{"gene":"MARCHF9","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2009,"finding":"MARCH9 is a RING-CH transmembrane E3 ubiquitin ligase that down-regulates multiple plasma membrane proteins in lymphoid cells, including CD4, MHC-I, ICAM-1, PTPRJ/CD148, CD32B, HLA-DQ, CD150, and CD155, as identified by SILAC-based quantitative plasma membrane proteomics combined with flow cytometry confirmation.","method":"SILAC quantitative mass spectrometry of plasma membrane proteome in MARCH9-expressing B cell line, confirmed by flow cytometry","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative proteomics with orthogonal flow cytometry confirmation, multiple substrates validated, high correlation (r²=0.93) between methods","pmids":["19457934"],"is_preprint":false},{"year":2010,"finding":"MARCH9 (and MARCH4) ubiquitinates the NKG2D ligand Mult1, suppressing its cell-surface expression; lysines in the cytoplasmic tail of Mult1 are essential for this repression. Heat-shock treatment dissociates Mult1 from MARCH9, reversing down-regulation and increasing Mult1 at the cell surface.","method":"Co-immunoprecipitation, flow cytometry, lysine mutant analysis, heat-shock functional assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, mutagenesis of acceptor lysines, and functional reversal by heat shock provide multiple orthogonal lines of evidence","pmids":["20870941"],"is_preprint":false},{"year":2012,"finding":"MARCH9 ubiquitinates HLA-DM via a single lysine in the cytoplasmic tail of the DMα chain, inducing loss of DM from the cell surface by a mechanism involving both direct ubiquitin-chain attachment to DMα and a functional tyrosine-based signal on DMβ.","method":"Transfection/overexpression, flow cytometry, mutational analysis of acceptor lysine and tyrosine-based signal","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of substrate lysine and co-expressed constructs, single lab, two orthogonal readouts","pmids":["22247549"],"is_preprint":false},{"year":2012,"finding":"MARCH9 substrate specificity for HLA-DQ (versus MARCH8 preference for HLA-DR) is determined by residues at the interface of the transmembrane domain and cytosol of the HLA β-chain; the acceptor lysine functions optimally at its natural position relative to the bilayer.","method":"Mutagenesis and chimeric construct analysis of HLA-DR/DQ transmembrane and cytoplasmic tail residues, flow cytometry readout","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with functional readout, single lab, defines molecular determinants of substrate recognition","pmids":["22761441"],"is_preprint":false},{"year":2017,"finding":"MARCH9 ubiquitinates the cytoplasmic tail lysine residues of MHC-I, redirecting MHC-I from the default secretory pathway to Syntaxin 6-positive endosomal compartments at the trans-Golgi network, thereby regulating MHC-I export from the TGN. MARCH9 also targets CD1a. MARCH9 expression is modulated by microbial pattern exposure in dendritic cells.","method":"Overexpression/knockdown of MARCH9, co-localization with Syntaxin 6 by microscopy, MHC-I ubiquitination assay, flow cytometry, stimulation of dendritic cells with microbial patterns","journal":"Immunology and cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ubiquitination assay, co-localization, flow cytometry, DC stimulation) in a single focused study establishing TGN sorting mechanism","pmids":["28559542"],"is_preprint":false},{"year":2018,"finding":"A single serine-to-alanine substitution in the first transmembrane domain (TM1) of human MARCH9 completely abolishes its ability to down-regulate HLA-A2 and CD4, identifying this serine as critical for substrate regulation. Solution NMR of the MARCH9 two-TM fragment shows that residues closest to the extracellular face of TM1 and TM2 define the key functional region.","method":"Site-directed mutagenesis of MARCH9 TM domains, flow cytometry functional assay, solution NMR of TM domain fragment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — combines NMR structure with mutagenesis and functional assay, single lab but multiple orthogonal methods","pmids":["30554144"],"is_preprint":false},{"year":2018,"finding":"MARCH9 interacts with ICAM-1 and overexpression of MARCH9 down-regulates ICAM-1 protein, attenuating migration and invasion of lung adenocarcinoma cells.","method":"Co-immunoprecipitation, overexpression in A549 and H1299 cells, flow cytometry/western blot, migration/invasion assays","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction plus functional phenotype, single lab, consistent with established substrate (ICAM-1) from prior proteomics","pmids":["30278450"],"is_preprint":false},{"year":2019,"finding":"MARCH9 (along with MARCH2, MARCH4, and an isoform of MARCH3) down-regulates the IL-6 receptor alpha chain (IL6Rα) at the cell surface; functional RING domain, transmembrane domains, and C-terminal domains are all required for substrate recognition and down-regulation.","method":"cDNA expression library screen, overexpression in M1 cells, domain deletion/mutagenesis constructs, flow cytometry","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — positive screen plus domain analysis, but MARCH9 is one of several proteins tested; mechanistic detail for MARCH9 specifically is moderate","pmids":["31488575"],"is_preprint":false},{"year":2021,"finding":"Tim-3 signaling in macrophages promotes MARCH9 expression, which in turn mediates K48-linked ubiquitination of MHC-I (HLA class I), leading to its proteasome-dependent degradation and reduced surface MHC-I presentation.","method":"Western blot, co-immunoprecipitation, ubiquitination assay (K48-linkage specific), Tim-3 antibody treatment and macrophage depletion in vivo","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and linkage-specific ubiquitination assay establish mechanism, but single lab without independent replication of the Tim-3→MARCH9→MHC-I axis","pmids":["34025669"],"is_preprint":false},{"year":2022,"finding":"Under heat stress, MARCH9 promotes K63-linked ubiquitination of ATG9A at the TGN, causing ubiquitinated ATG9A to disperse from the Golgi to the cytoplasm, inhibiting GRASP55 oligomerization and resulting in Golgi fragmentation. Knockout of MARCH9 prevents heat-stress-induced Golgi fragmentation.","method":"Co-immunoprecipitation, K63-specific ubiquitination assay, MARCH9 knockout cells, Golgi morphology imaging under heat stress","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, linkage-specific ubiquitination, genetic KO with morphological readout, and stress-reversal experiment across two papers from the same group","pmids":["35977480","36198086"],"is_preprint":false},{"year":2022,"finding":"MARCH9 (MARCHF9) promotes colorectal cancer cell proliferation and migration by downregulating the deubiquitinase CYLD and activating NF-κB (p65); knockdown of MARCH9 induces apoptosis and cell-cycle arrest, and suppresses xenograft tumor growth.","method":"MARCH9 knockdown/overexpression, western blot for CYLD and p65, apoptosis/cell cycle flow cytometry, xenograft in vivo model","journal":"Frontiers in oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect evidence of CYLD regulation by MARCH9 without direct ubiquitination assay of CYLD by MARCH9","pmids":["36185211"],"is_preprint":false},{"year":2023,"finding":"MARCH9 mediates ubiquitination and degradation of NADPH oxidase-2 (NOX2), reducing ROS accumulation and NLRP3 inflammasome activation, thereby suppressing pancreatic cell pyroptosis in acute pancreatitis.","method":"MARCH9 overexpression in AR42J cells and cerulein-induced rat model, flow cytometry for ROS, western blot for NLRP3/caspase-1/GSDMD, ubiquitination assay for NOX2","journal":"Pancreas","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ubiquitination assay plus in vitro and in vivo functional rescue, but single lab with limited mechanistic detail on ubiquitin linkage type","pmids":["37378901"],"is_preprint":false},{"year":2024,"finding":"LILRB2 promotes MARCH9–HLA-A (HLA-A ubiquitin ligase) interaction, facilitating ubiquitination and proteasomal degradation of HLA-A, thereby enabling breast cancer immune evasion by reducing surface MHC-I presentation to CD8+ T cells.","method":"Co-immunoprecipitation, histidine pulldown ubiquitination assay, western blot, syngeneic mouse tumor model","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and pulldown-based ubiquitination assay, in vivo model, single lab, moderate mechanistic detail","pmids":["38656573"],"is_preprint":false},{"year":2025,"finding":"MARCH9 interacts with NLRP3 and promotes its K48-linked polyubiquitination, leading to proteasomal degradation of NLRP3, thereby inhibiting inflammasome activation and pyroptosis during myocardial ischemia-reperfusion injury.","method":"Co-immunoprecipitation, K48-linkage-specific ubiquitination assay, western blot, mouse MI/R in vivo model, H9C2 and HEK293 cell in vitro studies","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — K48-linkage-specific Co-IP ubiquitination assay with in vivo validation, single lab, single publication","pmids":["41055760"],"is_preprint":false},{"year":2025,"finding":"ETV4 transcriptionally upregulates MARCH9, which then mediates K63-linked ubiquitination of the mitochondrial fusion protein Mfn2, targeting it for lysosomal degradation and impairing mitochondrial fusion in ovarian cancer cells.","method":"ETV4 silencing, MARCH9 overexpression rescue, western blot for Mfn2, lysosomal inhibitor (chloroquine) block, K63-ubiquitination assay","journal":"Cell biology and toxicology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ubiquitination assay with lysosomal inhibitor confirmation, in vitro only, single lab","pmids":["41114773"],"is_preprint":false},{"year":2026,"finding":"RBM10 recruits MARCHF9 to catalyze K33-linked ubiquitination of PEDV nonstructural protein 3 (Nsp3); the ubiquitinated Nsp3 is then recognized by the selective autophagy receptor p62 and delivered to autophagosomes for degradation, restricting viral replication.","method":"LC-MS/MS interaction screen, functional knockdown/overexpression assays, autophagic flux blockade, p62 knockdown, K33-linkage ubiquitination assay","journal":"Veterinary microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — linkage-specific ubiquitination assay plus genetic epistasis (p62 KD restores Nsp3), single lab, single publication; viral substrate differs from canonical mammalian substrates","pmids":["42229174"],"is_preprint":false}],"current_model":"MARCHF9 (MARCH9) is a membrane-anchored RING-CH E3 ubiquitin ligase whose two transmembrane domains (with a critical serine in TM1) mediate recognition of substrate transmembrane regions to catalyze ubiquitination of cytoplasmic tail lysines on diverse cell-surface and intracellular proteins—including MHC-I, MHC-II (HLA-DQ, HLA-DM), CD4, ICAM-1, IL6Rα, Mult1, ATG9A, NOX2, NLRP3, and Mfn2—thereby directing substrates to endosomal/lysosomal or proteasomal degradation pathways that regulate antigen presentation, innate immune signaling, Golgi integrity, and inflammasome activation."},"narrative":{"mechanistic_narrative":"MARCHF9 (MARCH9) is a membrane-anchored RING-CH E3 ubiquitin ligase that down-regulates a broad panel of cell-surface and intracellular membrane proteins by ubiquitinating lysines in their cytoplasmic tails, controlling antigen presentation, innate immune signaling, and organelle homeostasis [PMID:19457934, PMID:20870941]. Substrate recognition is governed by its two transmembrane domains: a single serine in TM1 is essential for activity, and solution NMR localizes the functional region to residues near the extracellular face of TM1 and TM2, while the RING and C-terminal domains are additionally required for substrate down-regulation [PMID:30554144, PMID:31488575]. Specificity among related substrates is dictated by residues at the transmembrane/cytosol interface of the target, as shown for its preference for HLA-DQ over HLA-DR, with the acceptor lysine acting optimally at its natural distance from the bilayer [PMID:22761441, PMID:22247549]. Functionally, MARCH9 routes MHC-I from the secretory pathway into Syntaxin 6-positive trans-Golgi-network endosomal compartments and drives proteasomal degradation of MHC-I/HLA-A via K48-linked ubiquitination, a mechanism co-opted in cancer immune evasion downstream of Tim-3 and LILRB2 signaling [PMID:28559542, PMID:34025669, PMID:38656573]. Beyond antigen presentation, MARCH9 ubiquitinates ATG9A in a K63-linked manner to promote heat-stress-induced Golgi fragmentation through loss of GRASP55 oligomerization [PMID:35977480, PMID:36198086], and degrades innate-immune and metabolic targets including NOX2 and NLRP3 to restrain ROS-driven inflammasome activation and pyroptosis [PMID:37378901, PMID:41055760], and Mfn2 to impair mitochondrial fusion [PMID:41114773]. The catalytic linkage type is substrate- and context-specific, spanning K48-, K63-, and K33-linked chains that route substrates to proteasomal, lysosomal, or selective-autophagy fates [PMID:35977480, PMID:36198086, PMID:41055760, PMID:42229174].","teleology":[{"year":2009,"claim":"Established MARCH9 as a transmembrane RING-CH E3 ligase with a broad surface-protein substrate repertoire, defining its core cellular role as a regulator of plasma membrane protein abundance.","evidence":"SILAC quantitative plasma membrane proteomics in a MARCH9-expressing B cell line with flow cytometry confirmation","pmids":["19457934"],"confidence":"High","gaps":["Does not distinguish direct ubiquitination substrates from indirect effects","No ubiquitin-linkage type or degradation route defined"]},{"year":2010,"claim":"Showed that MARCH9 directly engages substrate cytoplasmic-tail lysines and that the interaction is regulable, linking it to immune ligand control.","evidence":"Co-IP, acceptor-lysine mutagenesis, and heat-shock-induced dissociation of Mult1 from MARCH9 with flow cytometry","pmids":["20870941"],"confidence":"High","gaps":["Mechanism of heat-shock-induced dissociation not resolved","Shared activity with MARCH4 not deconvolved"]},{"year":2012,"claim":"Defined the molecular determinants of substrate selectivity, showing recognition resides at the substrate transmembrane/cytosol interface and that lysine position relative to the bilayer matters.","evidence":"Mutagenesis and chimeric HLA-DR/DQ and HLA-DM constructs with flow cytometry readout","pmids":["22247549","22761441"],"confidence":"Medium","gaps":["Single-lab structural model of the ligase-substrate interface absent","Discrimination from MARCH8 not structurally explained"]},{"year":2017,"claim":"Revealed that MARCH9 acts at the TGN to reroute MHC-I into endosomal compartments, establishing a sorting (not merely degradative) function in antigen presentation.","evidence":"Ubiquitination assay, Syntaxin 6 co-localization microscopy, and DC microbial-pattern stimulation","pmids":["28559542"],"confidence":"High","gaps":["Signal coupling MARCH9 expression to microbial sensing undefined","Fate of rerouted MHC-I (recycling vs degradation) not fully traced"]},{"year":2018,"claim":"Identified the TM1 serine as catalytically essential and mapped the functional region to the extracellular TM termini, providing a structural basis for substrate regulation.","evidence":"TM-domain site-directed mutagenesis with flow cytometry plus solution NMR of the two-TM fragment","pmids":["30554144"],"confidence":"High","gaps":["No full-length structure including the RING domain","Role of the TM1 serine in substrate contact vs ligase conformation unresolved"]},{"year":2018,"claim":"Extended the substrate range to ICAM-1 with a corresponding cancer cell-migration phenotype, connecting ligase activity to a tumor-relevant outcome.","evidence":"Co-IP, overexpression in lung adenocarcinoma lines, and migration/invasion assays","pmids":["30278450"],"confidence":"Medium","gaps":["Direct ubiquitination of ICAM-1 not demonstrated by linkage assay","Endogenous-level relevance not established"]},{"year":2021,"claim":"Placed MARCH9 in a defined signaling axis, showing Tim-3 induces MARCH9 to drive K48-linked proteasomal degradation of MHC-I in macrophages.","evidence":"K48-linkage-specific ubiquitination assay, Co-IP, and in vivo Tim-3 antibody/macrophage depletion","pmids":["34025669"],"confidence":"Medium","gaps":["Tim-3→MARCH9→MHC-I axis not independently replicated","Transcriptional vs post-translational induction of MARCH9 unclear"]},{"year":2022,"claim":"Demonstrated a non-immune organelle role, with MARCH9 K63-ubiquitinating ATG9A to disperse it from the Golgi and drive heat-stress Golgi fragmentation.","evidence":"Reciprocal Co-IP, K63-specific ubiquitination assay, MARCH9 knockout, and Golgi morphology imaging under heat stress (two papers, same group)","pmids":["35977480","36198086"],"confidence":"High","gaps":["Upstream stress sensor activating MARCH9 unknown","Whether ATG9A dispersal affects autophagy flux not resolved"]},{"year":2022,"claim":"Linked MARCH9 to NF-kB-driven colorectal cancer growth via downregulation of the deubiquitinase CYLD.","evidence":"MARCH9 knockdown/overexpression, western blot, apoptosis/cell-cycle flow cytometry, and xenograft model","pmids":["36185211"],"confidence":"Low","gaps":["No direct ubiquitination assay of CYLD by MARCH9","Mechanism connecting MARCH9 to CYLD loss undefined"]},{"year":2023,"claim":"Showed MARCH9 degrades NOX2 to limit ROS and NLRP3 inflammasome activation, defining an anti-pyroptotic role in acute pancreatitis.","evidence":"NOX2 ubiquitination assay, ROS flow cytometry, and cerulein-induced rat model with in vitro overexpression","pmids":["37378901"],"confidence":"Medium","gaps":["Ubiquitin-linkage type on NOX2 not determined","Direct vs ROS-mediated NLRP3 effects not separated"]},{"year":2024,"claim":"Established LILRB2 as a recruiter promoting MARCH9-mediated K48 degradation of HLA-A, providing a cancer immune-evasion mechanism.","evidence":"Co-IP, histidine-pulldown ubiquitination assay, and syngeneic mouse tumor model","pmids":["38656573"],"confidence":"Medium","gaps":["How LILRB2 enhances the MARCH9-HLA-A interaction structurally unknown","Single-lab finding"]},{"year":2025,"claim":"Extended degradative regulation to innate-immune and mitochondrial substrates, with MARCH9 K48-ubiquitinating NLRP3 and K63-ubiquitinating Mfn2 under transcriptional control of ETV4.","evidence":"Linkage-specific ubiquitination assays, lysosomal-inhibitor block, ETV4 silencing/rescue, and in vivo MI/R model","pmids":["41055760","41114773"],"confidence":"Medium","gaps":["Whether NLRP3 regulation generalizes beyond ischemia-reperfusion untested","Mfn2 work limited to in vitro ovarian cancer cells"]},{"year":2026,"claim":"Demonstrated antiviral function, with RBM10 recruiting MARCHF9 to K33-ubiquitinate a viral protein for p62-mediated selective autophagy, broadening both substrate scope and chain-type repertoire.","evidence":"LC-MS/MS interaction screen, K33-linkage ubiquitination assay, autophagic-flux blockade, and p62 knockdown epistasis","pmids":["42229174"],"confidence":"Medium","gaps":["Viral (PEDV Nsp3) substrate differs from canonical mammalian targets","Single lab, not independently confirmed"]},{"year":null,"claim":"How MARCH9 selects among K48-, K63-, and K33-linked chain outputs and routes substrates to proteasomal, lysosomal, or autophagic fates in a context-dependent manner remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the full-length ligase-substrate-E2 complex","Determinants selecting linkage type and degradative route unknown","Adaptors/recruiters governing substrate access only partially identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,9]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,8,9,13]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[4,9]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,4,8,12]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,9,13]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[9,14]}],"complexes":[],"partners":["ATG9A","NLRP3","NOX2","MFN2","ICAM-1","HLA-A","LILRB2","RBM10"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86YJ5","full_name":"E3 ubiquitin-protein ligase MARCHF9","aliases":["Membrane-associated RING finger protein 9","Membrane-associated RING-CH protein IX","MARCH-IX","RING finger protein 179","RING-type E3 ubiquitin transferase MARCHF9"],"length_aa":346,"mass_kda":37.8,"function":"E3 ubiquitin-protein ligase that may mediate ubiquitination of MHC-I, CD4 and ICAM1, and promote their subsequent endocytosis and sorting to lysosomes via multivesicular bodies. E3 ubiquitin ligases accept ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfer the ubiquitin to targeted substrates","subcellular_location":"Golgi apparatus membrane; Lysosome membrane","url":"https://www.uniprot.org/uniprotkb/Q86YJ5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MARCHF9","classification":"Common Essential","n_dependent_lines":313,"n_total_lines":1090,"dependency_fraction":0.2871559633027523},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MARCHF9","total_profiled":1310},"omim":[{"mim_id":"613336","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 9; MARCHF9","url":"https://www.omim.org/entry/613336"}],"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/MARCHF9"},"hgnc":{"alias_symbol":["RNF179","FLJ36578"],"prev_symbol":["MARCH9"]},"alphafold":{"accession":"Q86YJ5","domains":[{"cath_id":"3.30.40.10","chopping":"108-160","consensus_level":"medium","plddt":86.993,"start":108,"end":160},{"cath_id":"1.10.287","chopping":"169-257","consensus_level":"medium","plddt":85.1252,"start":169,"end":257}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86YJ5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86YJ5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86YJ5-F1-predicted_aligned_error_v6.png","plddt_mean":64.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MARCHF9","jax_strain_url":"https://www.jax.org/strain/search?query=MARCHF9"},"sequence":{"accession":"Q86YJ5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86YJ5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86YJ5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86YJ5"}},"corpus_meta":[{"pmid":"36343628","id":"PMC_36343628","title":"Tegument 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microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/42229174","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13118,"output_tokens":3958,"usd":0.049362,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11802,"output_tokens":4220,"usd":0.082255,"stage2_stop_reason":"end_turn"},"total_usd":0.131617,"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\": 2009,\n      \"finding\": \"MARCH9 is a RING-CH transmembrane E3 ubiquitin ligase that down-regulates multiple plasma membrane proteins in lymphoid cells, including CD4, MHC-I, ICAM-1, PTPRJ/CD148, CD32B, HLA-DQ, CD150, and CD155, as identified by SILAC-based quantitative plasma membrane proteomics combined with flow cytometry confirmation.\",\n      \"method\": \"SILAC quantitative mass spectrometry of plasma membrane proteome in MARCH9-expressing B cell line, confirmed by flow cytometry\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative proteomics with orthogonal flow cytometry confirmation, multiple substrates validated, high correlation (r²=0.93) between methods\",\n      \"pmids\": [\"19457934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MARCH9 (and MARCH4) ubiquitinates the NKG2D ligand Mult1, suppressing its cell-surface expression; lysines in the cytoplasmic tail of Mult1 are essential for this repression. Heat-shock treatment dissociates Mult1 from MARCH9, reversing down-regulation and increasing Mult1 at the cell surface.\",\n      \"method\": \"Co-immunoprecipitation, flow cytometry, lysine mutant analysis, heat-shock functional assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, mutagenesis of acceptor lysines, and functional reversal by heat shock provide multiple orthogonal lines of evidence\",\n      \"pmids\": [\"20870941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MARCH9 ubiquitinates HLA-DM via a single lysine in the cytoplasmic tail of the DMα chain, inducing loss of DM from the cell surface by a mechanism involving both direct ubiquitin-chain attachment to DMα and a functional tyrosine-based signal on DMβ.\",\n      \"method\": \"Transfection/overexpression, flow cytometry, mutational analysis of acceptor lysine and tyrosine-based signal\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of substrate lysine and co-expressed constructs, single lab, two orthogonal readouts\",\n      \"pmids\": [\"22247549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MARCH9 substrate specificity for HLA-DQ (versus MARCH8 preference for HLA-DR) is determined by residues at the interface of the transmembrane domain and cytosol of the HLA β-chain; the acceptor lysine functions optimally at its natural position relative to the bilayer.\",\n      \"method\": \"Mutagenesis and chimeric construct analysis of HLA-DR/DQ transmembrane and cytoplasmic tail residues, flow cytometry readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with functional readout, single lab, defines molecular determinants of substrate recognition\",\n      \"pmids\": [\"22761441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MARCH9 ubiquitinates the cytoplasmic tail lysine residues of MHC-I, redirecting MHC-I from the default secretory pathway to Syntaxin 6-positive endosomal compartments at the trans-Golgi network, thereby regulating MHC-I export from the TGN. MARCH9 also targets CD1a. MARCH9 expression is modulated by microbial pattern exposure in dendritic cells.\",\n      \"method\": \"Overexpression/knockdown of MARCH9, co-localization with Syntaxin 6 by microscopy, MHC-I ubiquitination assay, flow cytometry, stimulation of dendritic cells with microbial patterns\",\n      \"journal\": \"Immunology and cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ubiquitination assay, co-localization, flow cytometry, DC stimulation) in a single focused study establishing TGN sorting mechanism\",\n      \"pmids\": [\"28559542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A single serine-to-alanine substitution in the first transmembrane domain (TM1) of human MARCH9 completely abolishes its ability to down-regulate HLA-A2 and CD4, identifying this serine as critical for substrate regulation. Solution NMR of the MARCH9 two-TM fragment shows that residues closest to the extracellular face of TM1 and TM2 define the key functional region.\",\n      \"method\": \"Site-directed mutagenesis of MARCH9 TM domains, flow cytometry functional assay, solution NMR of TM domain fragment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — combines NMR structure with mutagenesis and functional assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"30554144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MARCH9 interacts with ICAM-1 and overexpression of MARCH9 down-regulates ICAM-1 protein, attenuating migration and invasion of lung adenocarcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, overexpression in A549 and H1299 cells, flow cytometry/western blot, migration/invasion assays\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction plus functional phenotype, single lab, consistent with established substrate (ICAM-1) from prior proteomics\",\n      \"pmids\": [\"30278450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MARCH9 (along with MARCH2, MARCH4, and an isoform of MARCH3) down-regulates the IL-6 receptor alpha chain (IL6Rα) at the cell surface; functional RING domain, transmembrane domains, and C-terminal domains are all required for substrate recognition and down-regulation.\",\n      \"method\": \"cDNA expression library screen, overexpression in M1 cells, domain deletion/mutagenesis constructs, flow cytometry\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — positive screen plus domain analysis, but MARCH9 is one of several proteins tested; mechanistic detail for MARCH9 specifically is moderate\",\n      \"pmids\": [\"31488575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Tim-3 signaling in macrophages promotes MARCH9 expression, which in turn mediates K48-linked ubiquitination of MHC-I (HLA class I), leading to its proteasome-dependent degradation and reduced surface MHC-I presentation.\",\n      \"method\": \"Western blot, co-immunoprecipitation, ubiquitination assay (K48-linkage specific), Tim-3 antibody treatment and macrophage depletion in vivo\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and linkage-specific ubiquitination assay establish mechanism, but single lab without independent replication of the Tim-3→MARCH9→MHC-I axis\",\n      \"pmids\": [\"34025669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Under heat stress, MARCH9 promotes K63-linked ubiquitination of ATG9A at the TGN, causing ubiquitinated ATG9A to disperse from the Golgi to the cytoplasm, inhibiting GRASP55 oligomerization and resulting in Golgi fragmentation. Knockout of MARCH9 prevents heat-stress-induced Golgi fragmentation.\",\n      \"method\": \"Co-immunoprecipitation, K63-specific ubiquitination assay, MARCH9 knockout cells, Golgi morphology imaging under heat stress\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, linkage-specific ubiquitination, genetic KO with morphological readout, and stress-reversal experiment across two papers from the same group\",\n      \"pmids\": [\"35977480\", \"36198086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCH9 (MARCHF9) promotes colorectal cancer cell proliferation and migration by downregulating the deubiquitinase CYLD and activating NF-κB (p65); knockdown of MARCH9 induces apoptosis and cell-cycle arrest, and suppresses xenograft tumor growth.\",\n      \"method\": \"MARCH9 knockdown/overexpression, western blot for CYLD and p65, apoptosis/cell cycle flow cytometry, xenograft in vivo model\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect evidence of CYLD regulation by MARCH9 without direct ubiquitination assay of CYLD by MARCH9\",\n      \"pmids\": [\"36185211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MARCH9 mediates ubiquitination and degradation of NADPH oxidase-2 (NOX2), reducing ROS accumulation and NLRP3 inflammasome activation, thereby suppressing pancreatic cell pyroptosis in acute pancreatitis.\",\n      \"method\": \"MARCH9 overexpression in AR42J cells and cerulein-induced rat model, flow cytometry for ROS, western blot for NLRP3/caspase-1/GSDMD, ubiquitination assay for NOX2\",\n      \"journal\": \"Pancreas\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ubiquitination assay plus in vitro and in vivo functional rescue, but single lab with limited mechanistic detail on ubiquitin linkage type\",\n      \"pmids\": [\"37378901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"LILRB2 promotes MARCH9–HLA-A (HLA-A ubiquitin ligase) interaction, facilitating ubiquitination and proteasomal degradation of HLA-A, thereby enabling breast cancer immune evasion by reducing surface MHC-I presentation to CD8+ T cells.\",\n      \"method\": \"Co-immunoprecipitation, histidine pulldown ubiquitination assay, western blot, syngeneic mouse tumor model\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and pulldown-based ubiquitination assay, in vivo model, single lab, moderate mechanistic detail\",\n      \"pmids\": [\"38656573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH9 interacts with NLRP3 and promotes its K48-linked polyubiquitination, leading to proteasomal degradation of NLRP3, thereby inhibiting inflammasome activation and pyroptosis during myocardial ischemia-reperfusion injury.\",\n      \"method\": \"Co-immunoprecipitation, K48-linkage-specific ubiquitination assay, western blot, mouse MI/R in vivo model, H9C2 and HEK293 cell in vitro studies\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — K48-linkage-specific Co-IP ubiquitination assay with in vivo validation, single lab, single publication\",\n      \"pmids\": [\"41055760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ETV4 transcriptionally upregulates MARCH9, which then mediates K63-linked ubiquitination of the mitochondrial fusion protein Mfn2, targeting it for lysosomal degradation and impairing mitochondrial fusion in ovarian cancer cells.\",\n      \"method\": \"ETV4 silencing, MARCH9 overexpression rescue, western blot for Mfn2, lysosomal inhibitor (chloroquine) block, K63-ubiquitination assay\",\n      \"journal\": \"Cell biology and toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ubiquitination assay with lysosomal inhibitor confirmation, in vitro only, single lab\",\n      \"pmids\": [\"41114773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RBM10 recruits MARCHF9 to catalyze K33-linked ubiquitination of PEDV nonstructural protein 3 (Nsp3); the ubiquitinated Nsp3 is then recognized by the selective autophagy receptor p62 and delivered to autophagosomes for degradation, restricting viral replication.\",\n      \"method\": \"LC-MS/MS interaction screen, functional knockdown/overexpression assays, autophagic flux blockade, p62 knockdown, K33-linkage ubiquitination assay\",\n      \"journal\": \"Veterinary microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — linkage-specific ubiquitination assay plus genetic epistasis (p62 KD restores Nsp3), single lab, single publication; viral substrate differs from canonical mammalian substrates\",\n      \"pmids\": [\"42229174\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MARCHF9 (MARCH9) is a membrane-anchored RING-CH E3 ubiquitin ligase whose two transmembrane domains (with a critical serine in TM1) mediate recognition of substrate transmembrane regions to catalyze ubiquitination of cytoplasmic tail lysines on diverse cell-surface and intracellular proteins—including MHC-I, MHC-II (HLA-DQ, HLA-DM), CD4, ICAM-1, IL6Rα, Mult1, ATG9A, NOX2, NLRP3, and Mfn2—thereby directing substrates to endosomal/lysosomal or proteasomal degradation pathways that regulate antigen presentation, innate immune signaling, Golgi integrity, and inflammasome activation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MARCHF9 (MARCH9) is a membrane-anchored RING-CH E3 ubiquitin ligase that down-regulates a broad panel of cell-surface and intracellular membrane proteins by ubiquitinating lysines in their cytoplasmic tails, controlling antigen presentation, innate immune signaling, and organelle homeostasis [#0, #1]. Substrate recognition is governed by its two transmembrane domains: a single serine in TM1 is essential for activity, and solution NMR localizes the functional region to residues near the extracellular face of TM1 and TM2, while the RING and C-terminal domains are additionally required for substrate down-regulation [#5, #7]. Specificity among related substrates is dictated by residues at the transmembrane/cytosol interface of the target, as shown for its preference for HLA-DQ over HLA-DR, with the acceptor lysine acting optimally at its natural distance from the bilayer [#3, #2]. Functionally, MARCH9 routes MHC-I from the secretory pathway into Syntaxin 6-positive trans-Golgi-network endosomal compartments and drives proteasomal degradation of MHC-I/HLA-A via K48-linked ubiquitination, a mechanism co-opted in cancer immune evasion downstream of Tim-3 and LILRB2 signaling [#4, #8, #12]. Beyond antigen presentation, MARCH9 ubiquitinates ATG9A in a K63-linked manner to promote heat-stress-induced Golgi fragmentation through loss of GRASP55 oligomerization [#9], and degrades innate-immune and metabolic targets including NOX2 and NLRP3 to restrain ROS-driven inflammasome activation and pyroptosis [#11, #13], and Mfn2 to impair mitochondrial fusion [#14]. The catalytic linkage type is substrate- and context-specific, spanning K48-, K63-, and K33-linked chains that route substrates to proteasomal, lysosomal, or selective-autophagy fates [#9, #13, #15].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established MARCH9 as a transmembrane RING-CH E3 ligase with a broad surface-protein substrate repertoire, defining its core cellular role as a regulator of plasma membrane protein abundance.\",\n      \"evidence\": \"SILAC quantitative plasma membrane proteomics in a MARCH9-expressing B cell line with flow cytometry confirmation\",\n      \"pmids\": [\"19457934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not distinguish direct ubiquitination substrates from indirect effects\", \"No ubiquitin-linkage type or degradation route defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed that MARCH9 directly engages substrate cytoplasmic-tail lysines and that the interaction is regulable, linking it to immune ligand control.\",\n      \"evidence\": \"Co-IP, acceptor-lysine mutagenesis, and heat-shock-induced dissociation of Mult1 from MARCH9 with flow cytometry\",\n      \"pmids\": [\"20870941\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of heat-shock-induced dissociation not resolved\", \"Shared activity with MARCH4 not deconvolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the molecular determinants of substrate selectivity, showing recognition resides at the substrate transmembrane/cytosol interface and that lysine position relative to the bilayer matters.\",\n      \"evidence\": \"Mutagenesis and chimeric HLA-DR/DQ and HLA-DM constructs with flow cytometry readout\",\n      \"pmids\": [\"22247549\", \"22761441\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab structural model of the ligase-substrate interface absent\", \"Discrimination from MARCH8 not structurally explained\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revealed that MARCH9 acts at the TGN to reroute MHC-I into endosomal compartments, establishing a sorting (not merely degradative) function in antigen presentation.\",\n      \"evidence\": \"Ubiquitination assay, Syntaxin 6 co-localization microscopy, and DC microbial-pattern stimulation\",\n      \"pmids\": [\"28559542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal coupling MARCH9 expression to microbial sensing undefined\", \"Fate of rerouted MHC-I (recycling vs degradation) not fully traced\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified the TM1 serine as catalytically essential and mapped the functional region to the extracellular TM termini, providing a structural basis for substrate regulation.\",\n      \"evidence\": \"TM-domain site-directed mutagenesis with flow cytometry plus solution NMR of the two-TM fragment\",\n      \"pmids\": [\"30554144\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length structure including the RING domain\", \"Role of the TM1 serine in substrate contact vs ligase conformation unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended the substrate range to ICAM-1 with a corresponding cancer cell-migration phenotype, connecting ligase activity to a tumor-relevant outcome.\",\n      \"evidence\": \"Co-IP, overexpression in lung adenocarcinoma lines, and migration/invasion assays\",\n      \"pmids\": [\"30278450\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of ICAM-1 not demonstrated by linkage assay\", \"Endogenous-level relevance not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed MARCH9 in a defined signaling axis, showing Tim-3 induces MARCH9 to drive K48-linked proteasomal degradation of MHC-I in macrophages.\",\n      \"evidence\": \"K48-linkage-specific ubiquitination assay, Co-IP, and in vivo Tim-3 antibody/macrophage depletion\",\n      \"pmids\": [\"34025669\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tim-3→MARCH9→MHC-I axis not independently replicated\", \"Transcriptional vs post-translational induction of MARCH9 unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated a non-immune organelle role, with MARCH9 K63-ubiquitinating ATG9A to disperse it from the Golgi and drive heat-stress Golgi fragmentation.\",\n      \"evidence\": \"Reciprocal Co-IP, K63-specific ubiquitination assay, MARCH9 knockout, and Golgi morphology imaging under heat stress (two papers, same group)\",\n      \"pmids\": [\"35977480\", \"36198086\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream stress sensor activating MARCH9 unknown\", \"Whether ATG9A dispersal affects autophagy flux not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked MARCH9 to NF-kB-driven colorectal cancer growth via downregulation of the deubiquitinase CYLD.\",\n      \"evidence\": \"MARCH9 knockdown/overexpression, western blot, apoptosis/cell-cycle flow cytometry, and xenograft model\",\n      \"pmids\": [\"36185211\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct ubiquitination assay of CYLD by MARCH9\", \"Mechanism connecting MARCH9 to CYLD loss undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed MARCH9 degrades NOX2 to limit ROS and NLRP3 inflammasome activation, defining an anti-pyroptotic role in acute pancreatitis.\",\n      \"evidence\": \"NOX2 ubiquitination assay, ROS flow cytometry, and cerulein-induced rat model with in vitro overexpression\",\n      \"pmids\": [\"37378901\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin-linkage type on NOX2 not determined\", \"Direct vs ROS-mediated NLRP3 effects not separated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established LILRB2 as a recruiter promoting MARCH9-mediated K48 degradation of HLA-A, providing a cancer immune-evasion mechanism.\",\n      \"evidence\": \"Co-IP, histidine-pulldown ubiquitination assay, and syngeneic mouse tumor model\",\n      \"pmids\": [\"38656573\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How LILRB2 enhances the MARCH9-HLA-A interaction structurally unknown\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended degradative regulation to innate-immune and mitochondrial substrates, with MARCH9 K48-ubiquitinating NLRP3 and K63-ubiquitinating Mfn2 under transcriptional control of ETV4.\",\n      \"evidence\": \"Linkage-specific ubiquitination assays, lysosomal-inhibitor block, ETV4 silencing/rescue, and in vivo MI/R model\",\n      \"pmids\": [\"41055760\", \"41114773\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NLRP3 regulation generalizes beyond ischemia-reperfusion untested\", \"Mfn2 work limited to in vitro ovarian cancer cells\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstrated antiviral function, with RBM10 recruiting MARCHF9 to K33-ubiquitinate a viral protein for p62-mediated selective autophagy, broadening both substrate scope and chain-type repertoire.\",\n      \"evidence\": \"LC-MS/MS interaction screen, K33-linkage ubiquitination assay, autophagic-flux blockade, and p62 knockdown epistasis\",\n      \"pmids\": [\"42229174\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Viral (PEDV Nsp3) substrate differs from canonical mammalian targets\", \"Single lab, not independently confirmed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MARCH9 selects among K48-, K63-, and K33-linked chain outputs and routes substrates to proteasomal, lysosomal, or autophagic fates in a context-dependent manner remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the full-length ligase-substrate-E2 complex\", \"Determinants selecting linkage type and degradative route unknown\", \"Adaptors/recruiters governing substrate access only partially identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 9]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 8, 9, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [4, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 4, 8, 12]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 9, 13]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [9, 14]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ATG9A\", \"NLRP3\", \"NOX2\", \"Mfn2\", \"ICAM-1\", \"HLA-A\", \"LILRB2\", \"RBM10\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}