{"gene":"MARCHF1","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2008,"finding":"MARCH1 (MARCH-I) is a RING-CH E3 ubiquitin ligase that promotes ubiquitination of the HLA-DR beta-chain in human monocyte-derived dendritic cells, driving surface internalization of mature HLA-DR complexes and reducing their stability and surface levels. Maturation-dependent down-regulation of MARCH1 upon LPS stimulation is a key event in MHC class II up-regulation at the DC surface.","method":"Ubiquitination assays, siRNA knockdown in primary human monocyte-derived DCs, flow cytometry for surface HLA-DR levels, LPS maturation experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional data (MARCH1 knockdown reverses IL-10-induced MHC-II downregulation), replicated across multiple labs","pmids":["18305173"],"is_preprint":false},{"year":2008,"finding":"MARCH1 is induced by IL-10 in human primary monocytes and mediates intracellular sequestration of MHC class II by ubiquitinating MHC-II molecules. Direct interaction between MHC-II and MARCH1 was demonstrated by co-immunoprecipitation. siRNA-mediated knockdown of MARCH1 reverses IL-10-induced MHC-II downregulation in primary monocytes.","method":"Co-immunoprecipitation, siRNA knockdown, flow cytometry, detection of mono- and poly-ubiquitinated MHC-II in IL-10-treated monocytes","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus siRNA rescue, independently replicated","pmids":["18389477"],"is_preprint":false},{"year":2009,"finding":"MARCH1 protein has a very short half-life (<30 min) in primary dendritic cells and APC cell lines, with degradation occurring partly in lysosomes via cysteine proteases. Mutational analysis defined discrete N-terminal endosomal sorting motifs and a C-terminal domain required for proper localization, functional interaction with substrates, and protein destabilization. Mutation of C-terminal tyrosine-based sorting signals reduced incorporation into exosomes. A C-terminal 221VQNC224 motif affects the spatial organization of the two cytoplasmic tails and MARCH1 activity.","method":"Pulse-chase half-life assays, lysosomal inhibitor treatment, cysteine protease inhibitor treatment, domain deletion/point mutagenesis, fusion reporter constructs, flow cytometry","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro functional mutagenesis plus pharmacological dissection, single lab with multiple orthogonal methods","pmids":["19880452"],"is_preprint":false},{"year":2009,"finding":"MARCH-I-mediated MHC II ubiquitination is necessary for the maintenance of conventional dendritic cell function in vivo. MARCH-I-deficient cDCs accumulate MHC II and B7-2 but exhibit impaired antigen-presenting ability and reduced cytokine production. Knockin mice carrying MHC II unable to be ubiquitinated phenocopy MARCH-I knockout, demonstrating the functional relevance is through MHC II ubiquitination specifically.","method":"MARCH-I knockout mice, MHC II ubiquitination knockin mice, in vivo antigen presentation assays, cytokine measurement","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis using both KO and ubiquitination-site knockin mice with defined phenotypic readout","pmids":["19917682"],"is_preprint":false},{"year":2011,"finding":"The transmembrane domain of CD83 blocks MARCH1 association with MHC class II, thereby preventing IL-10-driven, MARCH1-dependent ubiquitination and degradation of MHC class II and CD86 in dendritic cells. An ENU-induced mutation eliminating the CD83 TM region abolishes this protective effect.","method":"ENU-induced TM-domain mutant mice, flow cytometry for MHC II and CD86, IL-10 stimulation assays, genetic epistasis","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mouse model with defined molecular mechanism, replicated by independent study (PMID 40397676)","pmids":["21220452"],"is_preprint":false},{"year":2012,"finding":"MARCH1 is itself ubiquitinated via K48-linked polyubiquitin chains. A lysine-less N- and C-terminal mutant has reduced ubiquitination and prolonged half-life. An inactive catalytic mutant (M1WI) also shows reduced ubiquitination, consistent with autoubiquitination. MARCH1 homodimerizes and forms heterodimers with other MARCH family members; co-expression of wild-type MARCH1 decreases levels of the inactive M1WI mutant via transubiquitination.","method":"Co-immunoprecipitation, FRET/energy transfer, polyubiquitin chain-specific antibodies, site-directed mutagenesis, pulse-chase half-life, transfection in human cell lines","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, FRET, mutagenesis, chain-specific Abs) in single lab","pmids":["22508929"],"is_preprint":false},{"year":2012,"finding":"MARCH1 contains N-terminal endosomal sorting motifs and a functional C-terminal 221VQNC224 motif (also found in parkin) that affects the spatial organization of its cytoplasmic tails and E3 ligase activity. Mutation of C-terminal tyrosine-based sorting signals reduces MARCH1 incorporation into exosomes.","method":"Site-directed mutagenesis, type I/II transmembrane reporter fusions, exosome fractionation, functional substrate downregulation assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-function mutagenesis with multiple readouts in single lab","pmids":["23264739"],"is_preprint":false},{"year":2013,"finding":"Tollip reduces MARCH1 protein levels and competes with MARCH1 for binding to MHC II molecules. Knockdown of Tollip in HLA-DR-expressing cells increases HLA-DR surface expression and reduces CLIP-associated MHC II. Truncation of HLA-DR cytoplasmic tails abrogates Tollip's effect on MHC II expression.","method":"Tollip knockdown (siRNA), overexpression, co-immunoprecipitation, flow cytometry, reporter cell lines","journal":"Results in immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and knockdown/overexpression with functional readout, single lab, two complementary approaches","pmids":["24600555"],"is_preprint":false},{"year":2013,"finding":"MARCH1-mediated MHCII ubiquitination in dendritic cells is required for proper production of thymus-derived regulatory T cells (Treg) in vivo. DCs deficient in MARCH1 or carrying non-ubiquitinatable MHCII both fail to generate antigen-specific Treg cells despite increased surface MHCII, demonstrating that ubiquitin-dependent MHCII turnover, not MHCII surface levels per se, is critical.","method":"MARCH1 knockout mice, MHCII ubiquitination knockin mice, in vivo and in vitro Treg generation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with two independent mouse models (KO and ubiquitination-site knockin), defined cellular phenotype","pmids":["23712430"],"is_preprint":false},{"year":2015,"finding":"IL-10 stimulates expression of MARCH-I in activated macrophages (but not dendritic cells), thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells in a MARCH-I-dependent manner. In contrast, IL-10-mediated suppression of DC antigen presentation is MARCH-I-independent.","method":"IL-10 stimulation of macrophages vs. DCs, MARCH-I KO macrophages and DCs, flow cytometry, antigen-presentation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific KO comparison with defined functional readout, independently replicated concept","pmids":["26408197"],"is_preprint":false},{"year":2015,"finding":"March-I ubiquitination promotes degradation of newly synthesized pMHC-II and prevents pMHC-II recycling from the DC surface, routing internalized pMHC-II to lysosomes. Acute DC or B cell activation terminates March-I expression, which results in efficient pMHC-II recycling and prevention of lysosomal targeting, thereby stabilizing pMHC-II for antigen presentation.","method":"MHC-II ubiquitination mutant mice, biochemical recycling assays, lysosomal targeting assays, surface stability measurements in DCs and B cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mouse model with multiple biochemical assays and functional outcome, single lab with multiple orthogonal methods","pmids":["26240324"],"is_preprint":false},{"year":2016,"finding":"MARCH1 ubiquitinates the insulin receptor (INSR) to decrease cell surface INSR levels in the basal (non-stimulated) state, impairing insulin sensitivity. March1 loss-of-function enhances, and overexpression impairs, hepatic insulin sensitivity in mice. Unlike other INSR ubiquitin ligases, MARCH1 acts basally rather than after insulin stimulation.","method":"Large-scale RNAi screen, March1 KO mice, March1 overexpression mice, INSR ubiquitination assays, insulin clamp studies, flow cytometry for cell surface INSR","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi screen followed by KO and OE mouse models with multiple functional readouts, single rigorous study","pmids":["27577745"],"is_preprint":false},{"year":2018,"finding":"MARCH1 indirectly regulates MHC I surface expression through its ubiquitination of MHC II. In MARCH1-deficient B cells and DCs, the altered MHC II trafficking (not direct MARCH1 ubiquitination of MHC I) causes reduced surface MHC I and impaired cross-presentation to CD8+ T cells. Deletion of MHC II in March1-/- cells restores normal MHC I surface expression.","method":"MARCH1 KO mice, MHC II KO in March1-/- background, flow cytometry, antigen cross-presentation assays to CD8+ T cells","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with double KO rescue, multiple cell types, defined functional outcome","pmids":["30001419"],"is_preprint":false},{"year":2018,"finding":"MARCH1 promotes ubiquitin-dependent MHCII turnover that is required to maintain homeostasis of lipid rafts and the tetraspanin web in DC plasma membranes. Without MHCII ubiquitination, MHCII accumulates excessively in the plasma membrane, disrupting these membrane domains and impairing DC ability to engage thymocytes for Treg differentiation.","method":"MARCH1 KO and MHCII ubiquitination knockin mice, lipid raft fractionation, tetraspanin web analysis, Treg differentiation co-culture assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic mouse models with membrane fractionation and defined cellular phenotype","pmids":["29371232"],"is_preprint":false},{"year":2018,"finding":"March-I is ubiquitinated on non-lysine residues (lysine-less March-I is ubiquitinated similarly to wild-type). March-I E3 ligase activity is not required for its own ubiquitination, indicating it does not autoubiquitinate itself. The E2 ubiquitin-conjugating enzyme Ube2D1 is required for March-I ubiquitination; Ube2D1 knockdown impairs March-I ubiquitination, increases March-I expression, and enhances March-I-dependent MHC-II downregulation.","method":"Lysine-less March-I mutant, catalytically inactive March-I mutant, Ube2D1 siRNA knockdown, ubiquitination assays, flow cytometry","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis (lysine-less and catalytic mutants) plus siRNA knockdown with multiple functional readouts, single lab","pmids":["29414787"],"is_preprint":false},{"year":2018,"finding":"The March-I gene contains an APC-specific internal promoter driving expression of a distinct isoform in DCs and B cells. Downstream sequences in the first coding exon confer activation-induced down-regulation of March-I expression in DCs. These regulatory elements do not function in fibroblasts, kidney, or epithelial cells.","method":"March-I promoter-GFP reporter constructs, transfection in multiple cell types, LPS activation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assays with multiple cell types and activation conditions, single lab","pmids":["29378848"],"is_preprint":false},{"year":2020,"finding":"MARCH1 inhibits MAVS/STING/TRIF-induced type I IFN signaling in vitro and in vivo. In malaria-infected hosts, March1 deficiency paradoxically reduces IFN-I production by activating inhibitors (SOCS1, USP18, TRIM24) and altering immune cell populations, leading to increased IFN-γ and improved host survival that is reversed by T cell depletion or IFN-γ neutralization.","method":"March1 KO mice, Plasmodium yoelii infection model, transspecies eQTL genetic screen, MAVS/STING/TRIF reporter assays, cytokine measurements, T cell depletion, antibody neutralization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mice with multiple functional assays, single lab, in vitro and in vivo data","pmids":["32606244"],"is_preprint":false},{"year":2020,"finding":"MARCH1 ubiquitination of MHC-II in DCs regulates DC gene expression and functional fitness. MHC-II ubiquitination-deficient DCs are poor stimulators of naive CD4 T cells and secrete IL-12 poorly in response to LPS despite high surface MHC-II. Single-cell RNA sequencing reveals these DCs have an altered gene expression signature that is reversed by LPS activation.","method":"MHC-II ubiquitination mutant mice, naive CD4 T cell stimulation assays, IL-12 ELISA, single-cell RNA sequencing","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mouse model with multiple functional assays plus scRNA-seq, single rigorous study with orthogonal methods","pmids":["33318291"],"is_preprint":false},{"year":2021,"finding":"MARCH1 ubiquitin ligase regulates CD86 and MHC II in professional and atypical antigen presenting cells of hematopoietic origin (including neutrophils, eosinophils, and monocytes), whereas MARCH8 operates in non-hematopoietic cells. Unbiased plasma membrane proteomic profiling of primary cells from MARCH1-deficient mice identified CD86 and MHC II as the only substrates unequivocally regulated by MARCH1 in vivo.","method":"MARCH1 KO and MARCH8 KO mice, unbiased plasma membrane proteomics (mass spectrometry), flow cytometry across multiple cell types","journal":"Current research in immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — unbiased proteomic substrate identification in KO mice across multiple tissues, single rigorous study with multiple orthogonal methods","pmids":["35492398"],"is_preprint":false},{"year":2021,"finding":"MARCH1-mediated ubiquitination of MHCII and CD86 on lymph node-resident DCs is required for TH2 cell development. Mice with mutations in the ubiquitin acceptor sites of both MHCII and CD86 fail to develop TH2 cells, demonstrating that clearance of antigen-presenting and costimulatory molecules by MARCH1 controls TCR signaling strength during TH2 polarization.","method":"MARCH1 KO mice, MHCII and CD86 ubiquitin-site knockin mice, in vivo TH2 differentiation assays, GATA-3 expression analysis","journal":"Science immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple knockin mouse models and defined TH2 differentiation phenotype","pmids":["34652961"],"is_preprint":false},{"year":2021,"finding":"MARCH1 promotes ubiquitination and degradation of HDAC11, which relieves HDAC11/KLF4-dependent repression of the OX40L gene promoter, thereby increasing OX40L expression in allergen-stimulated dendritic cells.","method":"Co-immunoprecipitation, immunofluorescence co-localization, luciferase OX40L promoter reporter assay, MARCH1 overexpression/knockdown, Western blot","journal":"Journal of asthma and allergy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, reporter assay, and overexpression/knockdown in same study, single lab","pmids":["34385821"],"is_preprint":false},{"year":2021,"finding":"Human MARCH1 recognizes the CD86 transmembrane domain through a highly specific surface in the hydrophobic core featuring proline at position 254. This recognition mode is distinct from the viral MARCH homolog MIR2, which requires an aspartic acid in the CD86 extracellular juxtamembrane region and no specific TM sequences.","method":"Deep mutational scanning of CD86 TM domain, overexpression in cell lines, flow cytometry for surface CD86","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis (deep mutational scanning) with functional readout, single lab with comprehensive coverage","pmids":["34157285"],"is_preprint":false},{"year":2022,"finding":"MARCH1 ubiquitinates and promotes degradation of PHLPP2 in oral squamous cell carcinoma cells. Co-immunoprecipitation confirmed direct interaction between MARCH1 and PHLPP2. MARCH1 knockdown suppresses OSCC tumorigenicity in vivo and increases PHLPP2 protein levels.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, Western blot, in vivo xenograft model","journal":"Clinical & translational oncology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP plus KD/OE functional experiments with in vivo validation, single lab","pmids":["35122633"],"is_preprint":false},{"year":2022,"finding":"MARCH1 isoform 1 (MARCH1.1) restricts influenza A virus (IAV) replication/infectivity, whereas isoform 2 (MARCH1.2) does not. Deletion of the entire N-terminal cytoplasmic domain of MARCH1.2, or specifically the 16 N-terminal residues, restores IAV restriction, mapping the difference in antiviral activity to the N-CT of MARCH1.2.","method":"Doxycycline-inducible overexpression system, viral titer assays, sequential N-CT deletion mutagenesis, qPCR for isoform expression","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion mutagenesis with viral titer readout, single lab, multiple deletions tested","pmids":["36423158"],"is_preprint":false},{"year":2022,"finding":"MARCH1 ubiquitinates and promotes degradation of the transferrin receptor (TfR), thereby regulating cellular iron levels during human cytomegalovirus (HCMV) infection. MARCH1 knockdown decreases infectious HCMV titers, increases ROS, lipid peroxidation, and mitochondrial dysfunction, establishing a proviral role for MARCH1 in regulating iron metabolism.","method":"MARCH1 knockdown (siRNA), TfR expression analysis, labile iron pool measurement, ROS and lipid peroxidation assays, viral titer assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with multiple functional readouts, single lab, mechanistic connection established","pmids":["35045264"],"is_preprint":false},{"year":2023,"finding":"MARCHF1 acts as an E3 ubiquitin ligase for GPX4, promoting its ubiquitination and degradation, thereby increasing susceptibility to ferroptosis. BaP/BPDE exposure up-regulates MARCHF1 expression, leading to GPX4 protein degradation and consequent suppression of HUVEC angiogenesis and induction of miscarriage.","method":"Ubiquitination assays, MARCHF1 overexpression/knockdown in HUVECs, GPX4 protein level measurement, angiogenesis assays, mouse BaP exposure miscarriage model","journal":"Environment international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assays plus KD/OE with in vitro and in vivo functional readouts, single lab","pmids":["37802009"],"is_preprint":false},{"year":2024,"finding":"Human MARCH1 and MARCH2 retain Ebola virus (EBOV) glycoprotein (GP) at the trans-Golgi network, reduce its cell surface display, and impair EBOV GP-pseudotyped virion infectivity by blocking furin-mediated cleavage. The furin P domain is recognized by MARCH1/2/8 and is critical for their blocking activities. Host furin interacts with MARCH1/2 and EBOV GP intracellularly.","method":"Co-immunoprecipitation of furin with MARCH1/2 and EBOV GP, EBOV GP-pseudotyped viral infection assays, furin P domain mutagenesis, subcellular localization analysis","journal":"Journal of medical virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional viral assays with domain-level mechanistic dissection, single lab","pmids":["38299743"],"is_preprint":false},{"year":2024,"finding":"MARCH1 interacts with TBK1 and promotes K63-linked ubiquitination of TBK1, attenuating TBK1 interaction with mTOR and thereby inhibiting growth factor-induced mTOR signaling. MARCH1 deficiency accelerates breast cancer cell proliferation, which is reversed by inhibition of mTOR, STING, or TBK1.","method":"Co-immunoprecipitation, denatured Co-IP ubiquitination assays, Western blot, shRNA knockdown, lentiviral overexpression, scratch wound and colony assays","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays with multiple rescue experiments, single lab","pmids":["39061024"],"is_preprint":false},{"year":2024,"finding":"MARCH1 mediates ubiquitination and degradation of PCSK9, lowering PCSK9 protein expression, and thereby suppresses inflammation and pyroptosis in cerebral ischemia-reperfusion injury. Mechanistically, PCSK9 knockdown reduces brain damage, upregulates LDLR, and inactivates the NLRP3 inflammasome.","method":"Co-immunoprecipitation, cycloheximide degradation assay, MARCH1/PCSK9 overexpression/knockdown, MCAO/R mouse model, OGD/R neuron model","journal":"Mammalian genome","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and degradation assays with in vivo and in vitro functional validation, single lab","pmids":["39115562"],"is_preprint":false},{"year":2025,"finding":"CD83 suppresses endogenous March-I-dependent MHC-II ubiquitination, endocytosis, and degradation in mouse spleen DCs. Endogenous March-I protein is present at extremely low copy numbers (~500 molecules/cell in DCs, ~125 in B cells), has a very short half-life, and March-I mRNA, protein, and MHC-II ubiquitination are all rapidly terminated upon DC or B cell activation.","method":"V5-epitope knock-in into endogenous March-I locus, quantitative flow cytometry, MHC-II ubiquitination assays, pulse-chase half-life, LPS activation of primary spleen DCs and B cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous knock-in tagging with quantitative analysis and multiple orthogonal assays, single rigorous study","pmids":["40397676"],"is_preprint":false},{"year":2025,"finding":"MARCHF1 promotes GABAB receptor downregulation under ischemic/excitotoxic conditions by interacting with GABAB receptors and inhibiting fast receptor recycling at the plasma membrane, leading to progressive neuronal death. An interfering peptide blocking the MARCH1/GABAB receptor interaction restores receptor surface expression and prevents neuronal death.","method":"Co-immunoprecipitation, interfering peptide competition, surface GABAB receptor quantification in ischemic neurons, neuronal death assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and peptide competition with functional cell-death readout, single lab, mechanistically specific","pmids":["39779794"],"is_preprint":false},{"year":2025,"finding":"MARCHF1 interacts with SARS-CoV-2 M protein and mediates ubiquitination and degradation of GPX4, contributing to ferroptosis during SARS-CoV-2 infection. The M-GPX4 interaction occurs at the R72 residue of GPX4.","method":"Co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis of GPX4 R72, ferroptosis assays in infected cells","journal":"Journal of medical virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination with mutagenesis identifying specific interaction site, single lab","pmids":["40186530"],"is_preprint":false},{"year":2025,"finding":"MARCH1 interacts with MYCT1 (a candidate tumor suppressor) and promotes its K48-linked ubiquitination and degradation, facilitating AML cell proliferation and survival. POU2F2 transcription factor positively regulates MARCH1 transcription in AML. MYCT1 knockdown abolishes the inhibitory effects of MARCH1 knockdown on AML cell growth.","method":"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, gain- and loss-of-function experiments, in vivo AML mouse model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assays, and genetic rescue experiments with in vivo validation, single lab","pmids":["40533483"],"is_preprint":false},{"year":2025,"finding":"MARCHF1 ubiquitinates and promotes degradation of SULF1 in colon cancer cells, thereby blocking SULF1-THBS2 interaction and suppressing TGF-β1/SMAD2/3 pathway activation, which leads to reduced tumor metastasis and enhanced sensitivity to 5-FU.","method":"Co-immunoprecipitation, ubiquitination assays, MARCHF1 and SULF1 knockdown/overexpression, TGF-β pathway analysis, drug sensitivity assays","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination with pathway rescue experiments, single lab","pmids":["41550717"],"is_preprint":false},{"year":2025,"finding":"MARCHF1 interacts with and promotes ubiquitination and degradation of REST (repressor element-1 silencing transcription factor), reducing REST protein levels and consequently de-repressing TFAM transcription, promoting mitochondrial function and breast cancer cell proliferation.","method":"Co-immunoprecipitation, ubiquitination assays, MARCHF1 knockdown/overexpression, Western blot for REST and TFAM, in vivo xenograft model","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP with functional KD/OE readouts and in vivo validation, single lab","pmids":["39428668"],"is_preprint":false},{"year":2026,"finding":"MARCH1 directly binds to KLF15 (Krüppel-like factor 15) in white adipose tissue, contributing to HFpEF pathogenesis. MARCH1 deficiency induces WAT beiging and alleviates cardiac dysfunction in a HFpEF mouse model; overexpressing KLF15 blocks the beiging effects of MARCH1 KO.","method":"Co-immunoprecipitation, MARCH1 KO HFpEF mouse model, RNA sequencing, echocardiography, histology, KLF15 overexpression rescue","journal":"Comprehensive Physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with KO mouse model and genetic rescue, single lab","pmids":["41986908"],"is_preprint":false},{"year":2026,"finding":"MARCH1 promotes ubiquitination and degradation of SLC25A17, a mitochondrial transporter. MARCH1 overexpression suppresses macrophage M2 polarization and DDP resistance in lung adenocarcinoma; SLC25A17 overexpression reverses the DDP sensitivity enhancement caused by MARCH1 reintroduction.","method":"Co-immunoprecipitation, ubiquitination assays, MARCH1 overexpression/knockdown, SLC25A17 rescue overexpression, flow cytometry, ELISA, in vivo tumor model","journal":"Integrative biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays with genetic rescue experiments and in vivo validation, single lab","pmids":["41758657"],"is_preprint":false}],"current_model":"MARCHF1 (MARCH1) is a membrane-anchored RING-CH E3 ubiquitin ligase that constitutively ubiquitinates the cytoplasmic tail of MHC class II beta-chain and CD86 in resting antigen-presenting cells, routing these molecules to lysosomes for degradation and limiting surface antigen presentation; this activity is terminated by DC maturation signals (e.g., LPS) or CD83 engagement, stabilizing surface pMHC-II for efficient CD4 T cell activation, and MARCH1 is itself regulated by IL-10 (which induces its expression in macrophages and monocytes), by Ube2D1-mediated lysine-independent ubiquitination that controls its own turnover, and by its very short protein half-life via lysosomal/cysteine protease degradation. Beyond immune regulation, MARCH1 ubiquitinates the insulin receptor (setting basal insulin sensitivity), GPX4 (promoting ferroptosis), TBK1 (K63-linked, suppressing mTOR signaling), PCSK9 (suppressing NLRP3-mediated inflammation), HDAC11 (increasing OX40L expression), PHLPP2, REST, MYCT1, SULF1, SLC25A17, TfR, and GABAB receptors, placing it as a broad-spectrum membrane-localized E3 ligase that controls immunological tolerance, metabolic homeostasis, antiviral restriction, and neuronal survival."},"narrative":{"mechanistic_narrative":"MARCHF1 (MARCH1) is a membrane-anchored RING-CH E3 ubiquitin ligase that serves as a central rheostat of antigen presentation by constitutively ubiquitinating the cytoplasmic tails of MHC class II and the costimulatory molecule CD86 in antigen-presenting cells, routing these molecules to lysosomes and limiting their surface display [PMID:18305173, PMID:18389477, PMID:35492398]. By targeting newly synthesized peptide-MHC-II for degradation and preventing its recycling from the cell surface, MARCH1 keeps resting APCs in a low-presentation state; acute activation signals such as LPS rapidly terminate MARCH1 expression, stabilizing surface pMHC-II for CD4 T cell engagement [PMID:26240324, PMID:40397676]. This ubiquitin-dependent turnover — rather than steady-state surface MHC-II levels per se — is required for conventional DC fitness, thymic regulatory T cell generation, TH2 development, lipid raft/tetraspanin web homeostasis, and indirectly for MHC class I cross-presentation [PMID:19917682, PMID:23712430, PMID:29371232, PMID:34652961, PMID:30001419]. MARCH1 substrate recognition is encoded in the substrate transmembrane domain, exemplified by its requirement for proline-254 in the CD86 TM core [PMID:34157285]. The ligase is held in check by multiple layers of regulation: it is induced by IL-10 in monocytes and macrophages [PMID:18389477, PMID:26408197], its substrate engagement is blocked by the transmembrane domain of CD83 [PMID:21220452, PMID:40397676] and competed by Tollip [PMID:24600555], and the protein itself is extremely short-lived (~500 molecules/cell, half-life <30 min) and is turned over via lysosomal cysteine proteases and through Ube2D1-dependent lysine-independent ubiquitination [PMID:19880452, PMID:29414787, PMID:40397676]. Beyond immunity, MARCH1 ubiquitinates a broad set of membrane and intracellular targets including the insulin receptor to set basal insulin sensitivity [PMID:27577745], GPX4 to promote ferroptosis [PMID:37802009], and TBK1 via K63-linked chains to suppress mTOR signaling [PMID:39061024], and it contributes to antiviral restriction of influenza and Ebola glycoprotein trafficking [PMID:36423158, PMID:38299743].","teleology":[{"year":2008,"claim":"Established MARCH1 as the E3 ligase that ubiquitinates MHC class II and drives its surface internalization, defining a molecular basis for maturation-controlled antigen presentation.","evidence":"Ubiquitination assays, Co-IP, and siRNA knockdown in primary human monocyte-derived DCs and IL-10-treated monocytes with surface flow cytometry","pmids":["18305173","18389477"],"confidence":"High","gaps":["Substrate ubiquitin acceptor sites not yet mapped in these studies","Whether CD86 is a co-substrate not addressed"]},{"year":2009,"claim":"Showed in vivo that MHC-II ubiquitination specifically — not MARCH1 itself broadly — underlies DC function, using ubiquitination-site knockin mice that phenocopy the knockout.","evidence":"MARCH1 knockout and non-ubiquitinatable MHC-II knockin mice with in vivo antigen presentation and cytokine readouts; half-life and domain mutagenesis","pmids":["19917682","19880452"],"confidence":"High","gaps":["Mechanism linking MHC-II accumulation to impaired antigen presentation unresolved","C-terminal VQNC motif role in catalysis not structurally defined"]},{"year":2011,"claim":"Defined CD83 transmembrane domain as a physiological brake that blocks MARCH1 association with MHC-II, explaining how maturation signals protect surface MHC-II and CD86.","evidence":"ENU-induced CD83 TM-domain mutant mice with IL-10 stimulation and flow cytometry","pmids":["21220452"],"confidence":"High","gaps":["Structural basis of CD83-MARCH1 TM interference not defined","Whether CD83 acts as competitive decoy substrate untested"]},{"year":2013,"claim":"Connected MARCH1-dependent MHC-II turnover to immune tolerance by showing it is required for thymic Treg generation, and identified Tollip as a competing regulator.","evidence":"MARCH1 KO and ubiquitination knockin mice in Treg assays; Tollip knockdown/Co-IP in HLA-DR cells","pmids":["23712430","24600555"],"confidence":"High","gaps":["How ubiquitin turnover translates to Treg-inducing DC signals unclear","Tollip mechanism (single-lab) lacks reciprocal validation"]},{"year":2015,"claim":"Resolved the trafficking logic — MARCH1 degrades newly synthesized pMHC-II and blocks its surface recycling — and showed IL-10 induction of MARCH1 is cell-type specific (macrophages, not DCs).","evidence":"MHC-II ubiquitination mutant mice with recycling and lysosomal targeting assays; cell-type-specific KO comparison with IL-10 stimulation","pmids":["26240324","26408197"],"confidence":"High","gaps":["Sorting machinery routing ubiquitinated pMHC-II to lysosomes not fully identified","Transcriptional basis of cell-type-specific IL-10 responsiveness unresolved"]},{"year":2016,"claim":"Extended MARCH1 beyond immunity by identifying the insulin receptor as a basal-state substrate that sets metabolic insulin sensitivity, distinguishing MARCH1 from stimulus-dependent INSR ligases.","evidence":"RNAi screen, March1 KO and overexpression mice, INSR ubiquitination and insulin clamp studies","pmids":["27577745"],"confidence":"High","gaps":["INSR ubiquitination site not mapped","Tissue-specific contribution to systemic metabolism not fully partitioned"]},{"year":2018,"claim":"Refined the self-regulation and substrate-scope picture: MARCH1 is turned over by lysine-independent, Ube2D1-dependent ubiquitination (not autoubiquitination), and unbiased proteomics confirmed CD86 and MHC-II as its only unequivocal in vivo membrane substrates in hematopoietic cells.","evidence":"Lysine-less and catalytic mutants with Ube2D1 knockdown; plasma membrane proteomics in MARCH1 and MARCH8 KO mice; double-KO MHC-I rescue; APC-specific promoter reporters","pmids":["29414787","35492398","30001419","29378848"],"confidence":"High","gaps":["Earlier autoubiquitination model (idx 5) is contradicted — chain dependence on Ube2D1 vs MARCH1 activity needs reconciliation","Proteomics may miss low-abundance or non-membrane substrates later reported"]},{"year":2021,"claim":"Defined the molecular grammar of substrate recognition (CD86 TM proline-254) and broadened immune function to TH2 development and DC functional programming via MHC-II/CD86 clearance.","evidence":"Deep mutational scanning of CD86 TM; MHC-II/CD86 ubiquitin-site knockin mice in TH2 assays; scRNA-seq of ubiquitination-deficient DCs","pmids":["34157285","34652961","33318291"],"confidence":"High","gaps":["No structure of MARCH1-substrate TM complex","How surface molecule clearance tunes TCR signaling strength mechanistically unresolved"]},{"year":2024,"claim":"Established quantitatively that endogenous MARCH1 is present at extremely low copy number with rapid activation-induced termination, and expanded the non-immune substrate repertoire to TBK1 (K63-linked, suppressing mTOR).","evidence":"Endogenous V5 knock-in with quantitative flow cytometry and pulse-chase; Co-IP and denatured ubiquitination assays for TBK1 with mTOR/STING/TBK1 inhibitor rescue","pmids":["40397676","39061024"],"confidence":"High","gaps":["TBK1 finding (single-lab, Medium) lacks independent confirmation","Chain-type switching (K48 for MHC-II vs K63 for TBK1) determinants unknown"]},{"year":2025,"claim":"Reported a wave of context-specific substrates (GPX4, PCSK9, SULF1, MYCT1, REST, GABAB receptor, SLC25A17, KLF15) linking MARCH1 to ferroptosis, neuronal death, and tumor biology.","evidence":"Co-IP and ubiquitination/degradation assays with knockdown/overexpression and rescue across cancer, ischemia, and infection models","pmids":["37802009","39115562","41550717","40533483","39428668","39779794","40186530","41758657"],"confidence":"Medium","gaps":["Each substrate rests on single-lab Co-IP without reciprocal/structural validation","Most were not detected in the unbiased in vivo proteomic screen (idx 18), raising context-dependence questions","Direct vs indirect ubiquitination not always disambiguated"]},{"year":null,"claim":"How a single low-abundance, short-lived membrane ligase achieves such broad and context-specific substrate selectivity — and how chain-type and subcellular routing are determined — remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of MARCH1 with any substrate","Determinants selecting K48 vs K63 linkage across substrates unknown","Reconciliation of restricted in vivo proteomic substrate set with many reported disease-context substrates pending"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,11,25,27,32,33]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,11,25,27]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,5,14,21]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,10,18,21]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,6,10]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[26]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[0,2,10]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,3,8,9,18,19]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,11,25,27,33]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[10,30]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[11,24,36]}],"complexes":[],"partners":["CD74","CD86","CD83","TOLLIP","UBE2D1","INSR","GPX4","TBK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TCQ1","full_name":"E3 ubiquitin-protein ligase MARCHF1","aliases":["Membrane-associated RING finger protein 1","Membrane-associated RING-CH protein I","MARCH-I","RING finger protein 171","RING-type E3 ubiquitin transferase MARCHF1"],"length_aa":289,"mass_kda":32.3,"function":"E3 ubiquitin-protein ligase that mediates ubiquitination of TFRC, CD86, FAS and MHC class II proteins, such as HLA-DR alpha and beta, and promotes their subsequent endocytosis and sorting to lysosomes via multivesicular bodies (PubMed:18389477, PubMed:18305173, PubMed:21220452, PubMed:35045264). By constitutively ubiquitinating MHC class II proteins in immature dendritic cells, down-regulates their cell surface localization thus sequestering them in the intracellular endosomal system. Also regulates insulin sensitivity by controlling surface expression of the insulin receptor subunit beta/INSR by direct ubiquitination and degradation (PubMed:27577745) (Microbial infection) Plays a role in iron metabolism by regulating the levels of the transferrin receptor TFRC during human cytomegalovirus infection, subsequently contributing to a proviral effect","subcellular_location":"Golgi apparatus, trans-Golgi network membrane; Lysosome membrane; Cytoplasmic vesicle membrane; Late endosome membrane; Early endosome membrane; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q8TCQ1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MARCHF1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1090,"dependency_fraction":0.0009174311926605505},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MARCHF1","total_profiled":1310},"omim":[{"mim_id":"613331","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 1; MARCHF1","url":"https://www.omim.org/entry/613331"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Endoplasmic reticulum","reliability":"Additional"},{"location":"Golgi apparatus","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":29.3},{"tissue":"retina","ntpm":21.3}],"url":"https://www.proteinatlas.org/search/MARCHF1"},"hgnc":{"alias_symbol":["FLJ20668","MARCH-I","RNF171"],"prev_symbol":["MARCH1"]},"alphafold":{"accession":"Q8TCQ1","domains":[{"cath_id":"3.30.40.10","chopping":"77-131","consensus_level":"medium","plddt":94.1173,"start":77,"end":131},{"cath_id":"1.10.287","chopping":"150-236","consensus_level":"high","plddt":87.122,"start":150,"end":236}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCQ1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCQ1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCQ1-F1-predicted_aligned_error_v6.png","plddt_mean":68.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MARCHF1","jax_strain_url":"https://www.jax.org/strain/search?query=MARCHF1"},"sequence":{"accession":"Q8TCQ1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TCQ1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TCQ1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCQ1"}},"corpus_meta":[{"pmid":"18305173","id":"PMC_18305173","title":"MHC 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virology","url":"https://pubmed.ncbi.nlm.nih.gov/40186530","citation_count":4,"is_preprint":false},{"pmid":"32973799","id":"PMC_32973799","title":"Lack of the E3 Ubiquitin Ligase March1 Affects CD8 T Cell Fate and Exacerbates Insulin Resistance in Obese Mice.","date":"2020","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32973799","citation_count":4,"is_preprint":false},{"pmid":"36100368","id":"PMC_36100368","title":"MARCH1 Controls an Exhaustion-like Program of Effector CD4+ T Cells Promoting Allergic Airway Inflammation.","date":"2022","source":"ImmunoHorizons","url":"https://pubmed.ncbi.nlm.nih.gov/36100368","citation_count":4,"is_preprint":false},{"pmid":"36451862","id":"PMC_36451862","title":"Targeting psychological stress-steroid-MARCH1 signaling pathway promotes the efficacy of specific allergen immunotherapy.","date":"2022","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/36451862","citation_count":3,"is_preprint":false},{"pmid":"39779794","id":"PMC_39779794","title":"The E3 ubiquitin ligase MARCH1 mediates downregulation of plasma membrane GABAB receptors under ischemic conditions by inhibiting fast receptor recycling.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39779794","citation_count":3,"is_preprint":false},{"pmid":"40533483","id":"PMC_40533483","title":"MARCH1, transcriptionally regulated by POU2F2, facilitates acute myeloid leukemia progression via inducing MYCT1 degradation.","date":"2025","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/40533483","citation_count":2,"is_preprint":false},{"pmid":"38331309","id":"PMC_38331309","title":"MARCH-I: A negative regulator of dendritic cell maturation.","date":"2024","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/38331309","citation_count":2,"is_preprint":false},{"pmid":"34853711","id":"PMC_34853711","title":"A Novel 4q32.3 Deletion in a Child: Additional Signs and the Role of MARCH1.","date":"2021","source":"Journal of pediatric genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34853711","citation_count":2,"is_preprint":false},{"pmid":"35656075","id":"PMC_35656075","title":"Induced overexpression of MARCH-1 in human macrophages altered to M2 phenotype for suppressing inflammation process.","date":"2022","source":"Iranian journal of basic medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35656075","citation_count":2,"is_preprint":false},{"pmid":"40397676","id":"PMC_40397676","title":"CD83 suppresses endogenous March-I-dependent MHC class II ubiquitination, endocytosis, and degradation.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/40397676","citation_count":1,"is_preprint":false},{"pmid":"39428668","id":"PMC_39428668","title":"MARCHF1 promotes breast cancer through accelerating REST ubiquitylation and following TFAM transcription.","date":"2024","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/39428668","citation_count":1,"is_preprint":false},{"pmid":"33537243","id":"PMC_33537243","title":"RON Expression Mediates Lipopolysaccharide-Mediated Dendritic Cell Maturation via March-I.","date":"2021","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/33537243","citation_count":1,"is_preprint":false},{"pmid":"21385572","id":"PMC_21385572","title":"MARCH-I expression in cord blood CD34+KDR+ cells.","date":"2011","source":"Clinical biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21385572","citation_count":1,"is_preprint":false},{"pmid":"41986908","id":"PMC_41986908","title":"MARCH1 Deletion Attenuates HFpEF by Promoting Adipose Beiging.","date":"2026","source":"Comprehensive Physiology","url":"https://pubmed.ncbi.nlm.nih.gov/41986908","citation_count":0,"is_preprint":false},{"pmid":"41283654","id":"PMC_41283654","title":"HIV-2 glycoproteins upregulate microRNAs 25 and 93 to counter the MARCH1 antiviral effect in macrophages.","date":"2025","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/41283654","citation_count":0,"is_preprint":false},{"pmid":"41550717","id":"PMC_41550717","title":"MARCHF1-mediated SULF1 degradation blocks THBS2/TGF-β/SMAD2/3 signaling to reverse colon cancer metastasis and 5-FU resistance.","date":"2025","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/41550717","citation_count":0,"is_preprint":false},{"pmid":"41758657","id":"PMC_41758657","title":"MARCH1 attenuates lung adenocarcinoma by blocking macrophage M2 polarization and cisplatin resistance through reducing SLC25A17 stability.","date":"2026","source":"Integrative biology : quantitative biosciences from nano to macro","url":"https://pubmed.ncbi.nlm.nih.gov/41758657","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.10.25.24316158","title":"Gene-Excessive Sleepiness Interactions Suggest Treatment Targets for Obstructive Sleep Apnea Subtype","date":"2024-10-28","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.25.24316158","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":35945,"output_tokens":9486,"usd":0.125062,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19715,"output_tokens":4279,"usd":0.102775,"stage2_stop_reason":"end_turn"},"total_usd":0.227837,"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\": 2008,\n      \"finding\": \"MARCH1 (MARCH-I) is a RING-CH E3 ubiquitin ligase that promotes ubiquitination of the HLA-DR beta-chain in human monocyte-derived dendritic cells, driving surface internalization of mature HLA-DR complexes and reducing their stability and surface levels. Maturation-dependent down-regulation of MARCH1 upon LPS stimulation is a key event in MHC class II up-regulation at the DC surface.\",\n      \"method\": \"Ubiquitination assays, siRNA knockdown in primary human monocyte-derived DCs, flow cytometry for surface HLA-DR levels, LPS maturation experiments\",\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 functional data (MARCH1 knockdown reverses IL-10-induced MHC-II downregulation), replicated across multiple labs\",\n      \"pmids\": [\"18305173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MARCH1 is induced by IL-10 in human primary monocytes and mediates intracellular sequestration of MHC class II by ubiquitinating MHC-II molecules. Direct interaction between MHC-II and MARCH1 was demonstrated by co-immunoprecipitation. siRNA-mediated knockdown of MARCH1 reverses IL-10-induced MHC-II downregulation in primary monocytes.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, flow cytometry, detection of mono- and poly-ubiquitinated MHC-II in IL-10-treated monocytes\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus siRNA rescue, independently replicated\",\n      \"pmids\": [\"18389477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MARCH1 protein has a very short half-life (<30 min) in primary dendritic cells and APC cell lines, with degradation occurring partly in lysosomes via cysteine proteases. Mutational analysis defined discrete N-terminal endosomal sorting motifs and a C-terminal domain required for proper localization, functional interaction with substrates, and protein destabilization. Mutation of C-terminal tyrosine-based sorting signals reduced incorporation into exosomes. A C-terminal 221VQNC224 motif affects the spatial organization of the two cytoplasmic tails and MARCH1 activity.\",\n      \"method\": \"Pulse-chase half-life assays, lysosomal inhibitor treatment, cysteine protease inhibitor treatment, domain deletion/point mutagenesis, fusion reporter constructs, flow cytometry\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro functional mutagenesis plus pharmacological dissection, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19880452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MARCH-I-mediated MHC II ubiquitination is necessary for the maintenance of conventional dendritic cell function in vivo. MARCH-I-deficient cDCs accumulate MHC II and B7-2 but exhibit impaired antigen-presenting ability and reduced cytokine production. Knockin mice carrying MHC II unable to be ubiquitinated phenocopy MARCH-I knockout, demonstrating the functional relevance is through MHC II ubiquitination specifically.\",\n      \"method\": \"MARCH-I knockout mice, MHC II ubiquitination knockin mice, in vivo antigen presentation assays, cytokine measurement\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis using both KO and ubiquitination-site knockin mice with defined phenotypic readout\",\n      \"pmids\": [\"19917682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The transmembrane domain of CD83 blocks MARCH1 association with MHC class II, thereby preventing IL-10-driven, MARCH1-dependent ubiquitination and degradation of MHC class II and CD86 in dendritic cells. An ENU-induced mutation eliminating the CD83 TM region abolishes this protective effect.\",\n      \"method\": \"ENU-induced TM-domain mutant mice, flow cytometry for MHC II and CD86, IL-10 stimulation assays, genetic epistasis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mouse model with defined molecular mechanism, replicated by independent study (PMID 40397676)\",\n      \"pmids\": [\"21220452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MARCH1 is itself ubiquitinated via K48-linked polyubiquitin chains. A lysine-less N- and C-terminal mutant has reduced ubiquitination and prolonged half-life. An inactive catalytic mutant (M1WI) also shows reduced ubiquitination, consistent with autoubiquitination. MARCH1 homodimerizes and forms heterodimers with other MARCH family members; co-expression of wild-type MARCH1 decreases levels of the inactive M1WI mutant via transubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, FRET/energy transfer, polyubiquitin chain-specific antibodies, site-directed mutagenesis, pulse-chase half-life, transfection in human cell lines\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, FRET, mutagenesis, chain-specific Abs) in single lab\",\n      \"pmids\": [\"22508929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MARCH1 contains N-terminal endosomal sorting motifs and a functional C-terminal 221VQNC224 motif (also found in parkin) that affects the spatial organization of its cytoplasmic tails and E3 ligase activity. Mutation of C-terminal tyrosine-based sorting signals reduces MARCH1 incorporation into exosomes.\",\n      \"method\": \"Site-directed mutagenesis, type I/II transmembrane reporter fusions, exosome fractionation, functional substrate downregulation assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-function mutagenesis with multiple readouts in single lab\",\n      \"pmids\": [\"23264739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Tollip reduces MARCH1 protein levels and competes with MARCH1 for binding to MHC II molecules. Knockdown of Tollip in HLA-DR-expressing cells increases HLA-DR surface expression and reduces CLIP-associated MHC II. Truncation of HLA-DR cytoplasmic tails abrogates Tollip's effect on MHC II expression.\",\n      \"method\": \"Tollip knockdown (siRNA), overexpression, co-immunoprecipitation, flow cytometry, reporter cell lines\",\n      \"journal\": \"Results in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and knockdown/overexpression with functional readout, single lab, two complementary approaches\",\n      \"pmids\": [\"24600555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MARCH1-mediated MHCII ubiquitination in dendritic cells is required for proper production of thymus-derived regulatory T cells (Treg) in vivo. DCs deficient in MARCH1 or carrying non-ubiquitinatable MHCII both fail to generate antigen-specific Treg cells despite increased surface MHCII, demonstrating that ubiquitin-dependent MHCII turnover, not MHCII surface levels per se, is critical.\",\n      \"method\": \"MARCH1 knockout mice, MHCII ubiquitination knockin mice, in vivo and in vitro Treg generation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with two independent mouse models (KO and ubiquitination-site knockin), defined cellular phenotype\",\n      \"pmids\": [\"23712430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"IL-10 stimulates expression of MARCH-I in activated macrophages (but not dendritic cells), thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells in a MARCH-I-dependent manner. In contrast, IL-10-mediated suppression of DC antigen presentation is MARCH-I-independent.\",\n      \"method\": \"IL-10 stimulation of macrophages vs. DCs, MARCH-I KO macrophages and DCs, flow cytometry, antigen-presentation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific KO comparison with defined functional readout, independently replicated concept\",\n      \"pmids\": [\"26408197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"March-I ubiquitination promotes degradation of newly synthesized pMHC-II and prevents pMHC-II recycling from the DC surface, routing internalized pMHC-II to lysosomes. Acute DC or B cell activation terminates March-I expression, which results in efficient pMHC-II recycling and prevention of lysosomal targeting, thereby stabilizing pMHC-II for antigen presentation.\",\n      \"method\": \"MHC-II ubiquitination mutant mice, biochemical recycling assays, lysosomal targeting assays, surface stability measurements in DCs and B cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mouse model with multiple biochemical assays and functional outcome, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26240324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MARCH1 ubiquitinates the insulin receptor (INSR) to decrease cell surface INSR levels in the basal (non-stimulated) state, impairing insulin sensitivity. March1 loss-of-function enhances, and overexpression impairs, hepatic insulin sensitivity in mice. Unlike other INSR ubiquitin ligases, MARCH1 acts basally rather than after insulin stimulation.\",\n      \"method\": \"Large-scale RNAi screen, March1 KO mice, March1 overexpression mice, INSR ubiquitination assays, insulin clamp studies, flow cytometry for cell surface INSR\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi screen followed by KO and OE mouse models with multiple functional readouts, single rigorous study\",\n      \"pmids\": [\"27577745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MARCH1 indirectly regulates MHC I surface expression through its ubiquitination of MHC II. In MARCH1-deficient B cells and DCs, the altered MHC II trafficking (not direct MARCH1 ubiquitination of MHC I) causes reduced surface MHC I and impaired cross-presentation to CD8+ T cells. Deletion of MHC II in March1-/- cells restores normal MHC I surface expression.\",\n      \"method\": \"MARCH1 KO mice, MHC II KO in March1-/- background, flow cytometry, antigen cross-presentation assays to CD8+ T cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with double KO rescue, multiple cell types, defined functional outcome\",\n      \"pmids\": [\"30001419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MARCH1 promotes ubiquitin-dependent MHCII turnover that is required to maintain homeostasis of lipid rafts and the tetraspanin web in DC plasma membranes. Without MHCII ubiquitination, MHCII accumulates excessively in the plasma membrane, disrupting these membrane domains and impairing DC ability to engage thymocytes for Treg differentiation.\",\n      \"method\": \"MARCH1 KO and MHCII ubiquitination knockin mice, lipid raft fractionation, tetraspanin web analysis, Treg differentiation co-culture assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic mouse models with membrane fractionation and defined cellular phenotype\",\n      \"pmids\": [\"29371232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"March-I is ubiquitinated on non-lysine residues (lysine-less March-I is ubiquitinated similarly to wild-type). March-I E3 ligase activity is not required for its own ubiquitination, indicating it does not autoubiquitinate itself. The E2 ubiquitin-conjugating enzyme Ube2D1 is required for March-I ubiquitination; Ube2D1 knockdown impairs March-I ubiquitination, increases March-I expression, and enhances March-I-dependent MHC-II downregulation.\",\n      \"method\": \"Lysine-less March-I mutant, catalytically inactive March-I mutant, Ube2D1 siRNA knockdown, ubiquitination assays, flow cytometry\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis (lysine-less and catalytic mutants) plus siRNA knockdown with multiple functional readouts, single lab\",\n      \"pmids\": [\"29414787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The March-I gene contains an APC-specific internal promoter driving expression of a distinct isoform in DCs and B cells. Downstream sequences in the first coding exon confer activation-induced down-regulation of March-I expression in DCs. These regulatory elements do not function in fibroblasts, kidney, or epithelial cells.\",\n      \"method\": \"March-I promoter-GFP reporter constructs, transfection in multiple cell types, LPS activation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assays with multiple cell types and activation conditions, single lab\",\n      \"pmids\": [\"29378848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MARCH1 inhibits MAVS/STING/TRIF-induced type I IFN signaling in vitro and in vivo. In malaria-infected hosts, March1 deficiency paradoxically reduces IFN-I production by activating inhibitors (SOCS1, USP18, TRIM24) and altering immune cell populations, leading to increased IFN-γ and improved host survival that is reversed by T cell depletion or IFN-γ neutralization.\",\n      \"method\": \"March1 KO mice, Plasmodium yoelii infection model, transspecies eQTL genetic screen, MAVS/STING/TRIF reporter assays, cytokine measurements, T cell depletion, antibody neutralization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mice with multiple functional assays, single lab, in vitro and in vivo data\",\n      \"pmids\": [\"32606244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MARCH1 ubiquitination of MHC-II in DCs regulates DC gene expression and functional fitness. MHC-II ubiquitination-deficient DCs are poor stimulators of naive CD4 T cells and secrete IL-12 poorly in response to LPS despite high surface MHC-II. Single-cell RNA sequencing reveals these DCs have an altered gene expression signature that is reversed by LPS activation.\",\n      \"method\": \"MHC-II ubiquitination mutant mice, naive CD4 T cell stimulation assays, IL-12 ELISA, single-cell RNA sequencing\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mouse model with multiple functional assays plus scRNA-seq, single rigorous study with orthogonal methods\",\n      \"pmids\": [\"33318291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MARCH1 ubiquitin ligase regulates CD86 and MHC II in professional and atypical antigen presenting cells of hematopoietic origin (including neutrophils, eosinophils, and monocytes), whereas MARCH8 operates in non-hematopoietic cells. Unbiased plasma membrane proteomic profiling of primary cells from MARCH1-deficient mice identified CD86 and MHC II as the only substrates unequivocally regulated by MARCH1 in vivo.\",\n      \"method\": \"MARCH1 KO and MARCH8 KO mice, unbiased plasma membrane proteomics (mass spectrometry), flow cytometry across multiple cell types\",\n      \"journal\": \"Current research in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — unbiased proteomic substrate identification in KO mice across multiple tissues, single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"35492398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MARCH1-mediated ubiquitination of MHCII and CD86 on lymph node-resident DCs is required for TH2 cell development. Mice with mutations in the ubiquitin acceptor sites of both MHCII and CD86 fail to develop TH2 cells, demonstrating that clearance of antigen-presenting and costimulatory molecules by MARCH1 controls TCR signaling strength during TH2 polarization.\",\n      \"method\": \"MARCH1 KO mice, MHCII and CD86 ubiquitin-site knockin mice, in vivo TH2 differentiation assays, GATA-3 expression analysis\",\n      \"journal\": \"Science immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple knockin mouse models and defined TH2 differentiation phenotype\",\n      \"pmids\": [\"34652961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MARCH1 promotes ubiquitination and degradation of HDAC11, which relieves HDAC11/KLF4-dependent repression of the OX40L gene promoter, thereby increasing OX40L expression in allergen-stimulated dendritic cells.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, luciferase OX40L promoter reporter assay, MARCH1 overexpression/knockdown, Western blot\",\n      \"journal\": \"Journal of asthma and allergy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, reporter assay, and overexpression/knockdown in same study, single lab\",\n      \"pmids\": [\"34385821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Human MARCH1 recognizes the CD86 transmembrane domain through a highly specific surface in the hydrophobic core featuring proline at position 254. This recognition mode is distinct from the viral MARCH homolog MIR2, which requires an aspartic acid in the CD86 extracellular juxtamembrane region and no specific TM sequences.\",\n      \"method\": \"Deep mutational scanning of CD86 TM domain, overexpression in cell lines, flow cytometry for surface CD86\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis (deep mutational scanning) with functional readout, single lab with comprehensive coverage\",\n      \"pmids\": [\"34157285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCH1 ubiquitinates and promotes degradation of PHLPP2 in oral squamous cell carcinoma cells. Co-immunoprecipitation confirmed direct interaction between MARCH1 and PHLPP2. MARCH1 knockdown suppresses OSCC tumorigenicity in vivo and increases PHLPP2 protein levels.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, Western blot, in vivo xenograft model\",\n      \"journal\": \"Clinical & translational oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP plus KD/OE functional experiments with in vivo validation, single lab\",\n      \"pmids\": [\"35122633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCH1 isoform 1 (MARCH1.1) restricts influenza A virus (IAV) replication/infectivity, whereas isoform 2 (MARCH1.2) does not. Deletion of the entire N-terminal cytoplasmic domain of MARCH1.2, or specifically the 16 N-terminal residues, restores IAV restriction, mapping the difference in antiviral activity to the N-CT of MARCH1.2.\",\n      \"method\": \"Doxycycline-inducible overexpression system, viral titer assays, sequential N-CT deletion mutagenesis, qPCR for isoform expression\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion mutagenesis with viral titer readout, single lab, multiple deletions tested\",\n      \"pmids\": [\"36423158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCH1 ubiquitinates and promotes degradation of the transferrin receptor (TfR), thereby regulating cellular iron levels during human cytomegalovirus (HCMV) infection. MARCH1 knockdown decreases infectious HCMV titers, increases ROS, lipid peroxidation, and mitochondrial dysfunction, establishing a proviral role for MARCH1 in regulating iron metabolism.\",\n      \"method\": \"MARCH1 knockdown (siRNA), TfR expression analysis, labile iron pool measurement, ROS and lipid peroxidation assays, viral titer assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with multiple functional readouts, single lab, mechanistic connection established\",\n      \"pmids\": [\"35045264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MARCHF1 acts as an E3 ubiquitin ligase for GPX4, promoting its ubiquitination and degradation, thereby increasing susceptibility to ferroptosis. BaP/BPDE exposure up-regulates MARCHF1 expression, leading to GPX4 protein degradation and consequent suppression of HUVEC angiogenesis and induction of miscarriage.\",\n      \"method\": \"Ubiquitination assays, MARCHF1 overexpression/knockdown in HUVECs, GPX4 protein level measurement, angiogenesis assays, mouse BaP exposure miscarriage model\",\n      \"journal\": \"Environment international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assays plus KD/OE with in vitro and in vivo functional readouts, single lab\",\n      \"pmids\": [\"37802009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Human MARCH1 and MARCH2 retain Ebola virus (EBOV) glycoprotein (GP) at the trans-Golgi network, reduce its cell surface display, and impair EBOV GP-pseudotyped virion infectivity by blocking furin-mediated cleavage. The furin P domain is recognized by MARCH1/2/8 and is critical for their blocking activities. Host furin interacts with MARCH1/2 and EBOV GP intracellularly.\",\n      \"method\": \"Co-immunoprecipitation of furin with MARCH1/2 and EBOV GP, EBOV GP-pseudotyped viral infection assays, furin P domain mutagenesis, subcellular localization analysis\",\n      \"journal\": \"Journal of medical virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional viral assays with domain-level mechanistic dissection, single lab\",\n      \"pmids\": [\"38299743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCH1 interacts with TBK1 and promotes K63-linked ubiquitination of TBK1, attenuating TBK1 interaction with mTOR and thereby inhibiting growth factor-induced mTOR signaling. MARCH1 deficiency accelerates breast cancer cell proliferation, which is reversed by inhibition of mTOR, STING, or TBK1.\",\n      \"method\": \"Co-immunoprecipitation, denatured Co-IP ubiquitination assays, Western blot, shRNA knockdown, lentiviral overexpression, scratch wound and colony assays\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays with multiple rescue experiments, single lab\",\n      \"pmids\": [\"39061024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCH1 mediates ubiquitination and degradation of PCSK9, lowering PCSK9 protein expression, and thereby suppresses inflammation and pyroptosis in cerebral ischemia-reperfusion injury. Mechanistically, PCSK9 knockdown reduces brain damage, upregulates LDLR, and inactivates the NLRP3 inflammasome.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide degradation assay, MARCH1/PCSK9 overexpression/knockdown, MCAO/R mouse model, OGD/R neuron model\",\n      \"journal\": \"Mammalian genome\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and degradation assays with in vivo and in vitro functional validation, single lab\",\n      \"pmids\": [\"39115562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CD83 suppresses endogenous March-I-dependent MHC-II ubiquitination, endocytosis, and degradation in mouse spleen DCs. Endogenous March-I protein is present at extremely low copy numbers (~500 molecules/cell in DCs, ~125 in B cells), has a very short half-life, and March-I mRNA, protein, and MHC-II ubiquitination are all rapidly terminated upon DC or B cell activation.\",\n      \"method\": \"V5-epitope knock-in into endogenous March-I locus, quantitative flow cytometry, MHC-II ubiquitination assays, pulse-chase half-life, LPS activation of primary spleen DCs and B cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous knock-in tagging with quantitative analysis and multiple orthogonal assays, single rigorous study\",\n      \"pmids\": [\"40397676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCHF1 promotes GABAB receptor downregulation under ischemic/excitotoxic conditions by interacting with GABAB receptors and inhibiting fast receptor recycling at the plasma membrane, leading to progressive neuronal death. An interfering peptide blocking the MARCH1/GABAB receptor interaction restores receptor surface expression and prevents neuronal death.\",\n      \"method\": \"Co-immunoprecipitation, interfering peptide competition, surface GABAB receptor quantification in ischemic neurons, neuronal death assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and peptide competition with functional cell-death readout, single lab, mechanistically specific\",\n      \"pmids\": [\"39779794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCHF1 interacts with SARS-CoV-2 M protein and mediates ubiquitination and degradation of GPX4, contributing to ferroptosis during SARS-CoV-2 infection. The M-GPX4 interaction occurs at the R72 residue of GPX4.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis of GPX4 R72, ferroptosis assays in infected cells\",\n      \"journal\": \"Journal of medical virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination with mutagenesis identifying specific interaction site, single lab\",\n      \"pmids\": [\"40186530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH1 interacts with MYCT1 (a candidate tumor suppressor) and promotes its K48-linked ubiquitination and degradation, facilitating AML cell proliferation and survival. POU2F2 transcription factor positively regulates MARCH1 transcription in AML. MYCT1 knockdown abolishes the inhibitory effects of MARCH1 knockdown on AML cell growth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, gain- and loss-of-function experiments, in vivo AML mouse model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assays, and genetic rescue experiments with in vivo validation, single lab\",\n      \"pmids\": [\"40533483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCHF1 ubiquitinates and promotes degradation of SULF1 in colon cancer cells, thereby blocking SULF1-THBS2 interaction and suppressing TGF-β1/SMAD2/3 pathway activation, which leads to reduced tumor metastasis and enhanced sensitivity to 5-FU.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, MARCHF1 and SULF1 knockdown/overexpression, TGF-β pathway analysis, drug sensitivity assays\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination with pathway rescue experiments, single lab\",\n      \"pmids\": [\"41550717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCHF1 interacts with and promotes ubiquitination and degradation of REST (repressor element-1 silencing transcription factor), reducing REST protein levels and consequently de-repressing TFAM transcription, promoting mitochondrial function and breast cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, MARCHF1 knockdown/overexpression, Western blot for REST and TFAM, in vivo xenograft model\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP with functional KD/OE readouts and in vivo validation, single lab\",\n      \"pmids\": [\"39428668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MARCH1 directly binds to KLF15 (Krüppel-like factor 15) in white adipose tissue, contributing to HFpEF pathogenesis. MARCH1 deficiency induces WAT beiging and alleviates cardiac dysfunction in a HFpEF mouse model; overexpressing KLF15 blocks the beiging effects of MARCH1 KO.\",\n      \"method\": \"Co-immunoprecipitation, MARCH1 KO HFpEF mouse model, RNA sequencing, echocardiography, histology, KLF15 overexpression rescue\",\n      \"journal\": \"Comprehensive Physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with KO mouse model and genetic rescue, single lab\",\n      \"pmids\": [\"41986908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MARCH1 promotes ubiquitination and degradation of SLC25A17, a mitochondrial transporter. MARCH1 overexpression suppresses macrophage M2 polarization and DDP resistance in lung adenocarcinoma; SLC25A17 overexpression reverses the DDP sensitivity enhancement caused by MARCH1 reintroduction.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, MARCH1 overexpression/knockdown, SLC25A17 rescue overexpression, flow cytometry, ELISA, in vivo tumor model\",\n      \"journal\": \"Integrative biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays with genetic rescue experiments and in vivo validation, single lab\",\n      \"pmids\": [\"41758657\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MARCHF1 (MARCH1) is a membrane-anchored RING-CH E3 ubiquitin ligase that constitutively ubiquitinates the cytoplasmic tail of MHC class II beta-chain and CD86 in resting antigen-presenting cells, routing these molecules to lysosomes for degradation and limiting surface antigen presentation; this activity is terminated by DC maturation signals (e.g., LPS) or CD83 engagement, stabilizing surface pMHC-II for efficient CD4 T cell activation, and MARCH1 is itself regulated by IL-10 (which induces its expression in macrophages and monocytes), by Ube2D1-mediated lysine-independent ubiquitination that controls its own turnover, and by its very short protein half-life via lysosomal/cysteine protease degradation. Beyond immune regulation, MARCH1 ubiquitinates the insulin receptor (setting basal insulin sensitivity), GPX4 (promoting ferroptosis), TBK1 (K63-linked, suppressing mTOR signaling), PCSK9 (suppressing NLRP3-mediated inflammation), HDAC11 (increasing OX40L expression), PHLPP2, REST, MYCT1, SULF1, SLC25A17, TfR, and GABAB receptors, placing it as a broad-spectrum membrane-localized E3 ligase that controls immunological tolerance, metabolic homeostasis, antiviral restriction, and neuronal survival.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MARCHF1 (MARCH1) is a membrane-anchored RING-CH E3 ubiquitin ligase that serves as a central rheostat of antigen presentation by constitutively ubiquitinating the cytoplasmic tails of MHC class II and the costimulatory molecule CD86 in antigen-presenting cells, routing these molecules to lysosomes and limiting their surface display [#0, #1, #18]. By targeting newly synthesized peptide-MHC-II for degradation and preventing its recycling from the cell surface, MARCH1 keeps resting APCs in a low-presentation state; acute activation signals such as LPS rapidly terminate MARCH1 expression, stabilizing surface pMHC-II for CD4 T cell engagement [#10, #29]. This ubiquitin-dependent turnover — rather than steady-state surface MHC-II levels per se — is required for conventional DC fitness, thymic regulatory T cell generation, TH2 development, lipid raft/tetraspanin web homeostasis, and indirectly for MHC class I cross-presentation [#3, #8, #13, #19, #12]. MARCH1 substrate recognition is encoded in the substrate transmembrane domain, exemplified by its requirement for proline-254 in the CD86 TM core [#21]. The ligase is held in check by multiple layers of regulation: it is induced by IL-10 in monocytes and macrophages [#1, #9], its substrate engagement is blocked by the transmembrane domain of CD83 [#4, #29] and competed by Tollip [#7], and the protein itself is extremely short-lived (~500 molecules/cell, half-life <30 min) and is turned over via lysosomal cysteine proteases and through Ube2D1-dependent lysine-independent ubiquitination [#2, #14, #29]. Beyond immunity, MARCH1 ubiquitinates a broad set of membrane and intracellular targets including the insulin receptor to set basal insulin sensitivity [#11], GPX4 to promote ferroptosis [#25], and TBK1 via K63-linked chains to suppress mTOR signaling [#27], and it contributes to antiviral restriction of influenza and Ebola glycoprotein trafficking [#23, #26].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established MARCH1 as the E3 ligase that ubiquitinates MHC class II and drives its surface internalization, defining a molecular basis for maturation-controlled antigen presentation.\",\n      \"evidence\": \"Ubiquitination assays, Co-IP, and siRNA knockdown in primary human monocyte-derived DCs and IL-10-treated monocytes with surface flow cytometry\",\n      \"pmids\": [\"18305173\", \"18389477\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate ubiquitin acceptor sites not yet mapped in these studies\", \"Whether CD86 is a co-substrate not addressed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed in vivo that MHC-II ubiquitination specifically — not MARCH1 itself broadly — underlies DC function, using ubiquitination-site knockin mice that phenocopy the knockout.\",\n      \"evidence\": \"MARCH1 knockout and non-ubiquitinatable MHC-II knockin mice with in vivo antigen presentation and cytokine readouts; half-life and domain mutagenesis\",\n      \"pmids\": [\"19917682\", \"19880452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking MHC-II accumulation to impaired antigen presentation unresolved\", \"C-terminal VQNC motif role in catalysis not structurally defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined CD83 transmembrane domain as a physiological brake that blocks MARCH1 association with MHC-II, explaining how maturation signals protect surface MHC-II and CD86.\",\n      \"evidence\": \"ENU-induced CD83 TM-domain mutant mice with IL-10 stimulation and flow cytometry\",\n      \"pmids\": [\"21220452\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CD83-MARCH1 TM interference not defined\", \"Whether CD83 acts as competitive decoy substrate untested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected MARCH1-dependent MHC-II turnover to immune tolerance by showing it is required for thymic Treg generation, and identified Tollip as a competing regulator.\",\n      \"evidence\": \"MARCH1 KO and ubiquitination knockin mice in Treg assays; Tollip knockdown/Co-IP in HLA-DR cells\",\n      \"pmids\": [\"23712430\", \"24600555\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ubiquitin turnover translates to Treg-inducing DC signals unclear\", \"Tollip mechanism (single-lab) lacks reciprocal validation\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the trafficking logic — MARCH1 degrades newly synthesized pMHC-II and blocks its surface recycling — and showed IL-10 induction of MARCH1 is cell-type specific (macrophages, not DCs).\",\n      \"evidence\": \"MHC-II ubiquitination mutant mice with recycling and lysosomal targeting assays; cell-type-specific KO comparison with IL-10 stimulation\",\n      \"pmids\": [\"26240324\", \"26408197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sorting machinery routing ubiquitinated pMHC-II to lysosomes not fully identified\", \"Transcriptional basis of cell-type-specific IL-10 responsiveness unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended MARCH1 beyond immunity by identifying the insulin receptor as a basal-state substrate that sets metabolic insulin sensitivity, distinguishing MARCH1 from stimulus-dependent INSR ligases.\",\n      \"evidence\": \"RNAi screen, March1 KO and overexpression mice, INSR ubiquitination and insulin clamp studies\",\n      \"pmids\": [\"27577745\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"INSR ubiquitination site not mapped\", \"Tissue-specific contribution to systemic metabolism not fully partitioned\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Refined the self-regulation and substrate-scope picture: MARCH1 is turned over by lysine-independent, Ube2D1-dependent ubiquitination (not autoubiquitination), and unbiased proteomics confirmed CD86 and MHC-II as its only unequivocal in vivo membrane substrates in hematopoietic cells.\",\n      \"evidence\": \"Lysine-less and catalytic mutants with Ube2D1 knockdown; plasma membrane proteomics in MARCH1 and MARCH8 KO mice; double-KO MHC-I rescue; APC-specific promoter reporters\",\n      \"pmids\": [\"29414787\", \"35492398\", \"30001419\", \"29378848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Earlier autoubiquitination model (idx 5) is contradicted — chain dependence on Ube2D1 vs MARCH1 activity needs reconciliation\", \"Proteomics may miss low-abundance or non-membrane substrates later reported\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the molecular grammar of substrate recognition (CD86 TM proline-254) and broadened immune function to TH2 development and DC functional programming via MHC-II/CD86 clearance.\",\n      \"evidence\": \"Deep mutational scanning of CD86 TM; MHC-II/CD86 ubiquitin-site knockin mice in TH2 assays; scRNA-seq of ubiquitination-deficient DCs\",\n      \"pmids\": [\"34157285\", \"34652961\", \"33318291\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of MARCH1-substrate TM complex\", \"How surface molecule clearance tunes TCR signaling strength mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established quantitatively that endogenous MARCH1 is present at extremely low copy number with rapid activation-induced termination, and expanded the non-immune substrate repertoire to TBK1 (K63-linked, suppressing mTOR).\",\n      \"evidence\": \"Endogenous V5 knock-in with quantitative flow cytometry and pulse-chase; Co-IP and denatured ubiquitination assays for TBK1 with mTOR/STING/TBK1 inhibitor rescue\",\n      \"pmids\": [\"40397676\", \"39061024\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"TBK1 finding (single-lab, Medium) lacks independent confirmation\", \"Chain-type switching (K48 for MHC-II vs K63 for TBK1) determinants unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reported a wave of context-specific substrates (GPX4, PCSK9, SULF1, MYCT1, REST, GABAB receptor, SLC25A17, KLF15) linking MARCH1 to ferroptosis, neuronal death, and tumor biology.\",\n      \"evidence\": \"Co-IP and ubiquitination/degradation assays with knockdown/overexpression and rescue across cancer, ischemia, and infection models\",\n      \"pmids\": [\"37802009\", \"39115562\", \"41550717\", \"40533483\", \"39428668\", \"39779794\", \"40186530\", \"41758657\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Each substrate rests on single-lab Co-IP without reciprocal/structural validation\", \"Most were not detected in the unbiased in vivo proteomic screen (idx 18), raising context-dependence questions\", \"Direct vs indirect ubiquitination not always disambiguated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single low-abundance, short-lived membrane ligase achieves such broad and context-specific substrate selectivity — and how chain-type and subcellular routing are determined — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of MARCH1 with any substrate\", \"Determinants selecting K48 vs K63 linkage across substrates unknown\", \"Reconciliation of restricted in vivo proteomic substrate set with many reported disease-context substrates pending\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 11, 25, 27, 32, 33]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 11, 25, 27]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 5, 14, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 10, 18, 21]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 6, 10]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [26]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 2, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 3, 8, 9, 18, 19]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 11, 25, 27, 33]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [10, 30]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [11, 24, 36]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CD74\",\n      \"CD86\",\n      \"CD83\",\n      \"TOLLIP\",\n      \"UBE2D1\",\n      \"INSR\",\n      \"GPX4\",\n      \"TBK1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}