{"gene":"MARCHF2","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2005,"finding":"MARCH2 (MARCH-II) localizes to endosomal vesicles and the plasma membrane, directly associates with syntaxin 6 (established by immunoprecipitation and in vitro binding), redistributes syntaxin 6 and syntaxin-6-interacting SNAREs into MARCH2-positive vesicles upon overexpression, perturbs retrograde transport of TGN38 and furin to the TGN, reduces cell surface transferrin receptor expression and transferrin uptake, and depletion by siRNA disrupts TGN localization of syntaxin 6 and TGN38/46.","method":"Immunoprecipitation, in vitro binding assay, siRNA knockdown, immunofluorescence microscopy, transferrin uptake assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus in vitro binding, siRNA rescue, multiple orthogonal functional readouts in a single focused study","pmids":["15689499"],"is_preprint":false},{"year":2006,"finding":"MARCH2 (MARCH-II) associates with MARCH3 by immunoprecipitation, and both share endosomal localization and syntaxin 6 binding; the PDZ-binding motif and RING finger of MARCH3 are essential for its localization and function, providing comparative structural context for MARCH2 domain requirements.","method":"Immunoprecipitation, immunofluorescence, mutational analysis","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP for MARCH2–MARCH3 interaction; primary focus is MARCH3 characterization but MARCH2 interaction confirmed","pmids":["16428329"],"is_preprint":false},{"year":2007,"finding":"MARCH2 interacts with the PDZ scaffold protein DLG1 in a PDZ domain-dependent manner (identified by tandem affinity purification/mass spectrometry and confirmed by co-immunoprecipitation), co-localizes with DLG1 at sites of cell-cell contact, and promotes DLG1 ubiquitination in vivo ubiquitination assays. Loss of the MARCH2 PDZ-binding motif abolishes its localization to cell-cell contact sites.","method":"Tandem affinity purification, mass spectrometry, co-immunoprecipitation, in vivo ubiquitination assay, immunofluorescence","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TAP-MS identification confirmed by Co-IP, ubiquitination assay, and localization experiment; single lab","pmids":["17980554"],"is_preprint":false},{"year":2012,"finding":"MARCH2 interacts with carvedilol-bound β2-adrenergic receptor (β2AR) (identified by LC-MS/MS proteomics and confirmed by co-immunoprecipitation), and its E3 ligase activity mediates ubiquitination, endocytosis, and lysosomal degradation of β2AR specifically in response to carvedilol. siRNA-mediated knockdown of MARCH2 abolished carvedilol-induced β2AR ubiquitination, endocytosis, and degradation in vascular smooth muscle cells. The MARCH2–β2AR association is stabilized by carvedilol and does not require β-arrestin.","method":"LC-MS/MS proteomics, co-immunoprecipitation, siRNA knockdown, ubiquitination assay, cell surface receptor degradation assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS identification confirmed by Co-IP, siRNA rescue, and multiple functional readouts (ubiquitination, endocytosis, degradation) in both overexpression and endogenous systems","pmids":["23166351"],"is_preprint":false},{"year":2013,"finding":"MARCH2 co-immunoprecipitates and co-localizes with the adaptor proteins CAL and STX6; its binding to CAL is enhanced synergistically by STX6. MARCH2 ubiquitinates mature CFTR and promotes its lysosomal degradation in a catalytic RING-domain-dependent manner. A catalytically dead RING mutant of MARCH2 fails to degrade CFTR. MARCH2 has no effect on CFTR lacking its PDZ motif, and silencing of CAL abolishes MARCH2-mediated CFTR degradation. siRNA-mediated silencing of MARCH2 in CF epithelial cells increases mature CFTR abundance.","method":"Co-immunoprecipitation, co-localization (immunofluorescence), in vivo ubiquitination assay, RING domain mutagenesis, siRNA knockdown, bafilomycin A1 treatment","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — catalytic mutagenesis, co-IP, ubiquitination assay, PDZ-motif requirement, and siRNA rescue with multiple orthogonal methods in one study","pmids":["23818989"],"is_preprint":false},{"year":2016,"finding":"MARCH2 overexpression impairs autophagy (reduces LC3B-II levels, impairs autophagic substrate degradation) whereas MARCH2 loss promotes autophagy via the PIK3CA-AKT-MTOR signaling pathway. MARCH2 interacts with CFTR via its PDZ domain, promotes CFTR ubiquitination and degradation, and thereby inhibits CFTR-mediated autophagy in tumor cells.","method":"Overexpression, siRNA/knockout, LC3B-II western blot, autophagic flux assay, co-immunoprecipitation, ubiquitination assay, in vivo tumorigenicity assay","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and functional autophagy readouts; single lab with multiple orthogonal methods","pmids":["27308891"],"is_preprint":false},{"year":2017,"finding":"MARCH2 knockout (via CRISPR/Cas9) in HCT116 colon cancer cells suppresses proliferation and promotes autophagy, apoptosis, and G2/M arrest associated with activation of endoplasmic reticulum stress, establishing MARCH2 as a negative regulator of ER stress in this cellular context.","method":"CRISPR/Cas9 knockout, cell proliferation assay, flow cytometry (apoptosis, cell cycle), autophagy assay, ER stress marker analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined phenotypic readouts; single lab, no in vitro biochemical reconstitution of ER stress mechanism","pmids":["28749466"],"is_preprint":false},{"year":2018,"finding":"In Xenopus, March2 (RING-type E3 ubiquitin ligase) promotes ubiquitin-mediated lysosomal degradation of Dishevelled (Dsh/Dvl), thereby antagonizing Wnt signaling. Dapper1 (Dpr1) stabilizes the March2–Dsh interaction and provides regional specificity in the dorso-animal region to control head formation.","method":"Xenopus embryo assays, co-immunoprecipitation, ubiquitination assay, lysosomal degradation assay, loss-of-function/gain-of-function in embryos","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and in vivo embryological epistasis; single lab, ortholog model system","pmids":["29549110"],"is_preprint":false},{"year":2018,"finding":"MARCH2 inhibits HIV-1 production through E3 ligase activity-dependent envelope protein degradation and/or intracellular retention (translocation), a mechanism similar to MARCH8. MARCH2 expression is upregulated upon HIV-1 infection.","method":"Overexpression with RING-dead mutant comparison, viral titer assay, envelope protein localization","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RING-domain activity requirement demonstrated by mutant comparison, viral function assay; single lab","pmids":["29573664"],"is_preprint":false},{"year":2019,"finding":"MARCH2 ubiquitinates ERGIC3 at lysines 6 and 8, leading to its proteasomal degradation; MARCH2 depletion increases endogenous ERGIC3 levels. MARCH2-mediated ERGIC3 ubiquitination reduces secretion of ERGIC3 cargo proteins α1-antitrypsin and haptoglobin. An ERGIC3 K6R/K8R mutant resistant to ubiquitination rescues secretion, confirming ERGIC3 ubiquitination as the primary mechanism.","method":"Co-immunoprecipitation, site-directed mutagenesis (K6R/K8R), siRNA knockdown, secretion assay, ubiquitination assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — site-directed mutagenesis identifying specific ubiquitin acceptor lysines, rescue experiment with ubiquitination-resistant mutant, multiple cargo secretion assays; single lab with orthogonal methods","pmids":["31142615"],"is_preprint":false},{"year":2020,"finding":"MARCH2 directly interacts with NEMO (NF-κB essential modulator) during the late phase of bacterial/viral infection and catalyzes K48-linked ubiquitination of NEMO at Lys326, resulting in NEMO proteasomal degradation and suppression of NF-κB and interferon signaling. MARCH2 knockout mice show increased innate immune responses and resistance to bacterial/viral infection, but enhanced susceptibility to LPS-induced cytokine storm.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K326), MARCH2-/- mouse model, bacterial/viral infection assays, cytokine measurement","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, site-specific mutagenesis, K48-linkage determination, in vivo KO mouse model with multiple readouts; single lab with orthogonal methods","pmids":["32935379"],"is_preprint":false},{"year":2021,"finding":"MARCH2 (along with MARCH3, 4, and 9) downregulates cell surface expression of integrin complexes. Specifically, Integrin α4 was upregulated in mature B-lymphocytes of MARCH2 knockout mice, accompanied by decreased B-cell numbers in the spleen, establishing MARCH2 as a regulator of integrin α4β1 (VLA4) surface levels.","method":"Cell surface proteomics, MARCH2 knockout mouse analysis, flow cytometry","journal":"Proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell surface proteomics and KO mouse validation; single lab, broader screen study with specific MARCH2 finding for integrin α4","pmids":["33945654"],"is_preprint":false},{"year":2022,"finding":"MARCH2 and MARCH3 associate with IL-5 receptor α (IL-5Rα) and mediate its K27-linked polyubiquitination at K379 (by MARCH2) and K383 (by MARCH3), leading to lysosomal degradation. MARCH2/3 double knockout markedly increases IL-5Rα levels, enhances IL-5-induced signaling, increases eosinophil proportions in bone marrow and blood, and aggravates OVA-induced airway eosinophilia in mice.","method":"Co-immunoprecipitation, ubiquitination assay (K27-linkage), site-directed mutagenesis, MARCH2/3 double-knockout mouse model, flow cytometry, OVA challenge model","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, K27-linkage and site-specific ubiquitination, in vivo KO mouse model with multiple orthogonal readouts","pmids":["35982175"],"is_preprint":false},{"year":2024,"finding":"MARCH2 directly interacts with PGAM5 and promotes its K48-linked polyubiquitination and proteasomal degradation. This prevents PGAM5 from forming liquid-liquid phase separation condensates with MAVS and recruiting NLRP3, thereby inhibiting NLRP3 inflammasome activation and cardiomyocyte pyroptosis during myocardial ischemia-reperfusion injury. MARCH2 genetic ablation aggravates myocardial infarction; AAV-based MARCH2 re-introduction ameliorates I/R-induced cardiac dysfunction.","method":"Co-immunoprecipitation, K48-linked ubiquitination assay, MARCH2 knockout mouse, single-cell RNA-seq, AAV gene delivery, phase separation assay, cardiac function measurement","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, specific ubiquitin-linkage determination, phase separation assay, KO and AAV rescue in vivo; single lab with multiple orthogonal methods","pmids":["38409220"],"is_preprint":false},{"year":2024,"finding":"MARCH2 is a novel E3 ligase for SNAIL: its RING domain mediates SNAIL ubiquitination and degradation, and PTK6 tyrosine kinase inhibition promotes the MARCH2–SNAIL interaction. MARCH2 overexpression suppresses EMT, cell migration, anoikis resistance, and metastasis of triple-negative breast cancer cells, phenocopying effects of SNAIL downregulation.","method":"Co-immunoprecipitation, ubiquitination assay, RING domain mutagenesis, migration/invasion assay, in vivo metastasis model","journal":"Cancer research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RING domain mutagenesis, Co-IP, functional metastasis assays; single lab","pmids":["38457262"],"is_preprint":false},{"year":2024,"finding":"Human MARCH2 (but not mouse MARCH2) restricts HIV-1 infection in primary CD4+ T cells; a specific amino acid absent in mouse MARCH2 is critical for antiretroviral function. MARCH2 domains required for binding and restriction of HIV-1 envelope glycoproteins were mapped. MARCH2 is incorporated inside nascent virions and reduces particle infectivity by blocking virus entry in a RING-CH-independent manner. MARCH2 prevents cell-to-cell HIV-1 transmission.","method":"Domain mapping/mutagenesis, viral infectivity assay, virion incorporation assay, primary CD4+ T cell infection, cell-to-cell transmission assay","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis, multiple functional readouts in primary cells; single lab","pmids":["39074162"],"is_preprint":false},{"year":2024,"finding":"MARCH1 and MARCH2 inhibit pseudorabies virus (PRV) replication at the cell-to-cell fusion step by associating with the viral gB/gD/gH/gL fusion complex and trapping it in the trans-Golgi network rather than degrading the proteins; this mechanism requires E3 ligase activity for blocking gB furin cleavage, though blocking gB cleavage itself does not account for the antiviral activity.","method":"Co-immunoprecipitation, viral replication assay, TGN localization assay, RING-dead mutant comparison","journal":"Veterinary microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of MARCH2 with viral fusion complex, localization assay, RING activity requirement; single lab","pmids":["38936155"],"is_preprint":false},{"year":2025,"finding":"MARCH2 interacts with PKM2 and promotes its K33-linked polyubiquitination, stimulating PKM2 dimer-to-tetramer conversion. In MARCH2-deficient vascular smooth muscle cells, reduced PKM2 tetramerization leads to glucose metabolism reprogramming, increased histone H3K18 lactylation, and p53-driven apoptosis, exacerbating aortic aneurysm/dissection. The PKM2 activator TEPP-46 rescues MARCH2 deficiency-worsened pathology.","method":"Co-immunoprecipitation, K33-linkage ubiquitination assay, MARCH2-/- and SMC-specific KO mice, PKM2 tetramer assay, metabolomics/ChIP (CUT&TAG), pharmacological rescue","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, specific ubiquitin-linkage determination, in vivo KO and pharmacological rescue with mechanistic pathway placement","pmids":["40079144"],"is_preprint":false},{"year":2025,"finding":"MARCH2 mediates K63-linked polyubiquitination of ALK5 (TGF-β type I receptor) at lysines 342/343 at endosomes following TGF-β-induced endocytosis, enhancing ALK5 catalytic activity. ALK5 K342/343R mutations abolish SMAD2 phosphorylation and TGF-β responses and reduce lung metastasis in a mouse model.","method":"Co-immunoprecipitation, K63-linkage ubiquitination assay, site-directed mutagenesis (K342/343R), SMAD2 phosphorylation assay, cell migration assay, in vivo lung metastasis model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis, K63-linkage ubiquitination, functional catalytic activity and in vivo metastasis; single lab","pmids":["41213909"],"is_preprint":false},{"year":2025,"finding":"MARCH2 constitutively associates with TNF-R1 and mediates K27-linked polyubiquitination of TNF-R1 at K340, resulting in its proteasomal degradation and dampening of TNF-triggered signaling. The deubiquitinase USP22 antagonizes this by removing K27-linked ubiquitin from K340, stabilizing TNF-R1. MARCH2 deficiency promotes TNF-triggered signaling; in acute liver injury, MARCH2 deficiency exacerbates pathology.","method":"Co-immunoprecipitation, K27-linkage ubiquitination assay, site-directed mutagenesis (K340), deubiquitination assay, MARCH2-/- and USP22-/- mouse models, cytokine and liver injury assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, site-specific ubiquitination, deubiquitinase antagonism, and in vivo mouse models with multiple orthogonal readouts","pmids":["41961857"],"is_preprint":false},{"year":2025,"finding":"MARCH2 interacts with PTPRD and facilitates its K27-linked polyubiquitination and subsequent lysosomal degradation in a RING-domain-dependent manner, promoting PTPRD translocation from the cell membrane to lysosomes. This inhibits odontoblast differentiation. March2-deficient mice and odontoblast-specific March2 knockdown mice exhibit increased dentin thickness and elevated odontoblast marker expression. MARCH2/PTPRD double knockdown reverses the enhanced differentiation caused by MARCH2 knockdown alone.","method":"Co-immunoprecipitation, K27-linkage ubiquitination assay, RING-domain mutagenesis, March2 knockout and conditional knockdown mice, double knockdown epistasis, lysosome trafficking assay","journal":"International journal of oral science","confidence":"High","confidence_rationale":"Tier 2 / Strong — RING mutagenesis, site-specific ubiquitination, lysosomal trafficking, in vivo KO and double-knockdown epistasis; single lab with orthogonal methods","pmids":["41513627"],"is_preprint":false},{"year":2025,"finding":"MARCH2 mediates K48-linked polyubiquitination of TIM-1 at K338, causing proteasomal degradation of TIM-1. MARCH2/3 double knockout increases TIM-1 levels, enhancing Zika virus infectivity in a TIM-1-dependent manner. A ubiquitination-resistant TIM-1 K338R/K346R mutant increases ZIKV infectivity more than wild-type TIM-1.","method":"Co-immunoprecipitation, K48-linkage ubiquitination assay, site-directed mutagenesis (K338R/K346R), MARCH2/3 double-knockout mice, ZIKV infection assay","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, site-specific ubiquitination, ubiquitination-resistant mutant rescue, and in vivo KO mouse validation","pmids":["40817191"],"is_preprint":false},{"year":2025,"finding":"TNF stimulation induces MARCH2 dimerization; dimerized MARCH2 undergoes K63-linked autoubiquitination at lysines 127 and 238, which promotes MARCH2's ability to recognize and ubiquitinate NEMO for proteasomal degradation. In resting cells, MARCH2 interacts with MARCH8, which inhibits MARCH2 activation.","method":"Affinity purification, LC-MS/MS, co-immunoprecipitation, ubiquitination assay (K63-linked autoubiquitination), site-directed mutagenesis (K127/K238), MARCH2-/- colitis model","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS identification of PTMs, Co-IP, site-directed mutagenesis, MARCH2-/- in vivo model; single lab","pmids":["40450320"],"is_preprint":false},{"year":2026,"finding":"MARCH2 enhances protein stability of NR1H2 (LXRβ) via K27-linked polyubiquitination in cardiac macrophages, leading to upregulation of the efferocytosis receptor MERTK and promoting clearance of apoptotic cardiomyocytes (efferocytosis). MARCH2 global or macrophage-specific knockout impairs efferocytosis and worsens doxorubicin-induced cardiomyopathy.","method":"Co-immunoprecipitation, K27-linked ubiquitination assay, MARCH2-/- and CX3CR1Cre conditional KO mice, efferocytosis assay, cardiac function measurement","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, specific ubiquitin-linkage (stabilizing K27), conditional KO mice with multiple mechanistic readouts; single lab","pmids":["41963318"],"is_preprint":false},{"year":2026,"finding":"MARCH2 interacts with IL-2Rα and catalyzes its K27-linked polyubiquitination at K267, resulting in proteasomal degradation. K267 mutation impairs MARCH2-mediated ubiquitination. MARCH2 deficiency promotes IL-2-triggered STAT5 phosphorylation, effector gene expression, and T cell proliferation.","method":"Co-immunoprecipitation, K27-linkage ubiquitination assay, site-directed mutagenesis (K267R), MARCH2 knockout, STAT5 phosphorylation assay, T cell proliferation assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, site-specific ubiquitination with mutagenesis rescue, KO with signaling and proliferation readouts; single lab with orthogonal methods","pmids":["42026765"],"is_preprint":false},{"year":2026,"finding":"Intracellular LRG1 recruits MARCH2 to VE-cadherin and MARCH2 catalyzes K48-linked polyubiquitination of VE-cadherin at K633, leading to proteasomal degradation, endothelial barrier disruption, and acute lung injury in sepsis. LRG1 deletion or PROTAC-mediated degradation of LRG1 reduces VE-cadherin loss and mitigates ALI.","method":"Co-immunoprecipitation, K48-linkage ubiquitination assay, site-directed mutagenesis (K633), Lrg1 knockout mice, endothelial permeability assay, sepsis ALI model","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, site-specific ubiquitination, in vivo KO model; single lab","pmids":["42045382"],"is_preprint":false},{"year":2023,"finding":"In grass carp, the MARCH2 ortholog (Cimarch2) binds TBK1 and promotes its degradation via the proteasome pathway, thereby suppressing IFN activation induced by poly I:C, SVCV, and GCRV. Overexpression promotes viral replication while knockdown enhances IFN responses.","method":"Co-immunoprecipitation, proteasome inhibitor experiment, overexpression and knockdown, IFN reporter assay, viral replication assay","journal":"Fish & shellfish immunology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and functional assays in a teleost ortholog; single lab, uncertain conservation in mammalian MARCH2","pmids":["37490971"],"is_preprint":false}],"current_model":"MARCHF2 (MARCH2) is a transmembrane RING-CH E3 ubiquitin ligase that localizes to endosomal vesicles and the TGN, where it regulates vesicular trafficking by binding syntaxin 6 (and CAL/STX6 adaptors) and ubiquitinating a broad array of substrates — including β2-adrenergic receptor, CFTR, ERGIC3, NEMO (K48-linked, at K326), TNF-R1 (K27-linked, at K340), IL-5Rα (K27-linked), TIM-1 (K48-linked), IL-2Rα (K27-linked, at K267), PGAM5 (K48-linked), PKM2 (K33-linked), ALK5 (K63-linked, at K342/343, activating), SNAIL, Dishevelled, PTPRD (K27-linked), NR1H2 (K27-linked, stabilizing), and VE-cadherin (K48-linked, at K633) — directing them to proteasomal or lysosomal degradation (or, in the case of ALK5 and NR1H2, modulating activity/stability) and thereby controlling endocytic trafficking, innate immune signaling, Wnt pathway activity, autophagy, ER stress, and cell surface receptor homeostasis; its own activation is regulated by TNF-induced dimerization and K63-linked autoubiquitination at K127/K238, and is inhibited in resting cells by interaction with MARCH8."},"narrative":{"mechanistic_narrative":"MARCHF2 (MARCH2) is a transmembrane RING-CH E3 ubiquitin ligase that governs membrane-protein homeostasis by controlling vesicular trafficking and the stability of cell-surface receptors and signaling proteins [PMID:15689499, PMID:23818989]. It localizes to endosomal vesicles and the plasma membrane and directly binds syntaxin 6, redistributing syntaxin-6-containing SNAREs and perturbing retrograde TGN transport and transferrin-receptor surface levels [PMID:15689499]. Substrate engagement is frequently directed through PDZ-based scaffolds: MARCH2 binds and ubiquitinates the PDZ protein DLG1 at cell-cell contacts [PMID:17980554], and uses the CAL/STX6 adaptor system, in a RING-catalysis- and PDZ-motif-dependent manner, to ubiquitinate mature CFTR and route it to lysosomal degradation [PMID:23818989]. Across many contexts MARCH2 acts as a degradative regulator, ubiquitinating ERGIC3 to limit secretory cargo export [PMID:31142615], the β2-adrenergic receptor for ligand-induced lysosomal degradation [PMID:23166351], and a broad set of immune and signaling substrates using distinct ubiquitin linkages — K48-linked degradation of NEMO at K326 to suppress NF-κB/interferon signaling [PMID:32935379], K27-linked degradation of TNF-R1 at K340 [PMID:41961857], IL-5Rα [PMID:35982175], IL-2Rα at K267 [PMID:42026765], and PTPRD [PMID:41513627], and K48-linked degradation of PGAM5 [PMID:38409220], TIM-1 [PMID:40817191], and VE-cadherin at K633 [PMID:42045382]. Not all outputs are degradative: MARCH2 stabilizes NR1H2 (LXRβ) via K27-linked ubiquitination to promote macrophage efferocytosis [PMID:41963318], enhances ALK5 catalytic activity through endosomal K63-linked ubiquitination to potentiate TGF-β/SMAD2 signaling [PMID:41213909], and drives PKM2 tetramerization via K33-linked ubiquitination [PMID:40079144]. Through these activities MARCH2 shapes innate immune signaling, receptor surface levels, autophagy and ER stress, Wnt and EMT programs, and antiviral defense in vivo [PMID:32935379, PMID:27308891, PMID:28749466, PMID:38457262]. Its catalytic output is regulated: TNF triggers MARCH2 dimerization and K63-linked autoubiquitination at K127/K238 to license NEMO targeting, while MARCH8 binding restrains MARCH2 in resting cells [PMID:40450320].","teleology":[{"year":2005,"claim":"Established MARCH2's foundational cellular role by showing it is an endosomal/plasma-membrane protein that binds syntaxin 6 and controls retrograde TGN trafficking and surface receptor levels, framing it as a trafficking regulator.","evidence":"Immunoprecipitation, in vitro binding, siRNA knockdown, immunofluorescence, and transferrin uptake assays","pmids":["15689499"],"confidence":"High","gaps":["Did not establish E3 ligase catalytic activity on a defined substrate","Mechanism of substrate selection within the SNARE machinery unresolved"]},{"year":2007,"claim":"Connected MARCH2 substrate recruitment to PDZ-scaffold recognition, showing PDZ-dependent binding and ubiquitination of DLG1 at cell-cell contacts.","evidence":"TAP-MS, co-immunoprecipitation, in vivo ubiquitination assay, immunofluorescence","pmids":["17980554"],"confidence":"Medium","gaps":["Ubiquitin linkage type and fate of DLG1 not defined","Physiological consequence of DLG1 ubiquitination not established"]},{"year":2013,"claim":"Demonstrated catalytic RING- and PDZ-motif-dependent degradation of a physiological substrate (CFTR) via the CAL/STX6 adaptor, linking trafficking partners to substrate ubiquitination.","evidence":"Co-IP, RING-domain mutagenesis, in vivo ubiquitination, siRNA rescue in CF epithelial cells, bafilomycin treatment","pmids":["23818989"],"confidence":"High","gaps":["Acceptor lysines on CFTR not mapped","Relative contribution of lysosomal vs proteasomal routing not fully resolved"]},{"year":2012,"claim":"Showed ligand-conditional substrate targeting: carvedilol-bound β2AR is selectively ubiquitinated and degraded by MARCH2 independent of β-arrestin, revealing a biased-agonism-coupled degradation mechanism.","evidence":"LC-MS/MS, Co-IP, siRNA rescue, ubiquitination and receptor degradation assays in vascular smooth muscle cells","pmids":["23166351"],"confidence":"High","gaps":["Structural basis for carvedilol-stabilized binding unknown","β2AR acceptor lysines not mapped"]},{"year":2019,"claim":"Provided high-resolution substrate mechanism by mapping ERGIC3 acceptor lysines (K6/K8) and showing ubiquitination controls secretory cargo export.","evidence":"Co-IP, K6R/K8R site-directed mutagenesis with rescue, siRNA, secretion and ubiquitination assays","pmids":["31142615"],"confidence":"High","gaps":["Ubiquitin linkage type on ERGIC3 not specified","Generality across secretory cargo beyond AAT/haptoglobin untested"]},{"year":2020,"claim":"Defined MARCH2 as a brake on innate immune signaling by K48-linked degradation of NEMO at K326, with in vivo immune phenotypes.","evidence":"Co-IP, K326 mutagenesis, K48-linkage determination, MARCH2-/- mouse infection and cytokine studies","pmids":["32935379"],"confidence":"High","gaps":["Timing/trigger of late-phase recruitment to NEMO not fully mechanized","Relationship to other NF-κB regulators unresolved at this stage"]},{"year":2022,"claim":"Extended receptor-homeostasis role to cytokine receptors, showing K27-linked degradation of IL-5Rα controls eosinophil biology in vivo.","evidence":"Co-IP, K27-linkage and site-specific ubiquitination, MARCH2/3 double-KO mice, OVA airway model","pmids":["35982175"],"confidence":"High","gaps":["Functional redundancy versus distinct roles of MARCH2 and MARCH3 not separated","Trafficking route to lysosome for IL-5Rα not detailed"]},{"year":2025,"claim":"Revealed non-degradative, activating ubiquitination modes: K63-linked modification of ALK5 enhancing TGF-β signaling and K33-linked modification driving PKM2 tetramerization and metabolic control.","evidence":"Co-IP, linkage-specific ubiquitination, site-directed mutagenesis (ALK5 K342/343; PKM2), KO and pharmacological rescue, in vivo metastasis and aortic disease models","pmids":["41213909","40079144"],"confidence":"Medium","gaps":["How linkage specificity (K63 vs K33 vs K48) is determined remains unexplained","Structural determinants distinguishing activating from degradative outputs unknown"]},{"year":2025,"claim":"Elucidated regulation of MARCH2 itself, showing TNF-induced dimerization and K63-linked autoubiquitination at K127/K238 license NEMO targeting, with MARCH8 acting as an inhibitor in resting cells.","evidence":"AP-MS, Co-IP, K63-linked autoubiquitination assays, K127/K238 mutagenesis, MARCH2-/- colitis model","pmids":["40450320"],"confidence":"Medium","gaps":["Structural basis of dimerization and MARCH8 inhibition not resolved","Whether autoubiquitination gates substrates beyond NEMO untested"]},{"year":2026,"claim":"Showed MARCH2 can stabilize substrates, with K27-linked ubiquitination of NR1H2 (LXRβ) upregulating MERTK to drive macrophage efferocytosis and cardioprotection.","evidence":"Co-IP, K27-linkage ubiquitination, global and macrophage-specific KO mice, efferocytosis and cardiac function assays","pmids":["41963318"],"confidence":"High","gaps":["Mechanism by which K27 chains stabilize rather than degrade NR1H2 unresolved","Acceptor lysine(s) on NR1H2 not mapped"]},{"year":null,"claim":"It remains unresolved what molecular features dictate MARCH2's choice of ubiquitin-chain linkage (K27/K33/K48/K63) and degradative versus stabilizing/activating outcomes across its diverse substrate set.","evidence":"No single study in the corpus reconstitutes linkage-determination or substrate-discrimination rules","pmids":[],"confidence":"Low","gaps":["No structural model of MARCH2 with substrates or E2 partners","Determinants of substrate vs adaptor recognition across contexts not unified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,9,10,19,21]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[4,9,10]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[17,18,23]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,18]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[16]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[10,12,19,21,24]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,3,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,9,10,19]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[18,7,10]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,4]}],"complexes":[],"partners":["STX6","DLG1","CAL","CFTR","NEMO","TNF-R1","MARCH8","PGAM5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P0N8","full_name":"E3 ubiquitin-protein ligase MARCHF2","aliases":["Membrane-associated RING finger protein 2","Membrane-associated RING-CH protein II","MARCH-II","RING finger protein 172","RING-type E3 ubiquitin transferase MARCHF2"],"length_aa":246,"mass_kda":27.0,"function":"E3 ubiquitin-protein ligase that may mediate ubiquitination of TFRC and CD86, and promote their subsequent endocytosis and sorting to lysosomes via multivesicular bodies. E3 ubiquitin ligases accept ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfer the ubiquitin to targeted substrates (PubMed:14722266, PubMed:16428329). Together with GOPC/CAL mediates the ubiquitination and lysosomal degradation of CFTR (PubMed:23818989). Ubiquitinates and therefore mediates the degradation of DLG1 (PubMed:17980554). Regulates the intracellular trafficking and secretion of alpha1-antitrypsin/SERPINA1 and HP/haptoglobin via ubiquitination and degradation of the cargo receptor ERGIC3 (PubMed:31142615). Negatively regulates the antiviral and antibacterial immune response by repression of the NF-kB and type 1 IFN signaling pathways, via MARCHF2-mediated K48-linked polyubiquitination of IKBKG/NEMO, resulting in its proteasomal degradation (PubMed:32935379). May be involved in endosomal trafficking through interaction with STX6 (PubMed:15689499) (Microbial infection) Positively regulates the degradation of Vesicular stomatitis virus (VSV) G protein via the lysosomal degradation pathway (PubMed:29573664). Represses HIV-1 viral production and may inhibit the translocation of HIV-1 env to the cell surface, resulting in decreased viral cell-cell transmission (PubMed:29573664)","subcellular_location":"Endoplasmic reticulum membrane; Lysosome membrane; Endosome membrane; Golgi apparatus membrane; Cytoplasm; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9P0N8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MARCHF2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1090,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MARCHF2","total_profiled":1310},"omim":[{"mim_id":"613332","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 2; MARCHF2","url":"https://www.omim.org/entry/613332"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MARCHF2"},"hgnc":{"alias_symbol":["HSPC240","MARCH-II","RNF172"],"prev_symbol":["MARCH2"]},"alphafold":{"accession":"Q9P0N8","domains":[{"cath_id":"3.30.40.10","chopping":"75-115","consensus_level":"high","plddt":92.1561,"start":75,"end":115},{"cath_id":"1.10.287","chopping":"123-214","consensus_level":"high","plddt":85.1897,"start":123,"end":214}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P0N8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P0N8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P0N8-F1-predicted_aligned_error_v6.png","plddt_mean":71.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MARCHF2","jax_strain_url":"https://www.jax.org/strain/search?query=MARCHF2"},"sequence":{"accession":"Q9P0N8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P0N8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P0N8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P0N8"}},"corpus_meta":[{"pmid":"38409220","id":"PMC_38409220","title":"The E3 ubiquitin ligase MARCH2 protects against myocardial ischemia-reperfusion injury through inhibiting pyroptosis via negative regulation of PGAM5/MAVS/NLRP3 axis.","date":"2024","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/38409220","citation_count":90,"is_preprint":false},{"pmid":"15689499","id":"PMC_15689499","title":"MARCH-II is a syntaxin-6-binding protein involved in endosomal trafficking.","date":"2005","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/15689499","citation_count":78,"is_preprint":false},{"pmid":"23166351","id":"PMC_23166351","title":"MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β(2)-adrenergic receptors.","date":"2012","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/23166351","citation_count":56,"is_preprint":false},{"pmid":"23818989","id":"PMC_23818989","title":"Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23818989","citation_count":49,"is_preprint":false},{"pmid":"29573664","id":"PMC_29573664","title":"MARCH2 is upregulated in HIV-1 infection and inhibits HIV-1 production through envelope protein translocation or degradation.","date":"2018","source":"Virology","url":"https://pubmed.ncbi.nlm.nih.gov/29573664","citation_count":48,"is_preprint":false},{"pmid":"16428329","id":"PMC_16428329","title":"MARCH-III Is a novel component of endosomes with properties similar to those of MARCH-II.","date":"2006","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16428329","citation_count":45,"is_preprint":false},{"pmid":"27308891","id":"PMC_27308891","title":"MARCH2 regulates autophagy by promoting CFTR ubiquitination and degradation and PIK3CA-AKT-MTOR signaling.","date":"2016","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/27308891","citation_count":43,"is_preprint":false},{"pmid":"32935379","id":"PMC_32935379","title":"Negative regulation of NEMO signaling by the ubiquitin E3 ligase MARCH2.","date":"2020","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/32935379","citation_count":33,"is_preprint":false},{"pmid":"17980554","id":"PMC_17980554","title":"DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact.","date":"2007","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/17980554","citation_count":30,"is_preprint":false},{"pmid":"28749466","id":"PMC_28749466","title":"Knockout of MARCH2 inhibits the growth of HCT116 colon cancer cells by inducing endoplasmic reticulum stress.","date":"2017","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/28749466","citation_count":30,"is_preprint":false},{"pmid":"35982175","id":"PMC_35982175","title":"The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target IL-5 receptor alpha to negatively regulate eosinophilic airway inflammation.","date":"2022","source":"Cellular & molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35982175","citation_count":27,"is_preprint":false},{"pmid":"40079144","id":"PMC_40079144","title":"March2 Alleviates Aortic Aneurysm/Dissection by Regulating PKM2 Polymerization.","date":"2025","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/40079144","citation_count":19,"is_preprint":false},{"pmid":"31142615","id":"PMC_31142615","title":"The E3 ubiquitin ligase MARCH2 regulates ERGIC3-dependent trafficking of secretory proteins.","date":"2019","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/31142615","citation_count":18,"is_preprint":false},{"pmid":"29549110","id":"PMC_29549110","title":"Head formation requires Dishevelled degradation that is mediated by March2 in concert with Dapper1.","date":"2018","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/29549110","citation_count":17,"is_preprint":false},{"pmid":"38457262","id":"PMC_38457262","title":"MARCH2, a Novel Oncogene-regulated SNAIL E3 Ligase, Suppresses Triple-negative Breast Cancer Metastases.","date":"2024","source":"Cancer research communications","url":"https://pubmed.ncbi.nlm.nih.gov/38457262","citation_count":11,"is_preprint":false},{"pmid":"38936155","id":"PMC_38936155","title":"MARCH1 and MARCH2 inhibit pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in trans-Golgi network.","date":"2024","source":"Veterinary microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/38936155","citation_count":7,"is_preprint":false},{"pmid":"40817191","id":"PMC_40817191","title":"The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target TIM-1 to limit Zika virus infection.","date":"2025","source":"Cellular & molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40817191","citation_count":6,"is_preprint":false},{"pmid":"38848952","id":"PMC_38848952","title":"KLF15-activated MARCH2 boosts cell proliferation and epithelial-mesenchymal transition and presents diagnostic significance for hepatocellular carcinoma.","date":"2024","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/38848952","citation_count":6,"is_preprint":false},{"pmid":"39074162","id":"PMC_39074162","title":"MARCH2, a T cell specific factor that restricts HIV-1 infection.","date":"2024","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/39074162","citation_count":6,"is_preprint":false},{"pmid":"33945654","id":"PMC_33945654","title":"Proteomic analyses reveal that immune integrins are major targets for regulation by Membrane-Associated Ring-CH (MARCH) proteins MARCH2, 3, 4 and 9.","date":"2021","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/33945654","citation_count":6,"is_preprint":false},{"pmid":"36959742","id":"PMC_36959742","title":"Silencing MARCH2 Inhibits the Invasion, Migration, and EMT Transformation of Human Colorectal Cancer SW480 Cells.","date":"2023","source":"Technology in cancer research & treatment","url":"https://pubmed.ncbi.nlm.nih.gov/36959742","citation_count":4,"is_preprint":false},{"pmid":"37490971","id":"PMC_37490971","title":"march2 negatively regulates antiviral immune response by targeting tbk1 in grass carp (Ctenopharyngodon idella).","date":"2023","source":"Fish & shellfish immunology","url":"https://pubmed.ncbi.nlm.nih.gov/37490971","citation_count":4,"is_preprint":false},{"pmid":"40450320","id":"PMC_40450320","title":"The E3 ubiquitin ligase MARCH2 controls TNF-α mediated inflammation by autoubiquitination.","date":"2025","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/40450320","citation_count":1,"is_preprint":false},{"pmid":"37786722","id":"PMC_37786722","title":"Determining the antiviral mechanism of MARCH2.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37786722","citation_count":0,"is_preprint":false},{"pmid":"41513627","id":"PMC_41513627","title":"MARCH2 suppresses odontoblast differentiation by polyubiquitinating PTPRD.","date":"2026","source":"International journal of oral science","url":"https://pubmed.ncbi.nlm.nih.gov/41513627","citation_count":0,"is_preprint":false},{"pmid":"41334913","id":"PMC_41334913","title":"MARCH2 inhibits avian leukosis virus replication by targeting gp85 for ubiquitination and degradation.","date":"2025","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/41334913","citation_count":0,"is_preprint":false},{"pmid":"41961857","id":"PMC_41961857","title":"Reciprocal regulation of TNF receptor 1-mediated signaling and inflammatory damages by MARCH2 and USP22.","date":"2026","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41961857","citation_count":0,"is_preprint":false},{"pmid":"41963318","id":"PMC_41963318","title":"MARCH2 prevents doxorubicin-induced cardiomyopathy by stabilizing NR1H2 and promoting clearance of apoptotic cardiomyocytes.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41963318","citation_count":0,"is_preprint":false},{"pmid":"41213909","id":"PMC_41213909","title":"MARCH2-mediated Lys63-linked polyubiquitination promotes metastasis by modulating the catalytic activity of TGF-β type I receptor.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41213909","citation_count":0,"is_preprint":false},{"pmid":"42026765","id":"PMC_42026765","title":"MARCH2 mediates K27-Linked polyubiquitination of IL-2 receptor α to negatively regulate T cell proliferation.","date":"2026","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/42026765","citation_count":0,"is_preprint":false},{"pmid":"42045382","id":"PMC_42045382","title":"Intracellular LRG1 recruits MARCH2 to ubiquitinate and degrade endothelial VE-cadherin in septic lung injury.","date":"2026","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/42045382","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16979,"output_tokens":7779,"usd":0.083811,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":17247,"output_tokens":4176,"usd":0.095317,"stage2_stop_reason":"end_turn"},"total_usd":0.179128,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"MARCH2 (MARCH-II) localizes to endosomal vesicles and the plasma membrane, directly associates with syntaxin 6 (established by immunoprecipitation and in vitro binding), redistributes syntaxin 6 and syntaxin-6-interacting SNAREs into MARCH2-positive vesicles upon overexpression, perturbs retrograde transport of TGN38 and furin to the TGN, reduces cell surface transferrin receptor expression and transferrin uptake, and depletion by siRNA disrupts TGN localization of syntaxin 6 and TGN38/46.\",\n      \"method\": \"Immunoprecipitation, in vitro binding assay, siRNA knockdown, immunofluorescence microscopy, transferrin uptake assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus in vitro binding, siRNA rescue, multiple orthogonal functional readouts in a single focused study\",\n      \"pmids\": [\"15689499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MARCH2 (MARCH-II) associates with MARCH3 by immunoprecipitation, and both share endosomal localization and syntaxin 6 binding; the PDZ-binding motif and RING finger of MARCH3 are essential for its localization and function, providing comparative structural context for MARCH2 domain requirements.\",\n      \"method\": \"Immunoprecipitation, immunofluorescence, mutational analysis\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP for MARCH2–MARCH3 interaction; primary focus is MARCH3 characterization but MARCH2 interaction confirmed\",\n      \"pmids\": [\"16428329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"MARCH2 interacts with the PDZ scaffold protein DLG1 in a PDZ domain-dependent manner (identified by tandem affinity purification/mass spectrometry and confirmed by co-immunoprecipitation), co-localizes with DLG1 at sites of cell-cell contact, and promotes DLG1 ubiquitination in vivo ubiquitination assays. Loss of the MARCH2 PDZ-binding motif abolishes its localization to cell-cell contact sites.\",\n      \"method\": \"Tandem affinity purification, mass spectrometry, co-immunoprecipitation, in vivo ubiquitination assay, immunofluorescence\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TAP-MS identification confirmed by Co-IP, ubiquitination assay, and localization experiment; single lab\",\n      \"pmids\": [\"17980554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MARCH2 interacts with carvedilol-bound β2-adrenergic receptor (β2AR) (identified by LC-MS/MS proteomics and confirmed by co-immunoprecipitation), and its E3 ligase activity mediates ubiquitination, endocytosis, and lysosomal degradation of β2AR specifically in response to carvedilol. siRNA-mediated knockdown of MARCH2 abolished carvedilol-induced β2AR ubiquitination, endocytosis, and degradation in vascular smooth muscle cells. The MARCH2–β2AR association is stabilized by carvedilol and does not require β-arrestin.\",\n      \"method\": \"LC-MS/MS proteomics, co-immunoprecipitation, siRNA knockdown, ubiquitination assay, cell surface receptor degradation assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS identification confirmed by Co-IP, siRNA rescue, and multiple functional readouts (ubiquitination, endocytosis, degradation) in both overexpression and endogenous systems\",\n      \"pmids\": [\"23166351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MARCH2 co-immunoprecipitates and co-localizes with the adaptor proteins CAL and STX6; its binding to CAL is enhanced synergistically by STX6. MARCH2 ubiquitinates mature CFTR and promotes its lysosomal degradation in a catalytic RING-domain-dependent manner. A catalytically dead RING mutant of MARCH2 fails to degrade CFTR. MARCH2 has no effect on CFTR lacking its PDZ motif, and silencing of CAL abolishes MARCH2-mediated CFTR degradation. siRNA-mediated silencing of MARCH2 in CF epithelial cells increases mature CFTR abundance.\",\n      \"method\": \"Co-immunoprecipitation, co-localization (immunofluorescence), in vivo ubiquitination assay, RING domain mutagenesis, siRNA knockdown, bafilomycin A1 treatment\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — catalytic mutagenesis, co-IP, ubiquitination assay, PDZ-motif requirement, and siRNA rescue with multiple orthogonal methods in one study\",\n      \"pmids\": [\"23818989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MARCH2 overexpression impairs autophagy (reduces LC3B-II levels, impairs autophagic substrate degradation) whereas MARCH2 loss promotes autophagy via the PIK3CA-AKT-MTOR signaling pathway. MARCH2 interacts with CFTR via its PDZ domain, promotes CFTR ubiquitination and degradation, and thereby inhibits CFTR-mediated autophagy in tumor cells.\",\n      \"method\": \"Overexpression, siRNA/knockout, LC3B-II western blot, autophagic flux assay, co-immunoprecipitation, ubiquitination assay, in vivo tumorigenicity assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and functional autophagy readouts; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27308891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MARCH2 knockout (via CRISPR/Cas9) in HCT116 colon cancer cells suppresses proliferation and promotes autophagy, apoptosis, and G2/M arrest associated with activation of endoplasmic reticulum stress, establishing MARCH2 as a negative regulator of ER stress in this cellular context.\",\n      \"method\": \"CRISPR/Cas9 knockout, cell proliferation assay, flow cytometry (apoptosis, cell cycle), autophagy assay, ER stress marker analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined phenotypic readouts; single lab, no in vitro biochemical reconstitution of ER stress mechanism\",\n      \"pmids\": [\"28749466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Xenopus, March2 (RING-type E3 ubiquitin ligase) promotes ubiquitin-mediated lysosomal degradation of Dishevelled (Dsh/Dvl), thereby antagonizing Wnt signaling. Dapper1 (Dpr1) stabilizes the March2–Dsh interaction and provides regional specificity in the dorso-animal region to control head formation.\",\n      \"method\": \"Xenopus embryo assays, co-immunoprecipitation, ubiquitination assay, lysosomal degradation assay, loss-of-function/gain-of-function in embryos\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and in vivo embryological epistasis; single lab, ortholog model system\",\n      \"pmids\": [\"29549110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MARCH2 inhibits HIV-1 production through E3 ligase activity-dependent envelope protein degradation and/or intracellular retention (translocation), a mechanism similar to MARCH8. MARCH2 expression is upregulated upon HIV-1 infection.\",\n      \"method\": \"Overexpression with RING-dead mutant comparison, viral titer assay, envelope protein localization\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RING-domain activity requirement demonstrated by mutant comparison, viral function assay; single lab\",\n      \"pmids\": [\"29573664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MARCH2 ubiquitinates ERGIC3 at lysines 6 and 8, leading to its proteasomal degradation; MARCH2 depletion increases endogenous ERGIC3 levels. MARCH2-mediated ERGIC3 ubiquitination reduces secretion of ERGIC3 cargo proteins α1-antitrypsin and haptoglobin. An ERGIC3 K6R/K8R mutant resistant to ubiquitination rescues secretion, confirming ERGIC3 ubiquitination as the primary mechanism.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (K6R/K8R), siRNA knockdown, secretion assay, ubiquitination assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — site-directed mutagenesis identifying specific ubiquitin acceptor lysines, rescue experiment with ubiquitination-resistant mutant, multiple cargo secretion assays; single lab with orthogonal methods\",\n      \"pmids\": [\"31142615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MARCH2 directly interacts with NEMO (NF-κB essential modulator) during the late phase of bacterial/viral infection and catalyzes K48-linked ubiquitination of NEMO at Lys326, resulting in NEMO proteasomal degradation and suppression of NF-κB and interferon signaling. MARCH2 knockout mice show increased innate immune responses and resistance to bacterial/viral infection, but enhanced susceptibility to LPS-induced cytokine storm.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K326), MARCH2-/- mouse model, bacterial/viral infection assays, cytokine measurement\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, site-specific mutagenesis, K48-linkage determination, in vivo KO mouse model with multiple readouts; single lab with orthogonal methods\",\n      \"pmids\": [\"32935379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MARCH2 (along with MARCH3, 4, and 9) downregulates cell surface expression of integrin complexes. Specifically, Integrin α4 was upregulated in mature B-lymphocytes of MARCH2 knockout mice, accompanied by decreased B-cell numbers in the spleen, establishing MARCH2 as a regulator of integrin α4β1 (VLA4) surface levels.\",\n      \"method\": \"Cell surface proteomics, MARCH2 knockout mouse analysis, flow cytometry\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell surface proteomics and KO mouse validation; single lab, broader screen study with specific MARCH2 finding for integrin α4\",\n      \"pmids\": [\"33945654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCH2 and MARCH3 associate with IL-5 receptor α (IL-5Rα) and mediate its K27-linked polyubiquitination at K379 (by MARCH2) and K383 (by MARCH3), leading to lysosomal degradation. MARCH2/3 double knockout markedly increases IL-5Rα levels, enhances IL-5-induced signaling, increases eosinophil proportions in bone marrow and blood, and aggravates OVA-induced airway eosinophilia in mice.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K27-linkage), site-directed mutagenesis, MARCH2/3 double-knockout mouse model, flow cytometry, OVA challenge model\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, K27-linkage and site-specific ubiquitination, in vivo KO mouse model with multiple orthogonal readouts\",\n      \"pmids\": [\"35982175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCH2 directly interacts with PGAM5 and promotes its K48-linked polyubiquitination and proteasomal degradation. This prevents PGAM5 from forming liquid-liquid phase separation condensates with MAVS and recruiting NLRP3, thereby inhibiting NLRP3 inflammasome activation and cardiomyocyte pyroptosis during myocardial ischemia-reperfusion injury. MARCH2 genetic ablation aggravates myocardial infarction; AAV-based MARCH2 re-introduction ameliorates I/R-induced cardiac dysfunction.\",\n      \"method\": \"Co-immunoprecipitation, K48-linked ubiquitination assay, MARCH2 knockout mouse, single-cell RNA-seq, AAV gene delivery, phase separation assay, cardiac function measurement\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, specific ubiquitin-linkage determination, phase separation assay, KO and AAV rescue in vivo; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38409220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCH2 is a novel E3 ligase for SNAIL: its RING domain mediates SNAIL ubiquitination and degradation, and PTK6 tyrosine kinase inhibition promotes the MARCH2–SNAIL interaction. MARCH2 overexpression suppresses EMT, cell migration, anoikis resistance, and metastasis of triple-negative breast cancer cells, phenocopying effects of SNAIL downregulation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RING domain mutagenesis, migration/invasion assay, in vivo metastasis model\",\n      \"journal\": \"Cancer research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RING domain mutagenesis, Co-IP, functional metastasis assays; single lab\",\n      \"pmids\": [\"38457262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Human MARCH2 (but not mouse MARCH2) restricts HIV-1 infection in primary CD4+ T cells; a specific amino acid absent in mouse MARCH2 is critical for antiretroviral function. MARCH2 domains required for binding and restriction of HIV-1 envelope glycoproteins were mapped. MARCH2 is incorporated inside nascent virions and reduces particle infectivity by blocking virus entry in a RING-CH-independent manner. MARCH2 prevents cell-to-cell HIV-1 transmission.\",\n      \"method\": \"Domain mapping/mutagenesis, viral infectivity assay, virion incorporation assay, primary CD4+ T cell infection, cell-to-cell transmission assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis, multiple functional readouts in primary cells; single lab\",\n      \"pmids\": [\"39074162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCH1 and MARCH2 inhibit pseudorabies virus (PRV) replication at the cell-to-cell fusion step by associating with the viral gB/gD/gH/gL fusion complex and trapping it in the trans-Golgi network rather than degrading the proteins; this mechanism requires E3 ligase activity for blocking gB furin cleavage, though blocking gB cleavage itself does not account for the antiviral activity.\",\n      \"method\": \"Co-immunoprecipitation, viral replication assay, TGN localization assay, RING-dead mutant comparison\",\n      \"journal\": \"Veterinary microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of MARCH2 with viral fusion complex, localization assay, RING activity requirement; single lab\",\n      \"pmids\": [\"38936155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH2 interacts with PKM2 and promotes its K33-linked polyubiquitination, stimulating PKM2 dimer-to-tetramer conversion. In MARCH2-deficient vascular smooth muscle cells, reduced PKM2 tetramerization leads to glucose metabolism reprogramming, increased histone H3K18 lactylation, and p53-driven apoptosis, exacerbating aortic aneurysm/dissection. The PKM2 activator TEPP-46 rescues MARCH2 deficiency-worsened pathology.\",\n      \"method\": \"Co-immunoprecipitation, K33-linkage ubiquitination assay, MARCH2-/- and SMC-specific KO mice, PKM2 tetramer assay, metabolomics/ChIP (CUT&TAG), pharmacological rescue\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, specific ubiquitin-linkage determination, in vivo KO and pharmacological rescue with mechanistic pathway placement\",\n      \"pmids\": [\"40079144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH2 mediates K63-linked polyubiquitination of ALK5 (TGF-β type I receptor) at lysines 342/343 at endosomes following TGF-β-induced endocytosis, enhancing ALK5 catalytic activity. ALK5 K342/343R mutations abolish SMAD2 phosphorylation and TGF-β responses and reduce lung metastasis in a mouse model.\",\n      \"method\": \"Co-immunoprecipitation, K63-linkage ubiquitination assay, site-directed mutagenesis (K342/343R), SMAD2 phosphorylation assay, cell migration assay, in vivo lung metastasis model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis, K63-linkage ubiquitination, functional catalytic activity and in vivo metastasis; single lab\",\n      \"pmids\": [\"41213909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH2 constitutively associates with TNF-R1 and mediates K27-linked polyubiquitination of TNF-R1 at K340, resulting in its proteasomal degradation and dampening of TNF-triggered signaling. The deubiquitinase USP22 antagonizes this by removing K27-linked ubiquitin from K340, stabilizing TNF-R1. MARCH2 deficiency promotes TNF-triggered signaling; in acute liver injury, MARCH2 deficiency exacerbates pathology.\",\n      \"method\": \"Co-immunoprecipitation, K27-linkage ubiquitination assay, site-directed mutagenesis (K340), deubiquitination assay, MARCH2-/- and USP22-/- mouse models, cytokine and liver injury assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, site-specific ubiquitination, deubiquitinase antagonism, and in vivo mouse models with multiple orthogonal readouts\",\n      \"pmids\": [\"41961857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH2 interacts with PTPRD and facilitates its K27-linked polyubiquitination and subsequent lysosomal degradation in a RING-domain-dependent manner, promoting PTPRD translocation from the cell membrane to lysosomes. This inhibits odontoblast differentiation. March2-deficient mice and odontoblast-specific March2 knockdown mice exhibit increased dentin thickness and elevated odontoblast marker expression. MARCH2/PTPRD double knockdown reverses the enhanced differentiation caused by MARCH2 knockdown alone.\",\n      \"method\": \"Co-immunoprecipitation, K27-linkage ubiquitination assay, RING-domain mutagenesis, March2 knockout and conditional knockdown mice, double knockdown epistasis, lysosome trafficking assay\",\n      \"journal\": \"International journal of oral science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RING mutagenesis, site-specific ubiquitination, lysosomal trafficking, in vivo KO and double-knockdown epistasis; single lab with orthogonal methods\",\n      \"pmids\": [\"41513627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH2 mediates K48-linked polyubiquitination of TIM-1 at K338, causing proteasomal degradation of TIM-1. MARCH2/3 double knockout increases TIM-1 levels, enhancing Zika virus infectivity in a TIM-1-dependent manner. A ubiquitination-resistant TIM-1 K338R/K346R mutant increases ZIKV infectivity more than wild-type TIM-1.\",\n      \"method\": \"Co-immunoprecipitation, K48-linkage ubiquitination assay, site-directed mutagenesis (K338R/K346R), MARCH2/3 double-knockout mice, ZIKV infection assay\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, site-specific ubiquitination, ubiquitination-resistant mutant rescue, and in vivo KO mouse validation\",\n      \"pmids\": [\"40817191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TNF stimulation induces MARCH2 dimerization; dimerized MARCH2 undergoes K63-linked autoubiquitination at lysines 127 and 238, which promotes MARCH2's ability to recognize and ubiquitinate NEMO for proteasomal degradation. In resting cells, MARCH2 interacts with MARCH8, which inhibits MARCH2 activation.\",\n      \"method\": \"Affinity purification, LC-MS/MS, co-immunoprecipitation, ubiquitination assay (K63-linked autoubiquitination), site-directed mutagenesis (K127/K238), MARCH2-/- colitis model\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS identification of PTMs, Co-IP, site-directed mutagenesis, MARCH2-/- in vivo model; single lab\",\n      \"pmids\": [\"40450320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MARCH2 enhances protein stability of NR1H2 (LXRβ) via K27-linked polyubiquitination in cardiac macrophages, leading to upregulation of the efferocytosis receptor MERTK and promoting clearance of apoptotic cardiomyocytes (efferocytosis). MARCH2 global or macrophage-specific knockout impairs efferocytosis and worsens doxorubicin-induced cardiomyopathy.\",\n      \"method\": \"Co-immunoprecipitation, K27-linked ubiquitination assay, MARCH2-/- and CX3CR1Cre conditional KO mice, efferocytosis assay, cardiac function measurement\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, specific ubiquitin-linkage (stabilizing K27), conditional KO mice with multiple mechanistic readouts; single lab\",\n      \"pmids\": [\"41963318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MARCH2 interacts with IL-2Rα and catalyzes its K27-linked polyubiquitination at K267, resulting in proteasomal degradation. K267 mutation impairs MARCH2-mediated ubiquitination. MARCH2 deficiency promotes IL-2-triggered STAT5 phosphorylation, effector gene expression, and T cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, K27-linkage ubiquitination assay, site-directed mutagenesis (K267R), MARCH2 knockout, STAT5 phosphorylation assay, T cell proliferation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, site-specific ubiquitination with mutagenesis rescue, KO with signaling and proliferation readouts; single lab with orthogonal methods\",\n      \"pmids\": [\"42026765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Intracellular LRG1 recruits MARCH2 to VE-cadherin and MARCH2 catalyzes K48-linked polyubiquitination of VE-cadherin at K633, leading to proteasomal degradation, endothelial barrier disruption, and acute lung injury in sepsis. LRG1 deletion or PROTAC-mediated degradation of LRG1 reduces VE-cadherin loss and mitigates ALI.\",\n      \"method\": \"Co-immunoprecipitation, K48-linkage ubiquitination assay, site-directed mutagenesis (K633), Lrg1 knockout mice, endothelial permeability assay, sepsis ALI model\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, site-specific ubiquitination, in vivo KO model; single lab\",\n      \"pmids\": [\"42045382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In grass carp, the MARCH2 ortholog (Cimarch2) binds TBK1 and promotes its degradation via the proteasome pathway, thereby suppressing IFN activation induced by poly I:C, SVCV, and GCRV. Overexpression promotes viral replication while knockdown enhances IFN responses.\",\n      \"method\": \"Co-immunoprecipitation, proteasome inhibitor experiment, overexpression and knockdown, IFN reporter assay, viral replication assay\",\n      \"journal\": \"Fish & shellfish immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and functional assays in a teleost ortholog; single lab, uncertain conservation in mammalian MARCH2\",\n      \"pmids\": [\"37490971\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MARCHF2 (MARCH2) is a transmembrane RING-CH E3 ubiquitin ligase that localizes to endosomal vesicles and the TGN, where it regulates vesicular trafficking by binding syntaxin 6 (and CAL/STX6 adaptors) and ubiquitinating a broad array of substrates — including β2-adrenergic receptor, CFTR, ERGIC3, NEMO (K48-linked, at K326), TNF-R1 (K27-linked, at K340), IL-5Rα (K27-linked), TIM-1 (K48-linked), IL-2Rα (K27-linked, at K267), PGAM5 (K48-linked), PKM2 (K33-linked), ALK5 (K63-linked, at K342/343, activating), SNAIL, Dishevelled, PTPRD (K27-linked), NR1H2 (K27-linked, stabilizing), and VE-cadherin (K48-linked, at K633) — directing them to proteasomal or lysosomal degradation (or, in the case of ALK5 and NR1H2, modulating activity/stability) and thereby controlling endocytic trafficking, innate immune signaling, Wnt pathway activity, autophagy, ER stress, and cell surface receptor homeostasis; its own activation is regulated by TNF-induced dimerization and K63-linked autoubiquitination at K127/K238, and is inhibited in resting cells by interaction with MARCH8.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MARCHF2 (MARCH2) is a transmembrane RING-CH E3 ubiquitin ligase that governs membrane-protein homeostasis by controlling vesicular trafficking and the stability of cell-surface receptors and signaling proteins [#0, #4]. It localizes to endosomal vesicles and the plasma membrane and directly binds syntaxin 6, redistributing syntaxin-6-containing SNAREs and perturbing retrograde TGN transport and transferrin-receptor surface levels [#0]. Substrate engagement is frequently directed through PDZ-based scaffolds: MARCH2 binds and ubiquitinates the PDZ protein DLG1 at cell-cell contacts [#2], and uses the CAL/STX6 adaptor system, in a RING-catalysis- and PDZ-motif-dependent manner, to ubiquitinate mature CFTR and route it to lysosomal degradation [#4]. Across many contexts MARCH2 acts as a degradative regulator, ubiquitinating ERGIC3 to limit secretory cargo export [#9], the \\u03b22-adrenergic receptor for ligand-induced lysosomal degradation [#3], and a broad set of immune and signaling substrates using distinct ubiquitin linkages \\u2014 K48-linked degradation of NEMO at K326 to suppress NF-\\u03baB/interferon signaling [#10], K27-linked degradation of TNF-R1 at K340 [#19], IL-5R\\u03b1 [#12], IL-2R\\u03b1 at K267 [#24], and PTPRD [#20], and K48-linked degradation of PGAM5 [#13], TIM-1 [#21], and VE-cadherin at K633 [#25]. Not all outputs are degradative: MARCH2 stabilizes NR1H2 (LXR\\u03b2) via K27-linked ubiquitination to promote macrophage efferocytosis [#23], enhances ALK5 catalytic activity through endosomal K63-linked ubiquitination to potentiate TGF-\\u03b2/SMAD2 signaling [#18], and drives PKM2 tetramerization via K33-linked ubiquitination [#17]. Through these activities MARCH2 shapes innate immune signaling, receptor surface levels, autophagy and ER stress, Wnt and EMT programs, and antiviral defense in vivo [#10, #5, #6, #14]. Its catalytic output is regulated: TNF triggers MARCH2 dimerization and K63-linked autoubiquitination at K127/K238 to license NEMO targeting, while MARCH8 binding restrains MARCH2 in resting cells [#22].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established MARCH2's foundational cellular role by showing it is an endosomal/plasma-membrane protein that binds syntaxin 6 and controls retrograde TGN trafficking and surface receptor levels, framing it as a trafficking regulator.\",\n      \"evidence\": \"Immunoprecipitation, in vitro binding, siRNA knockdown, immunofluorescence, and transferrin uptake assays\",\n      \"pmids\": [\"15689499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish E3 ligase catalytic activity on a defined substrate\", \"Mechanism of substrate selection within the SNARE machinery unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected MARCH2 substrate recruitment to PDZ-scaffold recognition, showing PDZ-dependent binding and ubiquitination of DLG1 at cell-cell contacts.\",\n      \"evidence\": \"TAP-MS, co-immunoprecipitation, in vivo ubiquitination assay, immunofluorescence\",\n      \"pmids\": [\"17980554\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin linkage type and fate of DLG1 not defined\", \"Physiological consequence of DLG1 ubiquitination not established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated catalytic RING- and PDZ-motif-dependent degradation of a physiological substrate (CFTR) via the CAL/STX6 adaptor, linking trafficking partners to substrate ubiquitination.\",\n      \"evidence\": \"Co-IP, RING-domain mutagenesis, in vivo ubiquitination, siRNA rescue in CF epithelial cells, bafilomycin treatment\",\n      \"pmids\": [\"23818989\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Acceptor lysines on CFTR not mapped\", \"Relative contribution of lysosomal vs proteasomal routing not fully resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed ligand-conditional substrate targeting: carvedilol-bound \\u03b22AR is selectively ubiquitinated and degraded by MARCH2 independent of \\u03b2-arrestin, revealing a biased-agonism-coupled degradation mechanism.\",\n      \"evidence\": \"LC-MS/MS, Co-IP, siRNA rescue, ubiquitination and receptor degradation assays in vascular smooth muscle cells\",\n      \"pmids\": [\"23166351\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for carvedilol-stabilized binding unknown\", \"\\u03b22AR acceptor lysines not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided high-resolution substrate mechanism by mapping ERGIC3 acceptor lysines (K6/K8) and showing ubiquitination controls secretory cargo export.\",\n      \"evidence\": \"Co-IP, K6R/K8R site-directed mutagenesis with rescue, siRNA, secretion and ubiquitination assays\",\n      \"pmids\": [\"31142615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin linkage type on ERGIC3 not specified\", \"Generality across secretory cargo beyond AAT/haptoglobin untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined MARCH2 as a brake on innate immune signaling by K48-linked degradation of NEMO at K326, with in vivo immune phenotypes.\",\n      \"evidence\": \"Co-IP, K326 mutagenesis, K48-linkage determination, MARCH2-/- mouse infection and cytokine studies\",\n      \"pmids\": [\"32935379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Timing/trigger of late-phase recruitment to NEMO not fully mechanized\", \"Relationship to other NF-\\u03baB regulators unresolved at this stage\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended receptor-homeostasis role to cytokine receptors, showing K27-linked degradation of IL-5R\\u03b1 controls eosinophil biology in vivo.\",\n      \"evidence\": \"Co-IP, K27-linkage and site-specific ubiquitination, MARCH2/3 double-KO mice, OVA airway model\",\n      \"pmids\": [\"35982175\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional redundancy versus distinct roles of MARCH2 and MARCH3 not separated\", \"Trafficking route to lysosome for IL-5R\\u03b1 not detailed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed non-degradative, activating ubiquitination modes: K63-linked modification of ALK5 enhancing TGF-\\u03b2 signaling and K33-linked modification driving PKM2 tetramerization and metabolic control.\",\n      \"evidence\": \"Co-IP, linkage-specific ubiquitination, site-directed mutagenesis (ALK5 K342/343; PKM2), KO and pharmacological rescue, in vivo metastasis and aortic disease models\",\n      \"pmids\": [\"41213909\", \"40079144\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How linkage specificity (K63 vs K33 vs K48) is determined remains unexplained\", \"Structural determinants distinguishing activating from degradative outputs unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Elucidated regulation of MARCH2 itself, showing TNF-induced dimerization and K63-linked autoubiquitination at K127/K238 license NEMO targeting, with MARCH8 acting as an inhibitor in resting cells.\",\n      \"evidence\": \"AP-MS, Co-IP, K63-linked autoubiquitination assays, K127/K238 mutagenesis, MARCH2-/- colitis model\",\n      \"pmids\": [\"40450320\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of dimerization and MARCH8 inhibition not resolved\", \"Whether autoubiquitination gates substrates beyond NEMO untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed MARCH2 can stabilize substrates, with K27-linked ubiquitination of NR1H2 (LXR\\u03b2) upregulating MERTK to drive macrophage efferocytosis and cardioprotection.\",\n      \"evidence\": \"Co-IP, K27-linkage ubiquitination, global and macrophage-specific KO mice, efferocytosis and cardiac function assays\",\n      \"pmids\": [\"41963318\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which K27 chains stabilize rather than degrade NR1H2 unresolved\", \"Acceptor lysine(s) on NR1H2 not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved what molecular features dictate MARCH2's choice of ubiquitin-chain linkage (K27/K33/K48/K63) and degradative versus stabilizing/activating outcomes across its diverse substrate set.\",\n      \"evidence\": \"No single study in the corpus reconstitutes linkage-determination or substrate-discrimination rules\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of MARCH2 with substrates or E2 partners\", \"Determinants of substrate vs adaptor recognition across contexts not unified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0061630\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 9, 10, 19, 21]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [4, 9, 10]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [17, 18, 23]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 18]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [10, 12, 19, 21, 24]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 9, 10, 19]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [18, 7, 10]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"STX6\", \"DLG1\", \"CAL\", \"CFTR\", \"NEMO\", \"TNF-R1\", \"MARCH8\", \"PGAM5\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}