{"gene":"MARCHF8","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2003,"finding":"c-MIR (MARCHF8) was identified as a novel E3 ubiquitin ligase containing a BKS-PHD (RING-CH) domain. It targets B7-2 (CD86) for ubiquitination, rapid endocytosis, and lysosomal degradation by binding directly to B7-2. The BKS-PHD domain is functionally interchangeable with that of viral MIR1.","method":"Forced expression, ubiquitination assay, endocytosis and lysosomal degradation assays, domain-swap mutagenesis, binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (ubiquitination assay, binding assay, endocytosis/degradation assay, mutagenesis) in one study establishing enzymatic activity and substrate specificity","pmids":["12582153"],"is_preprint":false},{"year":2006,"finding":"c-MIR (MARCHF8) targets MHC class II (I-A beta-chain) for ubiquitination and lysosomal degradation. The ubiquitination depends on a single lysine residue (K225) in the cytoplasmic tail of the I-A beta-chain. In vivo, c-MIR overexpression in dendritic cells suppresses antigen presentation and impairs CD4 T cell development, and confers resistance to experimental autoimmune encephalomyelitis.","method":"Reconstitution in 293T cells, site-directed mutagenesis of ubiquitin acceptor lysine, forced expression in B cell lines, transgenic mouse model, flow cytometry, immunological assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution with mutagenesis of substrate lysine, confirmed in primary cells and transgenic mouse model with defined immunological phenotype","pmids":["16785530"],"is_preprint":false},{"year":2012,"finding":"MARCH8 interacts with IL-1 receptor accessory protein (IL1RAP) and catalyzes K48-linked polyubiquitination of IL1RAP at Lys512, leading to its degradation and suppression of IL-1β-induced NF-κB and MAPK activation. Overexpression of MARCH8 inhibits this signaling; knockdown has the opposite effect.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K512), overexpression and knockdown with NF-κB/MAPK reporter assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination assay, mutagenesis, functional signaling readout) in a single rigorous study","pmids":["22904187"],"is_preprint":false},{"year":2013,"finding":"MARCH-8 ubiquitinates TRAIL-R1 (death receptor 1) at the conserved membrane-proximal lysine K273 in its cytoplasmic tail, reducing TRAIL-R1 cell surface expression and attenuating TRAIL-induced apoptosis signaling. MARCH-8 ligase activity is required for this downregulation. Gene silencing confirmed MARCH-8 as the endogenous ligase responsible.","method":"Gene silencing (siRNA), ubiquitination assay, mutagenesis of K273, flow cytometry, apoptosis assay, interaction assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — endogenous ligase confirmed by gene silencing, mutagenesis of substrate acceptor lysine, and functional apoptosis readout in multiple experiments","pmids":["23300075"],"is_preprint":false},{"year":2015,"finding":"MARCH8 reduces HIV-1 infectivity in virus-producing cells by downregulating HIV-1 envelope glycoprotein (Env) from the cell surface through direct interaction, thereby reducing virion incorporation of Env. MARCH8 also broadly inhibits VSV-G pseudotyped viruses. Endogenous MARCH8 is highly expressed in monocyte-derived macrophages and dendritic cells; knockdown in macrophages increases virion infectivity.","method":"Ectopic expression, HIV-1 infection assay, flow cytometry (surface Env), Co-immunoprecipitation (MARCH8-Env interaction), siRNA/CRISPR knockout, macrophage differentiation experiments","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction, endogenous KO/KD with functional infectivity readout, confirmed in primary cells; replicated and extended in subsequent studies","pmids":["26523972"],"is_preprint":false},{"year":2016,"finding":"Salmonella effector SteD localizes to the Golgi and vesicles containing MARCH8. SteD acts as an adaptor that forces MARCH8-dependent ubiquitination of mature MHC-II (mMHCII), reducing surface mMHCII and B7.2 and suppressing T cell activation. The C-terminal cytoplasmic region of SteD binds mMHCII; its transmembrane domain binds MARCH8.","method":"Cellular localization (immunofluorescence), Co-immunoprecipitation, MARCH8-dependent ubiquitination assay, domain mapping by mutagenesis, T cell activation assay, mouse infection model","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mapping by mutagenesis, localization, Co-IP, functional immune readout, and in vivo mouse model all in one study","pmids":["27832589"],"is_preprint":false},{"year":2016,"finding":"In thymic cortical epithelial cells (cTECs), MARCH8 targets MHC-II for ubiquitination and turnover. CD83's transmembrane domain antagonizes MARCH8-mediated MHC-II ubiquitination in cTECs to stabilize MHC-II, which is required for CD4 T cell selection. Ablating March8 in Cd83-/- mice rescues CD4 T cell development. There is a division of labor: MARCH1 controls MHC-II in hematopoietic APCs; MARCH8 controls constitutive MHC-II in non-hematopoietic cTECs.","method":"Viral gene reconstitution in TECs, genetic epistasis (March8/Cd83 double knockout mice), ubiquitination-resistant MHC-II variant rescue, flow cytometry of T cell populations","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with double KO and rescue by ubiquitination-resistant variant, replicated functional T cell development phenotype","pmids":["27503071"],"is_preprint":false},{"year":2016,"finding":"KSHV lytic transactivator RTA upregulates MARCH8 transcription, and increased MARCH8 in turn downregulates HLA-DRα (MHC-II) surface expression, contributing to KSHV immune evasion. RTA can also directly bind and degrade HLA-DRα via the proteasome pathway independently.","method":"Overexpression, Western blot, flow cytometry, transcript analysis, KSHV de novo infection","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — overexpression and Western blot in multiple contexts, but mechanistic link between RTA-induced MARCH8 and HLA-DRα based on indirect evidence","pmids":["27356905"],"is_preprint":false},{"year":2017,"finding":"MARCH8 mediates K48-linked ubiquitination of DR4 (TRAIL-R1/death receptor 4) at K273, leading to its degradation. JWA upregulates MARCH8 expression to promote DR4 ubiquitination, thereby suppressing TRAIL-induced apoptosis in cisplatin-resistant gastric cancer cells.","method":"Ubiquitination assay, site-directed mutagenesis, Western blot, siRNA knockdown, apoptosis assay","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay and mutagenesis of substrate lysine, single lab, two orthogonal methods","pmids":["28671676"],"is_preprint":false},{"year":2017,"finding":"Yersinia pestis acetyltransferase YopJ acetylates MARCH8 at serine (S44, S71, S253) and lysine (K247, K252) residues, and this dual acetylation affects MARCH8 auto-ubiquitination. YopJ C172A mutant abolishing acetyltransferase activity reduces acetylation and diminishes MARCH8 auto-ubiquitination.","method":"Shotgun proteomics (label-free quantification), Western blot with site-specific antibodies, in vitro acetylation assay with purified YopJ, mutagenesis (C172A)","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro acetylation assay with mutagenesis and proteomics, single lab","pmids":["28103160"],"is_preprint":false},{"year":2019,"finding":"MARCH8 catalyzes K63-linked polyubiquitination of the HCV nonstructural protein NS2 both in vitro and in HCV-infected cells, and is required for HCV envelopment. MARCH8 was identified through NS2 interactome mapping with the ubiquitin-proteasome system.","method":"Mammalian cell-based interactome screen, in vitro ubiquitination assay, HCV infection assay, Co-immunoprecipitation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination assay combined with cellular infection assay and interactome screen, single lab with multiple orthogonal methods","pmids":["30759391"],"is_preprint":false},{"year":2019,"finding":"MARCH8 promotes ubiquitination and degradation of myosin light chain 2 (MLC2) in hippocampal neurons following TNF-α stimulation, contributing to caspase-3 activation and neuronal apoptosis. MARCH8 overexpression attenuates MLC2 levels; siRNA knockdown of MARCH8 blocks caspase-3 activation.","method":"In vivo intracerebroventricular TNF-α injection in rats, Western blot, siRNA knockdown, overexpression, apoptosis assay","journal":"Anatomical record","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single ubiquitination/degradation assay with limited mechanistic follow-up on substrate specificity","pmids":["31443122"],"is_preprint":false},{"year":2020,"finding":"MARCH8 reduces viral infectivity via two distinct mechanisms: (1) Ubiquitination-dependent downregulation of VSV-G cytoplasmic lysine residues (lysine mutant VSV-G is resistant); (2) A tyrosine motif (cytoplasmic tyrosine motif)-dependent mechanism for HIV-1 Env (MARCH8 tyrosine motif mutant loses ability to inhibit Env-mediated infection but retains VSV-G inhibition). These are two separate pathways.","method":"Mutagenesis of VSV-G cytoplasmic lysines, mutagenesis of MARCH8 tyrosine motif, pseudovirus infection assay, ubiquitination assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mechanistic dissection using multiple domain mutants and two substrates, clearly distinguishes two independent antiviral mechanisms","pmids":["32778221"],"is_preprint":false},{"year":2020,"finding":"MARCH8 blocks Ebola virus glycoprotein (EBOV GP) incorporation into virions by: (1) interacting with EBOV GP and furin; (2) retaining the GP/furin complex in the Golgi (BiFC assay); (3) inhibiting complex N-glycosylation of GP, O-glycosylation, and furin-mediated proteolytic cleavage. Only fully glycosylated GP is processed by furin and incorporated into virions. MARCH8 similarly blocks furin-mediated cleavage of HIV-1 Env (gp160) and H5N1 HA.","method":"Immunoprecipitation, bimolecular fluorescence complementation (BiFC) assay, glycosylation analysis, furin cleavage assay, virion incorporation assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal assays (BiFC, Co-IP, glycosylation analysis, cleavage assay) establishing Golgi-trapping and glycosylation-blocking mechanism","pmids":["32934085"],"is_preprint":false},{"year":2021,"finding":"MARCH8 mediates K63-linked polyubiquitination of influenza A virus M2 protein at lysine K78, redirecting M2 from the plasma membrane to lysosomes for degradation, thereby suppressing IAV release. A recombinant A/PR/8/34 virus carrying K78R M2 shows greater replication and pathogenicity. H1N1 IAV has evolved non-lysine residues at positions 78/79 to resist MARCH8-mediated restriction.","method":"In vitro and in vivo (mouse) infection assay, ubiquitination assay, site-directed mutagenesis (K78R), recombinant virus, lysosomal degradation assay, MARCH8 KO cells","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination, site-directed mutagenesis of substrate lysine, recombinant virus with confirmed phenotype in vivo, multiple orthogonal methods","pmids":["34285233"],"is_preprint":false},{"year":2021,"finding":"MARCH8 is identified as a positive regulator of type I IFN (IFN-I) signaling; SVA 2AB protein interacts with MARCHF8 and MAVS, forming a large complex that is degraded to deactivate IFN-I signaling. Additionally, 2AB degrades MARCHF8 and LC3 to inhibit autophagy.","method":"Co-immunoprecipitation, Western blot, siRNA knockdown, SVA infection assay, IFN signaling reporter assay","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and functional signaling readout, single lab; MARCHF8 role as positive IFN regulator is indirect (based on 2AB-mediated degradation phenotype)","pmids":["34964697"],"is_preprint":false},{"year":2021,"finding":"In primary immune cells in vivo, MARCH8 (but not MARCH1) operates specifically in non-hematopoietic cells including thymic and alveolar epithelial cells to regulate MHC-II and CD86 surface expression. Only CD86 and MHC-II were confirmed as MARCH8 substrates by unbiased proteomic profiling of plasma membranes from MARCH8-deficient primary cells.","method":"MARCH8-deficient mice, unbiased proteomic profiling of plasma membranes, flow cytometry, comparison with MARCH1-deficient mice","journal":"Current research in immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased plasma membrane proteomics in KO primary cells combined with flow cytometry across multiple cell types","pmids":["35492398"],"is_preprint":false},{"year":2021,"finding":"MARCH8 interacts with CD44 and mediates its lysosomal degradation. MARCH8 also ubiquitinates STAT3, a non-membrane protein, and promotes its proteasomal degradation, inducing apoptosis in breast cancer cells. Stable MARCH8 expression inhibits tumorigenesis and lung metastasis in vivo.","method":"Co-immunoprecipitation, ubiquitination assay, overexpression, xenograft mouse model, Western blot, apoptosis assay","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assays for two substrates, with in vivo confirmation, single lab","pmids":["34067416"],"is_preprint":false},{"year":2022,"finding":"MARCH8 targets cytoplasmic lysine residues of various viral envelope glycoproteins (rabies virus-G, LCMV glycoproteins, SARS-CoV and SARS-CoV-2 spike, Chikungunya E2, Ross River virus E2) for ubiquitination, intracellular degradation, and antiviral restriction. Lysine mutations in cytoplasmic tails of these glycoproteins confer resistance to MARCH8.","method":"Pseudovirus infection assay, site-directed mutagenesis of cytoplasmic lysines, immunofluorescence, ubiquitination assay","journal":"Microbiology spectrum","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis of substrate lysines combined with ubiquitination assays and infection readout across multiple viral glycoproteins","pmids":["35019698"],"is_preprint":false},{"year":2022,"finding":"MARCH8 interacts with the enzymatically active core of cGAS through its RING-CH domain and catalyzes K63-linked polyubiquitination of cGAS at K411. This ubiquitination inhibits cGAS DNA-binding ability and impairs cGAMP production, attenuating downstream innate immune responses. March8-deficient mice are less susceptible to HSV-1 infection.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K411), cGAMP production assay, DNA-binding assay, MARCH8 KO mice with HSV-1 infection","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ubiquitination, mutagenesis of substrate lysine, functional DNA-binding and cGAMP assays, in vivo mouse model","pmids":["35503863"],"is_preprint":false},{"year":2022,"finding":"MARCH8 interacts with ABCB1 (P-glycoprotein/MDR1) and promotes its ubiquitination and degradation. Rutaecarpine upregulates MARCH8 protein levels, leading to increased ABCB1 degradation and reversal of multidrug resistance in cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, Western blot, drug cytotoxicity assay, MARCH8 overexpression","journal":"Biomedicines","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay establishing substrate relationship, single lab","pmids":["34572328"],"is_preprint":false},{"year":2023,"finding":"MARCH8 interacts with PTEN and promotes its ubiquitination and proteasomal degradation, activating downstream AKT signaling and promoting HCC progression. MARCH8 overexpression promotes hepatic tumor growth in vivo via the AKT pathway.","method":"Co-immunoprecipitation, ubiquitination assay, Western blot, overexpression/knockdown, xenograft mouse model, flow cytometry","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay, in vivo tumor growth, single lab","pmids":["37098835"],"is_preprint":false},{"year":2023,"finding":"HPV E6-induced MYC/MAX transcriptional activation upregulates MARCHF8 expression in HPV-positive head and neck cancer. MARCHF8 directly interacts with and ubiquitinates FAS, TRAIL-R1 (DR4), and TRAIL-R2 (DR5), reducing their cell surface expression and inhibiting TNFRSF-mediated apoptosis. MARCHF8 KO in mouse oral cancer cells increases apoptosis and suppresses tumor growth in vivo.","method":"Promoter reporter assay, Co-immunoprecipitation, ubiquitination assay, flow cytometry, MARCHF8 KO in vivo tumor model, apoptosis assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple substrates confirmed by Co-IP and ubiquitination, in vivo tumor model with apoptosis phenotype, promoter mechanism defined","pmids":["36867660"],"is_preprint":false},{"year":2023,"finding":"MARCHF8 ubiquitinates and degrades CUL1 and UBE2L3, components of the SCF ubiquitin ligase complex responsible for HPV16 E7 degradation. By degrading CUL1 and UBE2L3, MARCHF8 reduces E7 ubiquitination, stabilizing the E7 oncoprotein in HPV+ head and neck cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression of CUL1/UBE2L3, in vivo tumor growth assay, Western blot","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus ubiquitination assays for CUL1 and UBE2L3, functional E7 stabilization shown, in vivo confirmation, single lab with multiple orthogonal methods","pmids":["38226814"],"is_preprint":false},{"year":2023,"finding":"EGR1 promotes SARS-CoV-2 N protein degradation via MARCH8 ubiquitin ligase activity and cargo receptor NDP52. MARCH8 mutants lacking ubiquitin ligase activity fail to degrade the N protein, confirming E3 activity is required.","method":"Co-immunoprecipitation, MARCH8 catalytic mutant, Western blot, SARS-CoV-2 replication assay, IFN reporter","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — catalytic mutant confirms E3 activity requirement, Co-IP, single lab","pmids":["37772822"],"is_preprint":false},{"year":2023,"finding":"MARCH8 interacts with PTPN4 and promotes its proteasomal ubiquitination and degradation, activating STAT3 phosphorylation (pSTAT3 Y705) and promoting pancreatic cancer growth and invasion. PTPN4 overexpression suppresses STAT3 activity.","method":"Co-immunoprecipitation, ubiquitination assay, Western blot, overexpression/knockdown, xenograft mouse model","journal":"Pancreas","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay, in vivo confirmation, single lab","pmids":["37747937"],"is_preprint":false},{"year":2024,"finding":"MARCH8 mediates K27-linked polyubiquitination of IL-7Rα at K265/K266, leading to lysosomal degradation of IL-7Rα. This negatively regulates IL-7-triggered STAT5 activation and T cell proliferation. MARCH8 deficiency increases IL-7-triggered signaling and splenic memory CD8+ T cell differentiation in mice.","method":"Co-immunoprecipitation, ubiquitination assay with K27-linkage specificity, site-directed mutagenesis (K265/K266), MARCH8 KO mice, STAT5 reporter, T cell proliferation assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — ubiquitination assay with linkage determination, mutagenesis of substrate acceptor lysines, in vivo MARCH8 KO mouse phenotype, multiple orthogonal methods","pmids":["39311660"],"is_preprint":false},{"year":2024,"finding":"MARCH8 inhibits pseudorabies virus (PRV) replication by trapping the viral cell-to-cell fusion complex (gB, gD, gH, gL) in the trans-Golgi network, independent of its E3 ubiquitin ligase activity. MARCH8 also blocks gB cleavage by recruiting furin (a ligase-dependent activity), but the furin-blocking activity alone does not inhibit viral infection in vitro.","method":"Protein interaction assay, subcellular localization (immunofluorescence/confocal), ligase-dead MARCH8 mutant, cell-to-cell fusion assay, viral replication assay","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ligase-dead mutant separates E3-independent from E3-dependent mechanisms, interaction and localization confirmed, single lab","pmids":["38944094"],"is_preprint":false},{"year":2024,"finding":"The antiviral function of MARCH8 requires both the RING-CH domain (for ubiquitination-dependent inhibition of VSV-G) and the C-terminal tyrosine motif (for inhibition of HIV-1 Env incorporation). Both N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, are critical for antiviral activity (determined by chimeric proteins between MARCH8 and non-antiviral MARCH3). MARCH8 is unable to block cell-to-cell HIV-1 infection.","method":"Chimeric protein domain analysis, mutagenesis of RING-CH and tyrosine motif, pseudovirus infection assay, cell-to-cell HIV-1 infection assay","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping by chimeric proteins and mutagenesis, single lab, multiple virus systems","pmids":["38667313"],"is_preprint":false},{"year":2024,"finding":"NLRP12 recruits MARCH8 (via its PYD domain) to the PRRSV glycoprotein GP2a, facilitating K48-linked polyubiquitination of GP2a at K128 and its lysosomal degradation via the MARCH8-NDP52 pathway, suppressing PRRSV replication.","method":"Co-immunoprecipitation (domain mapping), ubiquitination assay, site-directed mutagenesis (K128), overexpression/knockdown, viral replication assay","journal":"Veterinary microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-resolved Co-IP, mutagenesis of substrate lysine, single lab","pmids":["39362085"],"is_preprint":false},{"year":2024,"finding":"MARCHF8 interacts with MHC-I proteins, ubiquitinates them, and promotes their degradation in HPV-positive head and neck cancer, facilitating immune evasion. MARCHF8 KO restores MHC-I levels, suppresses tumor growth, and increases NK and CD8+ T cell infiltration. Combination of MARCHF8 KO with anti-PD-1 further enhances tumor suppression in ICI-refractory tumors.","method":"Co-immunoprecipitation, ubiquitination assay, flow cytometry, MARCHF8 KO mouse tumor model, immune cell infiltration analysis, anti-PD-1 combination treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ubiquitination assay, in vivo KO tumor model with immune phenotyping, combination immunotherapy experiment, confirmed in peer-reviewed publication","pmids":["41802050"],"is_preprint":false},{"year":2025,"finding":"MARCH8 interacts with and promotes K63-linked polyubiquitination of IFITM3 at K24, directing it from the plasma membrane to lysosomes for degradation. MARCH8-KO cells accumulate IFITM3 at the plasma membrane after interferon treatment. MARCH8 expression attenuates IFITM3-mediated restriction of VSV and influenza A virus entry.","method":"Co-immunoprecipitation coupled to LC-MS/MS, ubiquitination assay with K63 linkage determination, site-directed mutagenesis (K24), MARCH8-KO cells, flow cytometry, viral infection assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — CoIP-MS for substrate identification, ubiquitination assay with linkage specificity and mutagenesis of acceptor lysine, KO cells with functional viral infection readout","pmids":["41197719"],"is_preprint":false},{"year":2025,"finding":"MARCH8 ubiquitinates NSUN6 at K271 and K462, causing its proteasomal degradation. Reduced NSUN6 lowers m5C modification on PEX1 and PEX3 mRNAs, destabilizing them through loss of YBX1 binding, leading to decreased peroxisome synthesis, increased ROS, DNA damage, and increased cisplatin sensitivity. Elevated ROS enhances NSUN6-MARCH8 interaction, forming a positive feedback loop.","method":"Co-immunoprecipitation, ubiquitination assay with mutagenesis (K271/K462), m5C methylation analysis, RNA stability assay, peroxisome biogenesis assay, ROS measurement","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of substrate lysines, functional pathway analysis, single lab with multiple methods","pmids":["40683951"],"is_preprint":false},{"year":2025,"finding":"MARCH8 promotes ubiquitination and proteasomal degradation of SREBP1, suppressing key enzymes for fatty acid de novo synthesis and reducing lipid deposition in HCC. This mechanism is supported by RNA sequencing and untargeted metabolomics combined with in vivo/in vitro experiments.","method":"Co-immunoprecipitation, ubiquitination assay, RNA sequencing, untargeted metabolomics, in vivo xenograft and mouse liver cancer model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay for SREBP1, in vivo confirmation, single lab with metabolomics validation","pmids":["40379644"],"is_preprint":false},{"year":2025,"finding":"NFATc1 transcriptionally upregulates MARCH8 in metastatic pancreatic cancer cells (where MARCH8 promoter is hypomethylated), and MARCH8 physically interacts with Orai3 intracellular loop and ubiquitinates Orai3 at its N-terminus, leading to lysosomal degradation and suppression of pancreatic cancer metastasis. In non-metastatic cells, MARCH8 promoter is hypermethylated and MARCH8 is not induced.","method":"ChIP, promoter methylation analysis, Co-immunoprecipitation, ubiquitination assay with domain mapping, overexpression/knockdown, in vivo metastasis model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ChIP, promoter methylation, Co-IP, ubiquitination with domain mapping, in vivo metastasis assay), single lab","pmids":["41023307"],"is_preprint":false},{"year":2025,"finding":"MARCH8 ubiquitinates CEMIP and promotes its degradation, inhibiting CRC cell proliferation, metastasis, and inducing ferroptosis through inactivation of the PI3K/AKT pathway. Overexpression of CEMIP reverses these effects.","method":"Co-immunoprecipitation, ubiquitination assay, ferroptosis markers (ROS, Fe2+, GSH, MDA), Western blot (PI3K/AKT), xenograft tumor model","journal":"Pathology, research and practice","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay, in vivo tumor model, single lab","pmids":["40107011"],"is_preprint":false},{"year":2025,"finding":"MARCH8 promotes K33-linked polyubiquitination of FMDV capsid proteins VP1 (at K210), VP2 (at K63), and VP3 (at K118), leading to their proteasomal degradation, thereby restricting FMDV replication in a dose-dependent manner. MARCH8 interaction and degradation of a non-enveloped virus's structural proteins demonstrates activity beyond viral envelope glycoproteins.","method":"Co-immunoprecipitation, ubiquitination assay with linkage determination, site-directed mutagenesis of substrate lysines, MARCH8 overexpression/knockdown, viral replication assay","journal":"Veterinary research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — K33 linkage ubiquitination with mutagenesis of all three substrate lysines, single lab","pmids":["40307900"],"is_preprint":false},{"year":2025,"finding":"FOXA1 transcriptionally activates MARCH8 expression; MARCH8 then catalyzes K48-linked ubiquitination of HK2 at K763, leading to HK2 degradation and inhibition of PINK1/Parkin-mediated mitophagy, thereby protecting against PM2.5-induced lung injury.","method":"ChIP (FOXA1 binding to MARCH8 promoter), Co-immunoprecipitation, ubiquitination assay with site mutagenesis (K763), mitophagy assays (mt-Keima, MMP, mtROS), in vivo mouse lung injury model","journal":"Environment international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for transcriptional regulation, mutagenesis of substrate lysine, functional mitophagy readout, single lab","pmids":["42217319"],"is_preprint":false},{"year":2025,"finding":"MARCHF8 promotes the degradation of TGFBI via ubiquitination; TGFBI in turn promotes apoptosis and ECM breakdown in LPS-stimulated nucleus pulposus cells and activates the NF-κB signaling pathway. MARCHF8 controls TGFBI expression levels, modulating intervertebral disc degeneration.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, NF-κB signaling markers, Western blot, bioinformatics (WGCNA) to identify hub gene","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination assay, but limited mechanistic validation of MARCHF8 specificity; functional link to NF-κB indirect","pmids":["39752341"],"is_preprint":false},{"year":2025,"finding":"MARCH8 ubiquitinates and promotes proteasomal degradation of NLRP3, and this activity is upregulated by Orientin treatment, attenuating NLRP3 inflammasome activation and pyroptosis in endothelial cells.","method":"Co-immunoprecipitation, ubiquitination assay, Western blot, NLRP3 inflammasome activation markers, in vivo atherosclerosis mouse model","journal":"Mediators of inflammation","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination assay, functional inflammasome readout, single lab with limited mechanistic detail on MARCH8 substrate specificity","pmids":["41891145"],"is_preprint":false},{"year":2024,"finding":"MARCHF8 was identified as degrading MHC-I proteins via ubiquitination in HPV-positive head and neck cancer in a preprint study subsequently published (PMID 41802050). MARCHF8 knockdown restores MHC-I levels, increases NK and CD8+ T cell infiltration, and in combination with anti-PD-1 synergistically suppresses tumor growth in ICI-refractory tumors.","method":"Co-immunoprecipitation, ubiquitination assay, flow cytometry, MARCHF8 KO mouse tumor model, immune phenotyping, anti-PD-1 combination","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — preprint replicated in peer-reviewed publication; included here only as reference to preprint record; primary confidence derives from published version","pmids":["39677690"],"is_preprint":true},{"year":2025,"finding":"MARCH8 promotes lysosomal degradation of IFITM3 through K63-linked polyubiquitination at K24, facilitating trafficking from the plasma membrane to lysosomes; MARCH8-KO cells show plasma membrane accumulation of IFITM3 and enhanced antiviral restriction. MARCH8 expression thus counteracts IFITM3-mediated antiviral defense.","method":"Co-immunoprecipitation with LC-MS/MS, ubiquitination assay, mutagenesis (K24), flow cytometry (subcellular localization), MARCH8-KO cells, viral infection assay (VSV, IAV)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — substrate identification by CoIP-MS, mutagenesis of acceptor lysine, KO cells with localization and functional viral readout, single rigorous study with multiple orthogonal methods","pmids":["41197719"],"is_preprint":false}],"current_model":"MARCHF8 is a transmembrane RING-CH E3 ubiquitin ligase that constitutively downregulates diverse membrane proteins—including MHC-II, MHC-I, CD86/B7-2, TRAIL-R1/DR4, FAS, TRAIL-R2, IL1RAP, IL-7Rα, and transferrin receptor—by catalyzing their ubiquitination (with substrate-specific K27, K48, K63, or K33 linkages) and routing them to lysosomal or proteasomal degradation; it inhibits a broad spectrum of enveloped viruses either by ubiquitinating cytoplasmic lysines of viral envelope glycoproteins (e.g., VSV-G, IAV M2, SARS-CoV-2 spike) for lysosomal degradation, or by retaining viral fusion protein/furin complexes in the trans-Golgi network via a tyrosine-motif-dependent mechanism that blocks glycosylation and furin cleavage, thereby preventing glycoprotein incorporation into virions; MARCHF8 also ubiquitinates cytosolic and nuclear substrates (cGAS at K411, STAT3, PTEN, SREBP1, HK2, NSUN6, CUL1/UBE2L3) to regulate innate immunity, metabolism, and oncogenesis; its activity is modulated by CD83, acetylation (by bacterial YopJ), and miR-34a-5p, and it occupies a tissue-specific niche (non-hematopoietic cells) partly distinct from the related MARCH1 ligase."},"narrative":{"mechanistic_narrative":"MARCHF8 (c-MIR/MARCH8) is a transmembrane RING-CH E3 ubiquitin ligase that constitutively controls the surface abundance of membrane proteins and a growing set of cytosolic and nuclear substrates, coupling target ubiquitination to lysosomal or proteasomal degradation [PMID:12582153, PMID:16785530]. It was discovered through its ability to bind B7-2 (CD86) directly and route it to endocytosis and lysosomal degradation via a RING-CH (BKS-PHD) domain functionally interchangeable with viral MIR1 [PMID:12582153]. A central physiological role is immune modulation: MARCHF8 ubiquitinates the MHC-II beta-chain cytoplasmic tail at a single lysine to suppress antigen presentation [PMID:16785530], and in vivo it operates specifically in non-hematopoietic cells such as thymic and alveolar epithelia—distinct from MARCH1 in hematopoietic APCs—where CD86 and MHC-II are its confirmed endogenous substrates and where it shapes CD4 T cell selection [PMID:27503071, PMID:35492398]. It further tunes immune signaling by degrading immune receptors (IL1RAP via K48 linkage, IL-7Rα via K27 linkage, TRAIL-R1/DR4, FAS, TRAIL-R2/DR5) to dampen NF-κB/MAPK, IL-7/STAT5, and death-receptor apoptotic signaling [PMID:22904187, PMID:23300075, PMID:36867660, PMID:39311660]. MARCHF8 is a broad antiviral effector acting through two separable mechanisms encoded in distinct regions: RING-CH–dependent ubiquitination of cytoplasmic lysines on viral envelope glycoproteins (VSV-G, IAV M2 at K78, SARS-CoV-2 spike and others) targeting them for degradation, and a C-terminal tyrosine-motif–dependent activity that traps glycoprotein/furin complexes in the trans-Golgi network, blocking glycosylation, furin cleavage, and virion incorporation [PMID:32778221, PMID:32934085, PMID:34285233, PMID:35019698, PMID:38944094, PMID:38667313]. It also ubiquitinates cytosolic and nuclear regulators—cGAS at K411 (K63-linked, inhibiting cGAMP production), IFITM3 at K24, and metabolic and oncogenic factors including PTEN, STAT3, SREBP1, HK2, and NSUN6—placing it at the intersection of innate immunity, metabolism, and tumor biology [PMID:35503863, PMID:41197719, PMID:34067416, PMID:37098835, PMID:40379644]. In HPV-positive head and neck cancer, MARCHF8 is transcriptionally induced and drives immune evasion by ubiquitinating MHC-I, FAS, and TRAIL receptors and by stabilizing the E7 oncoprotein through degradation of the SCF components CUL1 and UBE2L3 [PMID:36867660, PMID:38226814, PMID:41802050]. Its activity is shaped by interacting regulators and modifications, including CD83 antagonism of MHC-II ubiquitination and bacterial YopJ-mediated acetylation affecting auto-ubiquitination [PMID:27503071, PMID:28103160].","teleology":[{"year":2003,"claim":"Established MARCHF8 as a bona fide E3 ubiquitin ligase and defined its enzymatic logic—direct substrate binding plus RING-CH-catalyzed ubiquitination driving endocytosis and lysosomal degradation.","evidence":"Forced expression, ubiquitination, endocytosis/degradation assays and domain-swap mutagenesis identifying B7-2 (CD86) as substrate","pmids":["12582153"],"confidence":"High","gaps":["Did not resolve which ubiquitin linkage types are used","Endogenous physiological context not addressed"]},{"year":2006,"claim":"Showed MARCHF8 controls antigen presentation by ubiquitinating MHC-II at a single defined cytoplasmic lysine, linking molecular activity to an organismal immune phenotype.","evidence":"Reconstitution with K225 mutagenesis of I-A beta-chain, transgenic mouse with impaired CD4 T cell development and EAE resistance","pmids":["16785530"],"confidence":"High","gaps":["Tissue/cell type where endogenous MARCH8 acts on MHC-II not yet defined","Did not distinguish MARCH8 from MARCH1 roles"]},{"year":2012,"claim":"Extended substrate range to signaling receptors, showing MARCHF8 uses K48-linked ubiquitination to degrade IL1RAP and suppress innate inflammatory signaling.","evidence":"Co-IP, K512 mutagenesis, NF-κB/MAPK reporter assays with overexpression and knockdown","pmids":["22904187"],"confidence":"High","gaps":["Whether IL1RAP degradation is lysosomal or proteasomal not fully resolved"]},{"year":2013,"claim":"Identified MARCHF8 as the endogenous ligase for death receptor TRAIL-R1, linking it to apoptosis regulation via a conserved membrane-proximal lysine.","evidence":"siRNA silencing, K273 mutagenesis, flow cytometry and apoptosis assays","pmids":["23300075"],"confidence":"High","gaps":["Physiological/disease context of TRAIL-R1 control not established in this study"]},{"year":2016,"claim":"Resolved the in vivo niche of MARCHF8—non-hematopoietic thymic epithelium—and revealed CD83 as an antagonist that stabilizes MHC-II for T cell selection, establishing a division of labor with MARCH1.","evidence":"TEC reconstitution, March8/Cd83 double-KO epistasis, ubiquitination-resistant MHC-II rescue, plus bacterial SteD adaptor co-opting MARCH8 at the Golgi","pmids":["27503071","27832589","27356905"],"confidence":"High","gaps":["Mechanism of CD83 transmembrane antagonism at atomic level unresolved","How pathogens redirect substrate specificity beyond adaptor binding unclear"]},{"year":2015,"claim":"Defined MARCHF8 as a host antiviral restriction factor that downregulates HIV-1 Env and broadly inhibits enveloped viruses in producer cells.","evidence":"Ectopic expression, infectivity assays, surface Env flow cytometry, Co-IP, siRNA/CRISPR KO in primary macrophages","pmids":["26523972"],"confidence":"High","gaps":["Molecular basis of Env restriction (ubiquitination vs trafficking) not yet separated"]},{"year":2020,"claim":"Dissected two mechanistically separable antiviral activities—RING-CH ubiquitination of glycoprotein cytoplasmic lysines versus a tyrosine-motif-dependent Golgi-trapping mechanism that blocks glycosylation and furin cleavage.","evidence":"VSV-G lysine mutants and MARCH8 tyrosine-motif mutants in pseudovirus assays; BiFC, glycosylation and furin cleavage assays for EBOV GP/HIV Env/H5N1 HA","pmids":["32778221","32934085"],"confidence":"High","gaps":["Structural basis of tyrosine-motif-mediated Golgi retention unknown","How the furin/glycoprotein complex is physically retained not defined"]},{"year":2021,"claim":"Confirmed substrate-specific ubiquitin linkage usage and antiviral targeting through unbiased and mechanistic approaches, including K63-linked degradation of IAV M2 and K48-linked HCV NS2 ubiquitination, while proteomics narrowed validated surface substrates to CD86 and MHC-II.","evidence":"In vivo recombinant IAV K78R virus, ubiquitination assays, MARCH8-KO mice with plasma membrane proteomics, HCV interactome screen","pmids":["34285233","30759391","35492398"],"confidence":"High","gaps":["How linkage specificity (K48 vs K63) is determined per substrate unresolved","Reconciling broad overexpression substrate lists with narrow endogenous proteomic hits"]},{"year":2022,"claim":"Expanded MARCHF8 substrates beyond membrane proteins to cytosolic innate immune sensors, showing K63-linked ubiquitination of cGAS at K411 inhibits DNA sensing and cGAMP production.","evidence":"Co-IP, ubiquitination assay, K411 mutagenesis, cGAMP and DNA-binding assays, March8-KO mice with HSV-1 infection","pmids":["35503863","35019698","34572328"],"confidence":"High","gaps":["Whether cGAS regulation is constitutive or stimulus-induced unclear","How a membrane-anchored ligase accesses cytosolic cGAS not addressed"]},{"year":2023,"claim":"Established MARCHF8 as a tumor-promoting effector in HPV-positive cancer that drives apoptosis resistance (FAS/TRAIL receptors) and stabilizes the E7 oncoprotein by degrading SCF components CUL1 and UBE2L3.","evidence":"Promoter reporter (MYC/MAX), Co-IP, ubiquitination assays, MARCHF8-KO in vivo tumor models, plus PTEN and PTPN4 degradation studies","pmids":["36867660","38226814","37098835","37747937"],"confidence":"High","gaps":["Whether oncogenic vs tumor-suppressive roles are context-dependent not unified","Direct vs indirect contribution of each substrate to tumor phenotype unclear"]},{"year":2024,"claim":"Demonstrated MARCHF8 mediates immune evasion via MHC-I degradation and links to checkpoint immunotherapy response, while defining K27-linked degradation of IL-7Rα controlling T cell memory.","evidence":"Co-IP, ubiquitination assays, MARCHF8-KO tumor models with immune phenotyping and anti-PD-1 combination; IL-7Rα K265/K266 mutagenesis with KO mouse T cell phenotype","pmids":["41802050","39311660","38667313","39362085"],"confidence":"High","gaps":["Determinants selecting MHC-I vs MHC-II vs other substrates unknown","Therapeutic targetability of MARCHF8 not established"]},{"year":2025,"claim":"Broadened the functional reach to metabolism, RNA modification, and additional antiviral/inflammatory pathways, including K63 ubiquitination of IFITM3, degradation of SREBP1, HK2, NSUN6, and K33-linked targeting of non-enveloped FMDV capsid proteins.","evidence":"CoIP-MS substrate ID, linkage-specific ubiquitination assays, mutagenesis of acceptor lysines, metabolomics, mitophagy and ferroptosis readouts across multiple single-lab studies","pmids":["41197719","40379644","42217319","40683951","40307900","41023307","40107011"],"confidence":"Medium","gaps":["Many cytosolic/nuclear substrates rest on single-lab Co-IP plus ubiquitination without reciprocal validation","Physiological relevance of the very broad substrate repertoire not reconciled"]},{"year":null,"claim":"How a single transmembrane RING-CH ligase achieves its remarkably diverse substrate selection and chooses among K27/K33/K48/K63 linkages and lysosomal versus proteasomal fates remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate recognition","No unifying rule linking substrate identity to ubiquitin linkage type or degradative route","Tissue-specific regulation of the enzyme's broad substrate set undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,2,3,19,26,31]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,19,26]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[0,12,14,36]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[5,13,27]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,14,16,31]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[0,14,17,31]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,6,16,19,26,30]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,12,14,18,22,30]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,3,17,23]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,8,17,22]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[32,33,37]}],"complexes":[],"partners":["CD86","MHC-II","IL1RAP","IL7R","CGAS","IFITM3","PTEN","STAT3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5T0T0","full_name":"E3 ubiquitin-protein ligase MARCHF8","aliases":["Cellular modulator of immune recognition","c-MIR","Membrane-associated RING finger protein 8","Membrane-associated RING-CH protein VIII","MARCH-VIII","RING finger protein 178","RING-type E3 ubiquitin transferase MARCHF8"],"length_aa":291,"mass_kda":33.0,"function":"E3 ubiquitin-protein ligase that plays several important roles in innate immunity and adaptive immunity (PubMed:34285233, PubMed:35019698, PubMed:35503863). Mediates ubiquitination of CD86 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:19117940, PubMed:19566897). Possesses a very broad antiviral activity by specifically inactivating different viral fusion proteins (PubMed:32934085). Targets and ubiquitinates cytoplasmic lysine residues of viral envelope glycoproteins with single transmembrane domains leading to their lysosomal degradation (PubMed:35019698). Therefore, shows broad-spectrum inhibition against many viruses including retroviruses, rhabdoviruses, arenaviruses, sarbecoviruses or influenzaviruses (PubMed:34285233, PubMed:35019698). Strongly blocks human immunodeficiency virus type 1 envelope glycoprotein incorporation into virions by down-regulating its cell surface expression. Also blocks ebola virus glycoprotein/GP incorporation via surface down-regulation (PubMed:32934085). Mediates 'Lys-63'-linked polyubiquitination of influenza M2 to target it to lysosome for degradation (PubMed:34285233). Mediates the regulation of constitutive ubiquitination and trafficking of the viral restriction factor BST2 within the endocytic pathway (PubMed:28320822). Plays a role in maintenance of immune tolerance to self by promoting the turnover and proteasomal degradation of PD-L1/CD274 via ubiquitination (PubMed:34183449). Catalyzes the 'Lys-63'-linked polyubiquitylation of cGAS thereby inhibiting its DNA binding ability and impairing its antiviral innate immunity (PubMed:35503863). Negatively regulates IL7-mediated T-cell homeostasis by mediating 'Lys-27'-linked polyubiquitination of IL7R, leading to its lysosomal degradation (PubMed:39311660) (Microbial infection) Mediates 'Lys-63'-linked polyubiquitination of hepatitis C virus/HCV protein NS2 which allows its binding to HGS, an ESCRT-0 complex component, and this interaction is essential for HCV envelopment","subcellular_location":"Golgi apparatus membrane; Endoplasmic reticulum membrane; Cytoplasmic vesicle membrane; Lysosome membrane; Early endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q5T0T0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MARCHF8","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1090,"dependency_fraction":0.006422018348623854},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CCDC47","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MARCHF8","total_profiled":1310},"omim":[{"mim_id":"613335","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 8; MARCHF8","url":"https://www.omim.org/entry/613335"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MARCHF8"},"hgnc":{"alias_symbol":["c-MIR","CMIR","MARCH-VIII","RNF178"],"prev_symbol":["MIR","MARCH8"]},"alphafold":{"accession":"Q5T0T0","domains":[{"cath_id":"3.30.40.10","chopping":"78-130","consensus_level":"medium","plddt":95.4855,"start":78,"end":130},{"cath_id":"1.10.287","chopping":"148-233","consensus_level":"high","plddt":88.4042,"start":148,"end":233}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T0T0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T0T0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T0T0-F1-predicted_aligned_error_v6.png","plddt_mean":70.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MARCHF8","jax_strain_url":"https://www.jax.org/strain/search?query=MARCHF8"},"sequence":{"accession":"Q5T0T0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5T0T0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5T0T0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T0T0"}},"corpus_meta":[{"pmid":"12582153","id":"PMC_12582153","title":"c-MIR, a human E3 ubiquitin ligase, is a functional homolog of herpesvirus proteins MIR1 and MIR2 and has similar activity.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12582153","citation_count":139,"is_preprint":false},{"pmid":"26523972","id":"PMC_26523972","title":"MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins.","date":"2015","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/26523972","citation_count":128,"is_preprint":false},{"pmid":"16785530","id":"PMC_16785530","title":"Inhibition of MHC class II expression and immune responses by c-MIR.","date":"2006","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/16785530","citation_count":115,"is_preprint":false},{"pmid":"22904187","id":"PMC_22904187","title":"The E3 ubiquitin ligase MARCH8 negatively regulates IL-1β-induced NF-κB activation by targeting the IL1RAP coreceptor for ubiquitination and degradation.","date":"2012","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/22904187","citation_count":103,"is_preprint":false},{"pmid":"27832589","id":"PMC_27832589","title":"The Salmonella Effector SteD Mediates MARCH8-Dependent Ubiquitination of MHC II Molecules and Inhibits T Cell Activation.","date":"2016","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/27832589","citation_count":89,"is_preprint":false},{"pmid":"34285233","id":"PMC_34285233","title":"MARCH8 inhibits influenza A virus infection by targeting viral M2 protein for ubiquitination-dependent degradation in lysosomes.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34285233","citation_count":77,"is_preprint":false},{"pmid":"23300075","id":"PMC_23300075","title":"Ubiquitination by the membrane-associated RING-CH-8 (MARCH-8) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptor 1.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23300075","citation_count":70,"is_preprint":false},{"pmid":"27503071","id":"PMC_27503071","title":"Thymic CD4 T cell selection requires attenuation of March8-mediated MHCII turnover in cortical epithelial cells through CD83.","date":"2016","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27503071","citation_count":67,"is_preprint":false},{"pmid":"30759391","id":"PMC_30759391","title":"MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment.","date":"2019","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/30759391","citation_count":53,"is_preprint":false},{"pmid":"32934085","id":"PMC_32934085","title":"MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope 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miR-30d-5p/MARCH8.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/39050450","citation_count":5,"is_preprint":false},{"pmid":"36715497","id":"PMC_36715497","title":"Duck MARCH8 Negatively Regulates the RLR Signaling Pathway through K29-Linked Polyubiquitination of MAVS.","date":"2023","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/36715497","citation_count":5,"is_preprint":false},{"pmid":"39881438","id":"PMC_39881438","title":"A pan-cancer analysis of MARCH8: molecular characteristics, clinical relevance, and immuno-oncology features.","date":"2025","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/39881438","citation_count":4,"is_preprint":false},{"pmid":"39772241","id":"PMC_39772241","title":"MARCH8 Restricts RSV Replication by Promoting Cellular Apoptosis Through Ubiquitin-Mediated Proteolysis of Viral SH 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Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/37661917","citation_count":2,"is_preprint":false},{"pmid":"38667313","id":"PMC_38667313","title":"Further Characterization of the Antiviral Transmembrane Protein MARCH8.","date":"2024","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/38667313","citation_count":2,"is_preprint":false},{"pmid":"41023307","id":"PMC_41023307","title":"NFATc1 drives Orai3 transcription and proteolysis by harnessing epigenome differences in the MARCH8 promoter.","date":"2025","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/41023307","citation_count":2,"is_preprint":false},{"pmid":"39677690","id":"PMC_39677690","title":"The membrane-associated ubiquitin ligase MARCHF8 promotes cancer immune evasion by degrading MHC class I proteins.","date":"2024","source":"bioRxiv : the preprint server for 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Protein Degradation and Inhibits Viral Replication Through the MARCHF8/NDP52 Autophagy Pathway.","date":"2025","source":"Microorganisms","url":"https://pubmed.ncbi.nlm.nih.gov/40871369","citation_count":0,"is_preprint":false},{"pmid":"40780411","id":"PMC_40780411","title":"The E3 ubiquitin ligase MARCHF8 restricts HSV-1 infection by inhibiting replication of the viral genome.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40780411","citation_count":0,"is_preprint":false},{"pmid":"37961092","id":"PMC_37961092","title":"The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37961092","citation_count":0,"is_preprint":false},{"pmid":"40645466","id":"PMC_40645466","title":"Porcine MARCH8 negatively regulates the RLR signaling pathway by targeting MAVS for protein degradation.","date":"2025","source":"Developmental and comparative immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40645466","citation_count":0,"is_preprint":false},{"pmid":"41802050","id":"PMC_41802050","title":"The membrane-associated ubiquitin ligase MARCHF8 degrades MHC-I in HPV-positive head and neck cancer for immune evasion.","date":"2026","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41802050","citation_count":0,"is_preprint":false},{"pmid":"41870734","id":"PMC_41870734","title":"EpCAM as a novel target of MARCH8: implications for esophageal squamous cell carcinoma progression.","date":"2026","source":"Journal of molecular histology","url":"https://pubmed.ncbi.nlm.nih.gov/41870734","citation_count":0,"is_preprint":false},{"pmid":"41891145","id":"PMC_41891145","title":"Orientin Mitigates High Glucose/Ox-LDL-Triggered Endothelial Cell Injury and Atherosclerosis by Regulating MARCH8-Mediated NLRP3 Inflammasome Activation.","date":"2026","source":"Mediators of inflammation","url":"https://pubmed.ncbi.nlm.nih.gov/41891145","citation_count":0,"is_preprint":false},{"pmid":"42217319","id":"PMC_42217319","title":"FOXA1 protects against PM2.5-induced chronic lung injury via MARCHF8-dependent mitophagy inhibition.","date":"2026","source":"Environment international","url":"https://pubmed.ncbi.nlm.nih.gov/42217319","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":41358,"output_tokens":11200,"usd":0.146037,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":22105,"output_tokens":5091,"usd":0.1189,"stage2_stop_reason":"end_turn"},"total_usd":0.264937,"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\": 2003,\n      \"finding\": \"c-MIR (MARCHF8) was identified as a novel E3 ubiquitin ligase containing a BKS-PHD (RING-CH) domain. It targets B7-2 (CD86) for ubiquitination, rapid endocytosis, and lysosomal degradation by binding directly to B7-2. The BKS-PHD domain is functionally interchangeable with that of viral MIR1.\",\n      \"method\": \"Forced expression, ubiquitination assay, endocytosis and lysosomal degradation assays, domain-swap mutagenesis, binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (ubiquitination assay, binding assay, endocytosis/degradation assay, mutagenesis) in one study establishing enzymatic activity and substrate specificity\",\n      \"pmids\": [\"12582153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"c-MIR (MARCHF8) targets MHC class II (I-A beta-chain) for ubiquitination and lysosomal degradation. The ubiquitination depends on a single lysine residue (K225) in the cytoplasmic tail of the I-A beta-chain. In vivo, c-MIR overexpression in dendritic cells suppresses antigen presentation and impairs CD4 T cell development, and confers resistance to experimental autoimmune encephalomyelitis.\",\n      \"method\": \"Reconstitution in 293T cells, site-directed mutagenesis of ubiquitin acceptor lysine, forced expression in B cell lines, transgenic mouse model, flow cytometry, immunological assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution with mutagenesis of substrate lysine, confirmed in primary cells and transgenic mouse model with defined immunological phenotype\",\n      \"pmids\": [\"16785530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MARCH8 interacts with IL-1 receptor accessory protein (IL1RAP) and catalyzes K48-linked polyubiquitination of IL1RAP at Lys512, leading to its degradation and suppression of IL-1β-induced NF-κB and MAPK activation. Overexpression of MARCH8 inhibits this signaling; knockdown has the opposite effect.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K512), overexpression and knockdown with NF-κB/MAPK reporter assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination assay, mutagenesis, functional signaling readout) in a single rigorous study\",\n      \"pmids\": [\"22904187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MARCH-8 ubiquitinates TRAIL-R1 (death receptor 1) at the conserved membrane-proximal lysine K273 in its cytoplasmic tail, reducing TRAIL-R1 cell surface expression and attenuating TRAIL-induced apoptosis signaling. MARCH-8 ligase activity is required for this downregulation. Gene silencing confirmed MARCH-8 as the endogenous ligase responsible.\",\n      \"method\": \"Gene silencing (siRNA), ubiquitination assay, mutagenesis of K273, flow cytometry, apoptosis assay, interaction assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — endogenous ligase confirmed by gene silencing, mutagenesis of substrate acceptor lysine, and functional apoptosis readout in multiple experiments\",\n      \"pmids\": [\"23300075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MARCH8 reduces HIV-1 infectivity in virus-producing cells by downregulating HIV-1 envelope glycoprotein (Env) from the cell surface through direct interaction, thereby reducing virion incorporation of Env. MARCH8 also broadly inhibits VSV-G pseudotyped viruses. Endogenous MARCH8 is highly expressed in monocyte-derived macrophages and dendritic cells; knockdown in macrophages increases virion infectivity.\",\n      \"method\": \"Ectopic expression, HIV-1 infection assay, flow cytometry (surface Env), Co-immunoprecipitation (MARCH8-Env interaction), siRNA/CRISPR knockout, macrophage differentiation experiments\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction, endogenous KO/KD with functional infectivity readout, confirmed in primary cells; replicated and extended in subsequent studies\",\n      \"pmids\": [\"26523972\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Salmonella effector SteD localizes to the Golgi and vesicles containing MARCH8. SteD acts as an adaptor that forces MARCH8-dependent ubiquitination of mature MHC-II (mMHCII), reducing surface mMHCII and B7.2 and suppressing T cell activation. The C-terminal cytoplasmic region of SteD binds mMHCII; its transmembrane domain binds MARCH8.\",\n      \"method\": \"Cellular localization (immunofluorescence), Co-immunoprecipitation, MARCH8-dependent ubiquitination assay, domain mapping by mutagenesis, T cell activation assay, mouse infection model\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mapping by mutagenesis, localization, Co-IP, functional immune readout, and in vivo mouse model all in one study\",\n      \"pmids\": [\"27832589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In thymic cortical epithelial cells (cTECs), MARCH8 targets MHC-II for ubiquitination and turnover. CD83's transmembrane domain antagonizes MARCH8-mediated MHC-II ubiquitination in cTECs to stabilize MHC-II, which is required for CD4 T cell selection. Ablating March8 in Cd83-/- mice rescues CD4 T cell development. There is a division of labor: MARCH1 controls MHC-II in hematopoietic APCs; MARCH8 controls constitutive MHC-II in non-hematopoietic cTECs.\",\n      \"method\": \"Viral gene reconstitution in TECs, genetic epistasis (March8/Cd83 double knockout mice), ubiquitination-resistant MHC-II variant rescue, flow cytometry of T cell populations\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with double KO and rescue by ubiquitination-resistant variant, replicated functional T cell development phenotype\",\n      \"pmids\": [\"27503071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"KSHV lytic transactivator RTA upregulates MARCH8 transcription, and increased MARCH8 in turn downregulates HLA-DRα (MHC-II) surface expression, contributing to KSHV immune evasion. RTA can also directly bind and degrade HLA-DRα via the proteasome pathway independently.\",\n      \"method\": \"Overexpression, Western blot, flow cytometry, transcript analysis, KSHV de novo infection\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — overexpression and Western blot in multiple contexts, but mechanistic link between RTA-induced MARCH8 and HLA-DRα based on indirect evidence\",\n      \"pmids\": [\"27356905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MARCH8 mediates K48-linked ubiquitination of DR4 (TRAIL-R1/death receptor 4) at K273, leading to its degradation. JWA upregulates MARCH8 expression to promote DR4 ubiquitination, thereby suppressing TRAIL-induced apoptosis in cisplatin-resistant gastric cancer cells.\",\n      \"method\": \"Ubiquitination assay, site-directed mutagenesis, Western blot, siRNA knockdown, apoptosis assay\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay and mutagenesis of substrate lysine, single lab, two orthogonal methods\",\n      \"pmids\": [\"28671676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Yersinia pestis acetyltransferase YopJ acetylates MARCH8 at serine (S44, S71, S253) and lysine (K247, K252) residues, and this dual acetylation affects MARCH8 auto-ubiquitination. YopJ C172A mutant abolishing acetyltransferase activity reduces acetylation and diminishes MARCH8 auto-ubiquitination.\",\n      \"method\": \"Shotgun proteomics (label-free quantification), Western blot with site-specific antibodies, in vitro acetylation assay with purified YopJ, mutagenesis (C172A)\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro acetylation assay with mutagenesis and proteomics, single lab\",\n      \"pmids\": [\"28103160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MARCH8 catalyzes K63-linked polyubiquitination of the HCV nonstructural protein NS2 both in vitro and in HCV-infected cells, and is required for HCV envelopment. MARCH8 was identified through NS2 interactome mapping with the ubiquitin-proteasome system.\",\n      \"method\": \"Mammalian cell-based interactome screen, in vitro ubiquitination assay, HCV infection assay, Co-immunoprecipitation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination assay combined with cellular infection assay and interactome screen, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30759391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MARCH8 promotes ubiquitination and degradation of myosin light chain 2 (MLC2) in hippocampal neurons following TNF-α stimulation, contributing to caspase-3 activation and neuronal apoptosis. MARCH8 overexpression attenuates MLC2 levels; siRNA knockdown of MARCH8 blocks caspase-3 activation.\",\n      \"method\": \"In vivo intracerebroventricular TNF-α injection in rats, Western blot, siRNA knockdown, overexpression, apoptosis assay\",\n      \"journal\": \"Anatomical record\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single ubiquitination/degradation assay with limited mechanistic follow-up on substrate specificity\",\n      \"pmids\": [\"31443122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MARCH8 reduces viral infectivity via two distinct mechanisms: (1) Ubiquitination-dependent downregulation of VSV-G cytoplasmic lysine residues (lysine mutant VSV-G is resistant); (2) A tyrosine motif (cytoplasmic tyrosine motif)-dependent mechanism for HIV-1 Env (MARCH8 tyrosine motif mutant loses ability to inhibit Env-mediated infection but retains VSV-G inhibition). These are two separate pathways.\",\n      \"method\": \"Mutagenesis of VSV-G cytoplasmic lysines, mutagenesis of MARCH8 tyrosine motif, pseudovirus infection assay, ubiquitination assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mechanistic dissection using multiple domain mutants and two substrates, clearly distinguishes two independent antiviral mechanisms\",\n      \"pmids\": [\"32778221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MARCH8 blocks Ebola virus glycoprotein (EBOV GP) incorporation into virions by: (1) interacting with EBOV GP and furin; (2) retaining the GP/furin complex in the Golgi (BiFC assay); (3) inhibiting complex N-glycosylation of GP, O-glycosylation, and furin-mediated proteolytic cleavage. Only fully glycosylated GP is processed by furin and incorporated into virions. MARCH8 similarly blocks furin-mediated cleavage of HIV-1 Env (gp160) and H5N1 HA.\",\n      \"method\": \"Immunoprecipitation, bimolecular fluorescence complementation (BiFC) assay, glycosylation analysis, furin cleavage assay, virion incorporation assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal assays (BiFC, Co-IP, glycosylation analysis, cleavage assay) establishing Golgi-trapping and glycosylation-blocking mechanism\",\n      \"pmids\": [\"32934085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MARCH8 mediates K63-linked polyubiquitination of influenza A virus M2 protein at lysine K78, redirecting M2 from the plasma membrane to lysosomes for degradation, thereby suppressing IAV release. A recombinant A/PR/8/34 virus carrying K78R M2 shows greater replication and pathogenicity. H1N1 IAV has evolved non-lysine residues at positions 78/79 to resist MARCH8-mediated restriction.\",\n      \"method\": \"In vitro and in vivo (mouse) infection assay, ubiquitination assay, site-directed mutagenesis (K78R), recombinant virus, lysosomal degradation assay, MARCH8 KO cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination, site-directed mutagenesis of substrate lysine, recombinant virus with confirmed phenotype in vivo, multiple orthogonal methods\",\n      \"pmids\": [\"34285233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MARCH8 is identified as a positive regulator of type I IFN (IFN-I) signaling; SVA 2AB protein interacts with MARCHF8 and MAVS, forming a large complex that is degraded to deactivate IFN-I signaling. Additionally, 2AB degrades MARCHF8 and LC3 to inhibit autophagy.\",\n      \"method\": \"Co-immunoprecipitation, Western blot, siRNA knockdown, SVA infection assay, IFN signaling reporter assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and functional signaling readout, single lab; MARCHF8 role as positive IFN regulator is indirect (based on 2AB-mediated degradation phenotype)\",\n      \"pmids\": [\"34964697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In primary immune cells in vivo, MARCH8 (but not MARCH1) operates specifically in non-hematopoietic cells including thymic and alveolar epithelial cells to regulate MHC-II and CD86 surface expression. Only CD86 and MHC-II were confirmed as MARCH8 substrates by unbiased proteomic profiling of plasma membranes from MARCH8-deficient primary cells.\",\n      \"method\": \"MARCH8-deficient mice, unbiased proteomic profiling of plasma membranes, flow cytometry, comparison with MARCH1-deficient mice\",\n      \"journal\": \"Current research in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased plasma membrane proteomics in KO primary cells combined with flow cytometry across multiple cell types\",\n      \"pmids\": [\"35492398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MARCH8 interacts with CD44 and mediates its lysosomal degradation. MARCH8 also ubiquitinates STAT3, a non-membrane protein, and promotes its proteasomal degradation, inducing apoptosis in breast cancer cells. Stable MARCH8 expression inhibits tumorigenesis and lung metastasis in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, overexpression, xenograft mouse model, Western blot, apoptosis assay\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assays for two substrates, with in vivo confirmation, single lab\",\n      \"pmids\": [\"34067416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCH8 targets cytoplasmic lysine residues of various viral envelope glycoproteins (rabies virus-G, LCMV glycoproteins, SARS-CoV and SARS-CoV-2 spike, Chikungunya E2, Ross River virus E2) for ubiquitination, intracellular degradation, and antiviral restriction. Lysine mutations in cytoplasmic tails of these glycoproteins confer resistance to MARCH8.\",\n      \"method\": \"Pseudovirus infection assay, site-directed mutagenesis of cytoplasmic lysines, immunofluorescence, ubiquitination assay\",\n      \"journal\": \"Microbiology spectrum\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis of substrate lysines combined with ubiquitination assays and infection readout across multiple viral glycoproteins\",\n      \"pmids\": [\"35019698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCH8 interacts with the enzymatically active core of cGAS through its RING-CH domain and catalyzes K63-linked polyubiquitination of cGAS at K411. This ubiquitination inhibits cGAS DNA-binding ability and impairs cGAMP production, attenuating downstream innate immune responses. March8-deficient mice are less susceptible to HSV-1 infection.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K411), cGAMP production assay, DNA-binding assay, MARCH8 KO mice with HSV-1 infection\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ubiquitination, mutagenesis of substrate lysine, functional DNA-binding and cGAMP assays, in vivo mouse model\",\n      \"pmids\": [\"35503863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCH8 interacts with ABCB1 (P-glycoprotein/MDR1) and promotes its ubiquitination and degradation. Rutaecarpine upregulates MARCH8 protein levels, leading to increased ABCB1 degradation and reversal of multidrug resistance in cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Western blot, drug cytotoxicity assay, MARCH8 overexpression\",\n      \"journal\": \"Biomedicines\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay establishing substrate relationship, single lab\",\n      \"pmids\": [\"34572328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MARCH8 interacts with PTEN and promotes its ubiquitination and proteasomal degradation, activating downstream AKT signaling and promoting HCC progression. MARCH8 overexpression promotes hepatic tumor growth in vivo via the AKT pathway.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Western blot, overexpression/knockdown, xenograft mouse model, flow cytometry\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay, in vivo tumor growth, single lab\",\n      \"pmids\": [\"37098835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HPV E6-induced MYC/MAX transcriptional activation upregulates MARCHF8 expression in HPV-positive head and neck cancer. MARCHF8 directly interacts with and ubiquitinates FAS, TRAIL-R1 (DR4), and TRAIL-R2 (DR5), reducing their cell surface expression and inhibiting TNFRSF-mediated apoptosis. MARCHF8 KO in mouse oral cancer cells increases apoptosis and suppresses tumor growth in vivo.\",\n      \"method\": \"Promoter reporter assay, Co-immunoprecipitation, ubiquitination assay, flow cytometry, MARCHF8 KO in vivo tumor model, apoptosis assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple substrates confirmed by Co-IP and ubiquitination, in vivo tumor model with apoptosis phenotype, promoter mechanism defined\",\n      \"pmids\": [\"36867660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MARCHF8 ubiquitinates and degrades CUL1 and UBE2L3, components of the SCF ubiquitin ligase complex responsible for HPV16 E7 degradation. By degrading CUL1 and UBE2L3, MARCHF8 reduces E7 ubiquitination, stabilizing the E7 oncoprotein in HPV+ head and neck cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, overexpression of CUL1/UBE2L3, in vivo tumor growth assay, Western blot\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus ubiquitination assays for CUL1 and UBE2L3, functional E7 stabilization shown, in vivo confirmation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38226814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EGR1 promotes SARS-CoV-2 N protein degradation via MARCH8 ubiquitin ligase activity and cargo receptor NDP52. MARCH8 mutants lacking ubiquitin ligase activity fail to degrade the N protein, confirming E3 activity is required.\",\n      \"method\": \"Co-immunoprecipitation, MARCH8 catalytic mutant, Western blot, SARS-CoV-2 replication assay, IFN reporter\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — catalytic mutant confirms E3 activity requirement, Co-IP, single lab\",\n      \"pmids\": [\"37772822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MARCH8 interacts with PTPN4 and promotes its proteasomal ubiquitination and degradation, activating STAT3 phosphorylation (pSTAT3 Y705) and promoting pancreatic cancer growth and invasion. PTPN4 overexpression suppresses STAT3 activity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Western blot, overexpression/knockdown, xenograft mouse model\",\n      \"journal\": \"Pancreas\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay, in vivo confirmation, single lab\",\n      \"pmids\": [\"37747937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCH8 mediates K27-linked polyubiquitination of IL-7Rα at K265/K266, leading to lysosomal degradation of IL-7Rα. This negatively regulates IL-7-triggered STAT5 activation and T cell proliferation. MARCH8 deficiency increases IL-7-triggered signaling and splenic memory CD8+ T cell differentiation in mice.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with K27-linkage specificity, site-directed mutagenesis (K265/K266), MARCH8 KO mice, STAT5 reporter, T cell proliferation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ubiquitination assay with linkage determination, mutagenesis of substrate acceptor lysines, in vivo MARCH8 KO mouse phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"39311660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCH8 inhibits pseudorabies virus (PRV) replication by trapping the viral cell-to-cell fusion complex (gB, gD, gH, gL) in the trans-Golgi network, independent of its E3 ubiquitin ligase activity. MARCH8 also blocks gB cleavage by recruiting furin (a ligase-dependent activity), but the furin-blocking activity alone does not inhibit viral infection in vitro.\",\n      \"method\": \"Protein interaction assay, subcellular localization (immunofluorescence/confocal), ligase-dead MARCH8 mutant, cell-to-cell fusion assay, viral replication assay\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ligase-dead mutant separates E3-independent from E3-dependent mechanisms, interaction and localization confirmed, single lab\",\n      \"pmids\": [\"38944094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The antiviral function of MARCH8 requires both the RING-CH domain (for ubiquitination-dependent inhibition of VSV-G) and the C-terminal tyrosine motif (for inhibition of HIV-1 Env incorporation). Both N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, are critical for antiviral activity (determined by chimeric proteins between MARCH8 and non-antiviral MARCH3). MARCH8 is unable to block cell-to-cell HIV-1 infection.\",\n      \"method\": \"Chimeric protein domain analysis, mutagenesis of RING-CH and tyrosine motif, pseudovirus infection assay, cell-to-cell HIV-1 infection assay\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping by chimeric proteins and mutagenesis, single lab, multiple virus systems\",\n      \"pmids\": [\"38667313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NLRP12 recruits MARCH8 (via its PYD domain) to the PRRSV glycoprotein GP2a, facilitating K48-linked polyubiquitination of GP2a at K128 and its lysosomal degradation via the MARCH8-NDP52 pathway, suppressing PRRSV replication.\",\n      \"method\": \"Co-immunoprecipitation (domain mapping), ubiquitination assay, site-directed mutagenesis (K128), overexpression/knockdown, viral replication assay\",\n      \"journal\": \"Veterinary microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-resolved Co-IP, mutagenesis of substrate lysine, single lab\",\n      \"pmids\": [\"39362085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCHF8 interacts with MHC-I proteins, ubiquitinates them, and promotes their degradation in HPV-positive head and neck cancer, facilitating immune evasion. MARCHF8 KO restores MHC-I levels, suppresses tumor growth, and increases NK and CD8+ T cell infiltration. Combination of MARCHF8 KO with anti-PD-1 further enhances tumor suppression in ICI-refractory tumors.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, flow cytometry, MARCHF8 KO mouse tumor model, immune cell infiltration analysis, anti-PD-1 combination treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ubiquitination assay, in vivo KO tumor model with immune phenotyping, combination immunotherapy experiment, confirmed in peer-reviewed publication\",\n      \"pmids\": [\"41802050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH8 interacts with and promotes K63-linked polyubiquitination of IFITM3 at K24, directing it from the plasma membrane to lysosomes for degradation. MARCH8-KO cells accumulate IFITM3 at the plasma membrane after interferon treatment. MARCH8 expression attenuates IFITM3-mediated restriction of VSV and influenza A virus entry.\",\n      \"method\": \"Co-immunoprecipitation coupled to LC-MS/MS, ubiquitination assay with K63 linkage determination, site-directed mutagenesis (K24), MARCH8-KO cells, flow cytometry, viral infection assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — CoIP-MS for substrate identification, ubiquitination assay with linkage specificity and mutagenesis of acceptor lysine, KO cells with functional viral infection readout\",\n      \"pmids\": [\"41197719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH8 ubiquitinates NSUN6 at K271 and K462, causing its proteasomal degradation. Reduced NSUN6 lowers m5C modification on PEX1 and PEX3 mRNAs, destabilizing them through loss of YBX1 binding, leading to decreased peroxisome synthesis, increased ROS, DNA damage, and increased cisplatin sensitivity. Elevated ROS enhances NSUN6-MARCH8 interaction, forming a positive feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with mutagenesis (K271/K462), m5C methylation analysis, RNA stability assay, peroxisome biogenesis assay, ROS measurement\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of substrate lysines, functional pathway analysis, single lab with multiple methods\",\n      \"pmids\": [\"40683951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH8 promotes ubiquitination and proteasomal degradation of SREBP1, suppressing key enzymes for fatty acid de novo synthesis and reducing lipid deposition in HCC. This mechanism is supported by RNA sequencing and untargeted metabolomics combined with in vivo/in vitro experiments.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RNA sequencing, untargeted metabolomics, in vivo xenograft and mouse liver cancer model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay for SREBP1, in vivo confirmation, single lab with metabolomics validation\",\n      \"pmids\": [\"40379644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NFATc1 transcriptionally upregulates MARCH8 in metastatic pancreatic cancer cells (where MARCH8 promoter is hypomethylated), and MARCH8 physically interacts with Orai3 intracellular loop and ubiquitinates Orai3 at its N-terminus, leading to lysosomal degradation and suppression of pancreatic cancer metastasis. In non-metastatic cells, MARCH8 promoter is hypermethylated and MARCH8 is not induced.\",\n      \"method\": \"ChIP, promoter methylation analysis, Co-immunoprecipitation, ubiquitination assay with domain mapping, overexpression/knockdown, in vivo metastasis model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ChIP, promoter methylation, Co-IP, ubiquitination with domain mapping, in vivo metastasis assay), single lab\",\n      \"pmids\": [\"41023307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH8 ubiquitinates CEMIP and promotes its degradation, inhibiting CRC cell proliferation, metastasis, and inducing ferroptosis through inactivation of the PI3K/AKT pathway. Overexpression of CEMIP reverses these effects.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ferroptosis markers (ROS, Fe2+, GSH, MDA), Western blot (PI3K/AKT), xenograft tumor model\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay, in vivo tumor model, single lab\",\n      \"pmids\": [\"40107011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH8 promotes K33-linked polyubiquitination of FMDV capsid proteins VP1 (at K210), VP2 (at K63), and VP3 (at K118), leading to their proteasomal degradation, thereby restricting FMDV replication in a dose-dependent manner. MARCH8 interaction and degradation of a non-enveloped virus's structural proteins demonstrates activity beyond viral envelope glycoproteins.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay with linkage determination, site-directed mutagenesis of substrate lysines, MARCH8 overexpression/knockdown, viral replication assay\",\n      \"journal\": \"Veterinary research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — K33 linkage ubiquitination with mutagenesis of all three substrate lysines, single lab\",\n      \"pmids\": [\"40307900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FOXA1 transcriptionally activates MARCH8 expression; MARCH8 then catalyzes K48-linked ubiquitination of HK2 at K763, leading to HK2 degradation and inhibition of PINK1/Parkin-mediated mitophagy, thereby protecting against PM2.5-induced lung injury.\",\n      \"method\": \"ChIP (FOXA1 binding to MARCH8 promoter), Co-immunoprecipitation, ubiquitination assay with site mutagenesis (K763), mitophagy assays (mt-Keima, MMP, mtROS), in vivo mouse lung injury model\",\n      \"journal\": \"Environment international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for transcriptional regulation, mutagenesis of substrate lysine, functional mitophagy readout, single lab\",\n      \"pmids\": [\"42217319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCHF8 promotes the degradation of TGFBI via ubiquitination; TGFBI in turn promotes apoptosis and ECM breakdown in LPS-stimulated nucleus pulposus cells and activates the NF-κB signaling pathway. MARCHF8 controls TGFBI expression levels, modulating intervertebral disc degeneration.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, NF-κB signaling markers, Western blot, bioinformatics (WGCNA) to identify hub gene\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination assay, but limited mechanistic validation of MARCHF8 specificity; functional link to NF-κB indirect\",\n      \"pmids\": [\"39752341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH8 ubiquitinates and promotes proteasomal degradation of NLRP3, and this activity is upregulated by Orientin treatment, attenuating NLRP3 inflammasome activation and pyroptosis in endothelial cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Western blot, NLRP3 inflammasome activation markers, in vivo atherosclerosis mouse model\",\n      \"journal\": \"Mediators of inflammation\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination assay, functional inflammasome readout, single lab with limited mechanistic detail on MARCH8 substrate specificity\",\n      \"pmids\": [\"41891145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARCHF8 was identified as degrading MHC-I proteins via ubiquitination in HPV-positive head and neck cancer in a preprint study subsequently published (PMID 41802050). MARCHF8 knockdown restores MHC-I levels, increases NK and CD8+ T cell infiltration, and in combination with anti-PD-1 synergistically suppresses tumor growth in ICI-refractory tumors.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, flow cytometry, MARCHF8 KO mouse tumor model, immune phenotyping, anti-PD-1 combination\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — preprint replicated in peer-reviewed publication; included here only as reference to preprint record; primary confidence derives from published version\",\n      \"pmids\": [\"39677690\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH8 promotes lysosomal degradation of IFITM3 through K63-linked polyubiquitination at K24, facilitating trafficking from the plasma membrane to lysosomes; MARCH8-KO cells show plasma membrane accumulation of IFITM3 and enhanced antiviral restriction. MARCH8 expression thus counteracts IFITM3-mediated antiviral defense.\",\n      \"method\": \"Co-immunoprecipitation with LC-MS/MS, ubiquitination assay, mutagenesis (K24), flow cytometry (subcellular localization), MARCH8-KO cells, viral infection assay (VSV, IAV)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — substrate identification by CoIP-MS, mutagenesis of acceptor lysine, KO cells with localization and functional viral readout, single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"41197719\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MARCHF8 is a transmembrane RING-CH E3 ubiquitin ligase that constitutively downregulates diverse membrane proteins—including MHC-II, MHC-I, CD86/B7-2, TRAIL-R1/DR4, FAS, TRAIL-R2, IL1RAP, IL-7Rα, and transferrin receptor—by catalyzing their ubiquitination (with substrate-specific K27, K48, K63, or K33 linkages) and routing them to lysosomal or proteasomal degradation; it inhibits a broad spectrum of enveloped viruses either by ubiquitinating cytoplasmic lysines of viral envelope glycoproteins (e.g., VSV-G, IAV M2, SARS-CoV-2 spike) for lysosomal degradation, or by retaining viral fusion protein/furin complexes in the trans-Golgi network via a tyrosine-motif-dependent mechanism that blocks glycosylation and furin cleavage, thereby preventing glycoprotein incorporation into virions; MARCHF8 also ubiquitinates cytosolic and nuclear substrates (cGAS at K411, STAT3, PTEN, SREBP1, HK2, NSUN6, CUL1/UBE2L3) to regulate innate immunity, metabolism, and oncogenesis; its activity is modulated by CD83, acetylation (by bacterial YopJ), and miR-34a-5p, and it occupies a tissue-specific niche (non-hematopoietic cells) partly distinct from the related MARCH1 ligase.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MARCHF8 (c-MIR/MARCH8) is a transmembrane RING-CH E3 ubiquitin ligase that constitutively controls the surface abundance of membrane proteins and a growing set of cytosolic and nuclear substrates, coupling target ubiquitination to lysosomal or proteasomal degradation [#0, #1]. It was discovered through its ability to bind B7-2 (CD86) directly and route it to endocytosis and lysosomal degradation via a RING-CH (BKS-PHD) domain functionally interchangeable with viral MIR1 [#0]. A central physiological role is immune modulation: MARCHF8 ubiquitinates the MHC-II beta-chain cytoplasmic tail at a single lysine to suppress antigen presentation [#1], and in vivo it operates specifically in non-hematopoietic cells such as thymic and alveolar epithelia\\u2014distinct from MARCH1 in hematopoietic APCs\\u2014where CD86 and MHC-II are its confirmed endogenous substrates and where it shapes CD4 T cell selection [#6, #16]. It further tunes immune signaling by degrading immune receptors (IL1RAP via K48 linkage, IL-7R\\u03b1 via K27 linkage, TRAIL-R1/DR4, FAS, TRAIL-R2/DR5) to dampen NF-\\u03baB/MAPK, IL-7/STAT5, and death-receptor apoptotic signaling [#2, #3, #22, #26]. MARCHF8 is a broad antiviral effector acting through two separable mechanisms encoded in distinct regions: RING-CH\\u2013dependent ubiquitination of cytoplasmic lysines on viral envelope glycoproteins (VSV-G, IAV M2 at K78, SARS-CoV-2 spike and others) targeting them for degradation, and a C-terminal tyrosine-motif\\u2013dependent activity that traps glycoprotein/furin complexes in the trans-Golgi network, blocking glycosylation, furin cleavage, and virion incorporation [#12, #13, #14, #18, #27, #28]. It also ubiquitinates cytosolic and nuclear regulators\\u2014cGAS at K411 (K63-linked, inhibiting cGAMP production), IFITM3 at K24, and metabolic and oncogenic factors including PTEN, STAT3, SREBP1, HK2, and NSUN6\\u2014placing it at the intersection of innate immunity, metabolism, and tumor biology [#19, #31, #17, #21, #33]. In HPV-positive head and neck cancer, MARCHF8 is transcriptionally induced and drives immune evasion by ubiquitinating MHC-I, FAS, and TRAIL receptors and by stabilizing the E7 oncoprotein through degradation of the SCF components CUL1 and UBE2L3 [#22, #23, #30]. Its activity is shaped by interacting regulators and modifications, including CD83 antagonism of MHC-II ubiquitination and bacterial YopJ-mediated acetylation affecting auto-ubiquitination [#6, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established MARCHF8 as a bona fide E3 ubiquitin ligase and defined its enzymatic logic\\u2014direct substrate binding plus RING-CH-catalyzed ubiquitination driving endocytosis and lysosomal degradation.\",\n      \"evidence\": \"Forced expression, ubiquitination, endocytosis/degradation assays and domain-swap mutagenesis identifying B7-2 (CD86) as substrate\",\n      \"pmids\": [\"12582153\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which ubiquitin linkage types are used\", \"Endogenous physiological context not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed MARCHF8 controls antigen presentation by ubiquitinating MHC-II at a single defined cytoplasmic lysine, linking molecular activity to an organismal immune phenotype.\",\n      \"evidence\": \"Reconstitution with K225 mutagenesis of I-A beta-chain, transgenic mouse with impaired CD4 T cell development and EAE resistance\",\n      \"pmids\": [\"16785530\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue/cell type where endogenous MARCH8 acts on MHC-II not yet defined\", \"Did not distinguish MARCH8 from MARCH1 roles\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended substrate range to signaling receptors, showing MARCHF8 uses K48-linked ubiquitination to degrade IL1RAP and suppress innate inflammatory signaling.\",\n      \"evidence\": \"Co-IP, K512 mutagenesis, NF-\\u03baB/MAPK reporter assays with overexpression and knockdown\",\n      \"pmids\": [\"22904187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether IL1RAP degradation is lysosomal or proteasomal not fully resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified MARCHF8 as the endogenous ligase for death receptor TRAIL-R1, linking it to apoptosis regulation via a conserved membrane-proximal lysine.\",\n      \"evidence\": \"siRNA silencing, K273 mutagenesis, flow cytometry and apoptosis assays\",\n      \"pmids\": [\"23300075\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological/disease context of TRAIL-R1 control not established in this study\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the in vivo niche of MARCHF8\\u2014non-hematopoietic thymic epithelium\\u2014and revealed CD83 as an antagonist that stabilizes MHC-II for T cell selection, establishing a division of labor with MARCH1.\",\n      \"evidence\": \"TEC reconstitution, March8/Cd83 double-KO epistasis, ubiquitination-resistant MHC-II rescue, plus bacterial SteD adaptor co-opting MARCH8 at the Golgi\",\n      \"pmids\": [\"27503071\", \"27832589\", \"27356905\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of CD83 transmembrane antagonism at atomic level unresolved\", \"How pathogens redirect substrate specificity beyond adaptor binding unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined MARCHF8 as a host antiviral restriction factor that downregulates HIV-1 Env and broadly inhibits enveloped viruses in producer cells.\",\n      \"evidence\": \"Ectopic expression, infectivity assays, surface Env flow cytometry, Co-IP, siRNA/CRISPR KO in primary macrophages\",\n      \"pmids\": [\"26523972\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of Env restriction (ubiquitination vs trafficking) not yet separated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Dissected two mechanistically separable antiviral activities\\u2014RING-CH ubiquitination of glycoprotein cytoplasmic lysines versus a tyrosine-motif-dependent Golgi-trapping mechanism that blocks glycosylation and furin cleavage.\",\n      \"evidence\": \"VSV-G lysine mutants and MARCH8 tyrosine-motif mutants in pseudovirus assays; BiFC, glycosylation and furin cleavage assays for EBOV GP/HIV Env/H5N1 HA\",\n      \"pmids\": [\"32778221\", \"32934085\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of tyrosine-motif-mediated Golgi retention unknown\", \"How the furin/glycoprotein complex is physically retained not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Confirmed substrate-specific ubiquitin linkage usage and antiviral targeting through unbiased and mechanistic approaches, including K63-linked degradation of IAV M2 and K48-linked HCV NS2 ubiquitination, while proteomics narrowed validated surface substrates to CD86 and MHC-II.\",\n      \"evidence\": \"In vivo recombinant IAV K78R virus, ubiquitination assays, MARCH8-KO mice with plasma membrane proteomics, HCV interactome screen\",\n      \"pmids\": [\"34285233\", \"30759391\", \"35492398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How linkage specificity (K48 vs K63) is determined per substrate unresolved\", \"Reconciling broad overexpression substrate lists with narrow endogenous proteomic hits\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded MARCHF8 substrates beyond membrane proteins to cytosolic innate immune sensors, showing K63-linked ubiquitination of cGAS at K411 inhibits DNA sensing and cGAMP production.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, K411 mutagenesis, cGAMP and DNA-binding assays, March8-KO mice with HSV-1 infection\",\n      \"pmids\": [\"35503863\", \"35019698\", \"34572328\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cGAS regulation is constitutive or stimulus-induced unclear\", \"How a membrane-anchored ligase accesses cytosolic cGAS not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established MARCHF8 as a tumor-promoting effector in HPV-positive cancer that drives apoptosis resistance (FAS/TRAIL receptors) and stabilizes the E7 oncoprotein by degrading SCF components CUL1 and UBE2L3.\",\n      \"evidence\": \"Promoter reporter (MYC/MAX), Co-IP, ubiquitination assays, MARCHF8-KO in vivo tumor models, plus PTEN and PTPN4 degradation studies\",\n      \"pmids\": [\"36867660\", \"38226814\", \"37098835\", \"37747937\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether oncogenic vs tumor-suppressive roles are context-dependent not unified\", \"Direct vs indirect contribution of each substrate to tumor phenotype unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated MARCHF8 mediates immune evasion via MHC-I degradation and links to checkpoint immunotherapy response, while defining K27-linked degradation of IL-7R\\u03b1 controlling T cell memory.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, MARCHF8-KO tumor models with immune phenotyping and anti-PD-1 combination; IL-7R\\u03b1 K265/K266 mutagenesis with KO mouse T cell phenotype\",\n      \"pmids\": [\"41802050\", \"39311660\", \"38667313\", \"39362085\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants selecting MHC-I vs MHC-II vs other substrates unknown\", \"Therapeutic targetability of MARCHF8 not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Broadened the functional reach to metabolism, RNA modification, and additional antiviral/inflammatory pathways, including K63 ubiquitination of IFITM3, degradation of SREBP1, HK2, NSUN6, and K33-linked targeting of non-enveloped FMDV capsid proteins.\",\n      \"evidence\": \"CoIP-MS substrate ID, linkage-specific ubiquitination assays, mutagenesis of acceptor lysines, metabolomics, mitophagy and ferroptosis readouts across multiple single-lab studies\",\n      \"pmids\": [\"41197719\", \"40379644\", \"42217319\", \"40683951\", \"40307900\", \"41023307\", \"40107011\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Many cytosolic/nuclear substrates rest on single-lab Co-IP plus ubiquitination without reciprocal validation\", \"Physiological relevance of the very broad substrate repertoire not reconciled\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single transmembrane RING-CH ligase achieves its remarkably diverse substrate selection and chooses among K27/K33/K48/K63 linkages and lysosomal versus proteasomal fates remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of substrate recognition\", \"No unifying rule linking substrate identity to ubiquitin linkage type or degradative route\", \"Tissue-specific regulation of the enzyme's broad substrate set undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 2, 3, 19, 26, 31]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 19, 26]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [0, 12, 14, 36]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [5, 13, 27]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 14, 16, 31]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 14, 17, 31]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 6, 16, 19, 26, 30]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 12, 14, 18, 22, 30]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 3, 17, 23]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 8, 17, 22]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [32, 33, 37]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CD86\", \"MHC-II\", \"IL1RAP\", \"IL7R\", \"cGAS\", \"IFITM3\", \"PTEN\", \"STAT3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}