizes the Golgi, dilates the rough ER, and impairs VWF processing and secretion [#11]. Beyond classical fusion, the longin domain pairs SEC22B with plasma-membrane syntaxins (Stx1, Stx4) at ER-PM contact sites, stabilized by extended synaptotagmins, to drive neurite outgrowth [#10] and to support unconventional secretion of cytosolic cargo loaded into SEC22B+ vesicles that tether to the PM via SNAP23-containing Q-SNARE complexes [#14]. In phagocytes, SEC22B pairs with phagosomal syntaxin 4 to deliver ER components to maturing phagosomes, sustain MHC class I cross-presentation, and tether ER-phagosome contact sites independently of STIM1; this tethering recruits the lipid-exchange protein ORP8 to set phagosomal PI(4)P, PS and PI(3)P content and restrain phagolysosome fusion and antigen degradation [#6, #15]. SEC22B is also required for megakaryocyte α-granule biogenesis through its interaction with NBEAL2 [#9] and for plasma cell maintenance and efficient antibody secretion [#17]. Intracellular Legionella exploits SEC22B by recruiting ER-derived vesicles to the Legionella-containing vacuole and driving non-canonical SEC22B pairing with plasma-membrane syntaxins; the bacterial effectors Lug15 and SdeA ubiquitinate SEC22B—SdeA installing phosphoribosyl-ubiquitin at serine 137 followed by canonical polyubiquitination—to promote these non-canonical complexes [#2, #5, #16, #19]. The same residue is targeted physiologically: glucagon signaling phosphorylates SEC22B at S137 in hepatocytes to control glycogen, lipid, and amino acid metabolism [#18].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established SEC22B as a functional component of early secretory traffic by showing it acts at the pre-Golgi intermediate compartment before the docking/fusion step of ER-to-Golgi transport.\",\n      \"evidence\": \"Immunofluorescence localization and antibody-inhibited semi-intact cell ER-Golgi transport reconstitution\",\n      \"pmids\": [\"9950687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the cognate Q-SNARE partners\", \"Mechanism of vesicle docking versus fusion not resolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolved the architecture of the SEC22B N-terminal domain, revealing a longin (profilin/GAF-like) fold that is structurally distinct from syntaxin 1A and does not govern SNARE assembly rate, reframing the domain's role away from kinetic regulation of fusion.\",\n      \"evidence\": \"X-ray crystallography at 2.4 Å plus in vitro SNARE assembly kinetics\",\n      \"pmids\": [\"11309394\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the longin fold left undefined\", \"No partner identified for the domain at this stage\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the molecular logic of canonical ER SNARE assembly, showing SEC22B binding to syntaxin 18 induces α-helicity that creates high-affinity sites for BNIP1 and p31/Use1.\",\n      \"evidence\": \"Pulldown assays and CD spectroscopy of SNARE motif α-helicity changes\",\n      \"pmids\": [\"17979832\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro biophysics not validated by reconstituted fusion\", \"Single-lab biophysical analysis\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed SEC22B abundance negatively tunes phagocytosis through its R-SNARE motif, implicating it in regulating Q-SNARE availability in phagocytes.\",\n      \"evidence\": \"Overexpression, shRNA knockdown, and domain mapping with phagocytosis assays in macrophages\",\n      \"pmids\": [\"19710423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Titration model of free syntaxin 18/D12 not directly demonstrated\", \"Single cell-line context\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected SEC22B to immune membrane traffic by showing it pairs with phagosomal syntaxin 4 to deliver ER components to phagosomes and enable MHC class I cross-presentation.\",\n      \"evidence\": \"siRNA knockdown, Co-IP of SEC22B-syntaxin 4, phagosome maturation and cross-presentation assays in dendritic cells\",\n      \"pmids\": [\"22153078\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SEC22B mediates fusion versus non-fusogenic tethering at phagosomes unresolved here\", \"Did not address contact-site lipid transfer\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Tested the genetic requirement for SEC22B in cross-presentation in vivo, yielding conflicting conclusions on whether SEC22B is essential for DC cross-priming.\",\n      \"evidence\": \"Two independent DC-specific conditional knockout mouse lines with ex vivo/in vivo cross-presentation assays; one used shRNA-in-KO controls\",\n      \"pmids\": [\"28663435\", \"28658614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The two conditional KO studies reach opposite conclusions\", \"shRNA off-target effects confound earlier knockdown data\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended SEC22B function beyond bulk traffic, establishing it as a longin-domain-mediated organizer at ER-PM contacts (with E-Syts and Stx1 for neurite growth) and as an essential factor for megakaryocyte α-granule biogenesis via NBEAL2.\",\n      \"evidence\": \"Co-IP with domain and disease-variant mapping, CRISPR knockout, and neurite/α-granule morphology assays\",\n      \"pmids\": [\"32843578\", \"32384141\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the NBEAL2 and E-Syt pathways intersect mechanistically is unknown\", \"Stoichiometry of non-fusogenic SEC22B-Stx1 complexes not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked SEC22B-dependent anterograde transport to specialized secretory organelle biology (Weibel-Palade body morphology and VWF processing) and broadened its immune signaling role to NO/cytokine production and NF-κB shuttling.\",\n      \"evidence\": \"shRNA knockdown with EM, VWF secretion assays in endothelial cells, and siRNA with NO/cytokine and NF-κB translocation readouts in dendritic cells\",\n      \"pmids\": [\"32336681\", \"34580108\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"NF-κB shuttling link is correlative\", \"Direct SNARE partners at WPBs not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified SEC22B as a regulator of autophagosome retrograde transport and as a mediator of unconventional cytosolic protein secretion through SEC22B+ vesicles tethering to the PM at ER-PM contacts.\",\n      \"evidence\": \"Knockdown/overexpression with Atg7 epistasis in an MCAO/R model; Co-IP, fractionation, proximity ligation and cargo-E-Syt1 inhibition in liver cancer cells\",\n      \"pmids\": [\"35654605\", \"36044553\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Opposing SEC22B/Ykt6 regulation mechanism not biochemically defined\", \"Cargo selection into SEC22B+ vesicles unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined the mechanistic basis of SEC22B at ER-phagosome contacts as a tether that recruits ORP8 to set phagosomal phospholipid composition, and established non-redundant requirements in plasma cell maintenance and antibody secretion.\",\n      \"evidence\": \"siRNA, Co-IP with ORP8, MAPPER artificial-tether and P33 MCS-disrupting mutant rescue, lipid profiling and calcium imaging; conditional KO mice with ELISA and organelle morphology\",\n      \"pmids\": [\"37794132\", \"36595686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How tethering is coordinated with SNARE pairing at the same membrane is unclear\", \"Transcriptional identity link in plasma cells is correlative\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed that Legionella effectors hijack SEC22B by ubiquitinating it (via the E3 ligase Lug15) to drive recruitment to the LCV and non-canonical pairing with plasma-membrane syntaxin 3.\",\n      \"evidence\": \"In vitro ubiquitination, Co-IP, and intracellular bacterial growth assays\",\n      \"pmids\": [\"37882795\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination site not defined in this study\", \"Host enzymatic background not excluded\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified SEC22B serine 137 as a physiological glucagon-regulated phosphosite controlling hepatic glycogen, lipid, and amino acid metabolism, expanding SEC22B's role into metabolic signaling.\",\n      \"evidence\": \"Time-resolved liver phosphoproteomics, hepatocyte-specific KO/overexpression mice, and Co-IP with phospho-site variants\",\n      \"pmids\": [\"39333498\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phospho-dependent binding partners not fully resolved\", \"Link between SNARE/trafficking activity and metabolic output not mechanistically closed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the chemistry of bacterial SEC22B ubiquitination, showing SdeA installs phosphoribosyl-ubiquitin at S137 followed by canonical polyubiquitination, with the deubiquitinase LotB reversing the syntaxin 3 pairing.\",\n      \"evidence\": \"In vitro ubiquitination, S137 mutagenesis, Co-IP, and LotB deubiquitinase assay\",\n      \"pmids\": [\"41669755\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Temporal interplay of Lug15 and SdeA modifications not defined\", \"Single-lab in vitro reconstitution\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Preprint-stage evidence extends SEC22B to ClC-5 secretory trafficking, secretory autophagy of UBB+1, and NRZ-coupled axonal ER-to-PM delivery of locally translated proteins.\",\n      \"evidence\": \"Co-IP and localization (ClC-5), siRNA secretion/fusion assays (UBB+1), live imaging and dominant-negative constructs (axonal NRZ-SEC22B)\",\n      \"pmids\": [\"bio_10.1101_2025.11.03.686312\", \"bio_10.1101_2024.12.31.630908\", \"bio_10.1101_2025.09.09.674816\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"All three are preprints lacking in vitro reconstitution\", \"Single-lab observations not independently confirmed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SEC22B switches between fusogenic ER-to-Golgi transport and non-fusogenic tethering at ER-PM/ER-phagosome contacts, and how S137 modification states integrate metabolic, infection, and trafficking outputs, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model linking longin-domain tethering to SNARE-zippering decisions\", \"Regulatory hierarchy of S137 phosphorylation versus ubiquitination unknown\", \"Contradictory cross-presentation KO data still unreconciled\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 3, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 15]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 11, 15]},\n      {\"term_id\": \"GO:0005793\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [10, 14]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [2, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 11]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 14]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 17]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"complexes\": [\n      \"SEC22B-syntaxin18-BNIP1-Use1 ER SNARE complex\",\n      \"NRZ tethering complex\"\n    ],\n    \"partners\": [\n      \"STX18\",\n      \"BNIP1\",\n      \"USE1\",\n      \"STX4\",\n      \"STX1\",\n      \"ORP8\",\n      \"NBEAL2\",\n      \"SNAP23\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}