{"gene":"MARCHF6","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2005,"finding":"Human TEB4 (MARCHF6) is an ER-resident ubiquitin ligase with a C4HC3 RING finger at its N-terminus located in the cytosol. The isolated RING domain catalyzes ubiquitin ligation in vitro in a Lys48-specific manner involving UBC7 as the E2. TEB4 promotes its own proteasomal degradation in a RING finger-dependent manner (autoubiquitination).","method":"In vitro ubiquitin ligation assay with isolated RING domain; mutational analysis of RING finger; proteasomal inhibitor experiments; subcellular fractionation/topology analysis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of E3 activity with defined E2 (UBC7), Lys48 specificity shown, RING mutant controls, multiple orthogonal methods in single study","pmids":["15673284"],"is_preprint":false},{"year":2005,"finding":"Yeast Doa10 (ortholog of human TEB4/MARCHF6) contains 14 transmembrane helices with both its N-terminal RING-CH domain and C-terminus facing the cytosol. Biochemical evidence supports a similar topology for human TEB4 (MARCHF6). The yeast Derlins are not required for degradation of Doa10 membrane substrates.","method":"Dual-topology reporter fusions at 16 positions in Doa10; protease digestion of yeast microsomes; bioinformatic topology prediction; in silico mutagenesis; topology comparison with human TEB4","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic topology mapping with 16 reporter fusions plus protease protection, replicated across yeast and comparative analysis with human ortholog","pmids":["16373356"],"is_preprint":false},{"year":2009,"finding":"TEB4 (MARCHF6) interacts with and mediates ubiquitination and degradation of type 2 iodothyronine deiodinase (D2). TEB4 knockdown decreases D2 ubiquitination and increases D2 activity and protein levels ~4-fold, prolonging D2 half-life. The effect is specific to D2 and requires a critical instability domain in D2; the other deiodinase D1 and a truncated D2 lacking the instability domain are unaffected.","method":"Co-immunoprecipitation; TEB4 overexpression (activity assay); siRNA knockdown of TEB4 (>90% reduction); measurement of D2 activity, ubiquitination, and protein levels; lentivirus-based knockdown in D2-expressing MSTO-211 cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, knockdown with multiple readouts (activity, ubiquitination, half-life), substrate specificity tested with controls, replicated in two cell lines","pmids":["19651899"],"is_preprint":false},{"year":2013,"finding":"The yeast Doa10 (and its mammalian ortholog Teb4/MARCHF6) mediates sterol-dependent ubiquitination and degradation of squalene monooxygenase (SM), a sterol-specific step in the mevalonate pathway. This constitutes an evolutionarily conserved feedback system for sterol homeostasis, distinct from and complementary to HMGR (Hrd1 branch) regulation.","method":"Genetic epistasis in yeast (doa10 deletion); sterol-dependent degradation assays; lipidomics; complementation with mammalian Teb4","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in yeast plus complementation with mammalian ortholog, multiple orthogonal methods, independently corroborated by subsequent mammalian studies","pmids":["23898401"],"is_preprint":false},{"year":2014,"finding":"MARCH6 (TEB4) physically interacts with squalene monooxygenase (SM) and acts as the E3 ligase controlling its cholesterol-dependent proteasomal degradation. MARCH6 overexpression reduces SM abundance in a RING-dependent manner; MARCH6 knockdown increases SM protein and activity and prevents its cholesterol-regulated degradation. MARCH6 knockdown also increases HMGCR levels in hepatocytes, establishing MARCH6 as a regulator of both SM and HMGCR.","method":"Co-immunoprecipitation; MARCH6 overexpression with RING mutant control; siRNA knockdown; immunoblotting; SM activity assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, RING-domain mutant control, knockdown with multiple substrates tested, independently replicated across multiple labs","pmids":["24449766"],"is_preprint":false},{"year":2014,"finding":"USP19, an ER-anchored deubiquitinating enzyme, interacts with MARCH6 and stabilizes it by deubiquitination, protecting MARCH6 from p97-dependent proteasomal degradation. USP19 overexpression delays MARCH6 degradation and reduces its ubiquitination; USP19 knockdown decreases MARCH6 levels and increases ubiquitination of MARCH6. Loss of USP19 also increases levels of the ERAD substrate ABCB11, consistent with MARCH6 being the mediating ligase.","method":"Co-immunoprecipitation; USP19 overexpression and siRNA knockdown; immunoblotting for MARCH6 ubiquitination and stability; p97 inhibition experiments","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction, gain- and loss-of-function with mechanistic readout (deubiquitination, p97 dependence), single lab","pmids":["25088257"],"is_preprint":false},{"year":2015,"finding":"MARCH6 acts as an endogenous inhibitor of the SREBP transcriptional program. Loss of MARCH6 increases SREBP-regulated gene expression (cholesterol biosynthesis and lipoprotein uptake genes) but paradoxically decreases cellular lipoprotein uptake due to enhanced lysosomal LDLR degradation. This is mediated by MARCH6-loss-induced upregulation of the E3 ligase IDOL, which drives LDLR degradation. Thus, MARCH6 uncouples cholesterol synthesis from lipoprotein uptake.","method":"Genetic knockdown/knockout of MARCH6; gene expression analysis; lipoprotein uptake assays; IDOL knockdown epistasis experiments","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (MARCH6 loss → IDOL induction → LDLR degradation), multiple pathway readouts, single lab","pmids":["26527619"],"is_preprint":false},{"year":2016,"finding":"A conserved C-terminal element (CTE), a 16-residue cytosol-facing motif after the final TM helix of Doa10/MARCH6, is required for degradation of a subset of substrates. Mutation of the conserved asparagine in the MARCH6 CTE (N890A) stabilizes MARCH6 itself to the same degree as a catalytically inactivating RING mutation (C9A), indicating the CTE is required for MARCH6 autoregulation. CRISPR/Cas9 endogenous tagging confirmed MARCH6 localizes to the ER.","method":"Alanine/asparagine mutagenesis of CTE; yeast ubiquitylation and degradation assays; MARCH6 autoregulation assays in human cells; CRISPR/Cas9 endogenous epitope tagging for ER localization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis at defined residue with multiple substrate readouts, endogenous localization by CRISPR tagging, single lab","pmids":["27068744"],"is_preprint":false},{"year":2018,"finding":"MARCH6 and TRC8 are both required for proteasomal degradation of misfolded cytosolic/ER-membrane substrates (mCherry-CL1 reporter); complete stabilization requires double knockout of both E3 ligases. MARCH6 and TRC8 both associate with the intramembrane protease SPP and cooperate to degrade tail-anchored heme oxygenase-1 (HO-1) following intramembrane proteolysis. The two ligases act independently of each other on these substrates.","method":"Forward genetic screens in human cells; CRISPR double knockout; quantitative mass spectrometry for protein turnover; Co-immunoprecipitation of SPP association; HO-1 degradation assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — forward genetic screen, double KO, reciprocal Co-IP with SPP, quantitative MS, multiple substrates tested, rigorous controls","pmids":["29519897"],"is_preprint":false},{"year":2018,"finding":"Cholesterol stabilizes MARCH6 protein by preventing its autodegradation, likely through a conformational change. This stabilization requires functional VCP/p97-dependent membrane extraction and proteasomal degradation, is absent in MARCH6 autodegradation-deficient mutants, and leads to increased degradation of at least three known MARCH6 substrates (SM and others). A putative sterol-sensing domain in MARCH6 is not required for this cholesterol-mediated stabilization.","method":"CRISPR/Cas9 gene editing; MARCH6 overexpression; immunoblotting; VCP/p97 inhibition; proteasome inhibition; MARCH6 autodegradation mutants; chemical chaperone treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO, multiple mutant controls, pharmacological dissection of pathway, single lab with orthogonal approaches","pmids":["30545937"],"is_preprint":false},{"year":2018,"finding":"MARCH6-dependent ERAD mediates proteasomal degradation of misfolded I1061T NPC1. This pathway acts complementarily with FAM134B-dependent selective ER autophagy (ER-phagy) to regulate I1061T NPC1 turnover.","method":"siRNA knockdown of MARCH6; proteasome inhibitor treatment; subcellular fractionation; in vivo mouse tissue analysis; identification of FAM134B-dependent ER-phagy pathway as parallel route","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MARCH6 knockdown with proteasomal readout, complemented by in vivo mouse model, single lab","pmids":["30202070"],"is_preprint":false},{"year":2019,"finding":"The transcription factor Sp1 binds to three Sp1 binding sites located ~100 bp downstream of the MARCH6 transcriptional start site and upregulates MARCH6 gene expression. Pharmacological and genetic inhibition of Sp1 reduces MARCH6 expression, which in turn affects stability of its substrate squalene monooxygenase.","method":"Luciferase reporter assays; qRT-PCR; pharmacological Sp1 inhibition; siRNA knockdown of Sp1; ChIP-seq data mining; promoter deletion analysis","journal":"Biochimica et biophysica acta. Molecular and cell biology of lipids","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase reporter, genetic and pharmacological inhibition of Sp1, functional downstream readout (SM stability), single lab","pmids":["31422115"],"is_preprint":false},{"year":2020,"finding":"MARCH6 controls levels of lanosterol 14α-demethylase (LDM) and 24-dehydrocholesterol reductase (DHCR24) by promoting their degradation; this degradation is not triggered by sterols. MARCH6 thereby targets multiple steps in the cholesterol synthesis pathway, representing the first E3 ligase known to control multiple enzymes in a single biochemical pathway.","method":"siRNA knockdown of MARCH6; immunoblotting for LDM and DHCR24 stability; sterol manipulation experiments; cell-based degradation assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with multiple substrate readouts and sterol treatment controls, single lab","pmids":["31904814"],"is_preprint":false},{"year":2020,"finding":"Ablation of MARCHF6 in endothelial cells increases SQLE protein and cholesterol load, leading to altered membrane order, disorganized VE-cadherin-based adherens junctions, decreased endothelial barrier function, and impaired SQLE-dependent sprouting angiogenesis. This positions MARCH6-mediated SQLE degradation as a determinant of endothelial integrity.","method":"siRNA/CRISPR-mediated MARCHF6 ablation in endothelial cells; SQLE overexpression; cholesterol measurement; membrane order assays; barrier function assays; sprouting angiogenesis assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with defined cellular phenotypes, SQLE overexpression phenocopy, single lab","pmids":["32755570"],"is_preprint":false},{"year":2021,"finding":"MARCH6 interacts with and promotes degradation (destabilization) of DHX9 in thyroid cancer cells. Mechanistically, MARCH6-mediated DHX9 destabilization activates the AKT/mTOR signaling pathway. DHX9 knockdown phenocopies MARCH6 overexpression in promoting proliferation and migration.","method":"Co-immunoprecipitation; MARCH6 overexpression and knockdown; DHX9 knockdown; AKT/mTOR pathway immunoblotting; cell proliferation and migration assays","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction, epistasis via DHX9 knockdown rescue, downstream pathway readout, single lab","pmids":["34512155"],"is_preprint":false},{"year":2022,"finding":"MARCHF6 recognizes NADPH through its C-terminal regulatory region; this NADPH binding upregulates MARCHF6 E3 ligase activity. MARCHF6 mediates ubiquitin-dependent degradation of the pro-ferroptotic effectors ACSL4 and p53. Loss of MARCHF6 increases ferroptosis sensitivity; inhibiting ferroptosis rescues growth of MARCHF6-deficient tumors and perinatal lethality of Marchf6-/- mice.","method":"NADPH binding assays; MARCHF6 ligase activity assays; ubiquitination assays for ACSL4 and p53; MARCHF6 knockout mice; tumor growth rescue experiments; ferroptosis inhibitor treatment","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — biochemical NADPH binding, in vitro/in vivo E3 activity, knockout mouse model with rescue, multiple substrates identified with orthogonal methods","pmids":["35941365"],"is_preprint":false},{"year":2023,"finding":"SC4MOL (the first acting enzyme of the C4-demethylation complex in cholesterol synthesis) is a substrate of MARCHF6; SC4MOL is rapidly turned over and sensitive to sterols. MARCHF6 thereby controls at least five enzymes in the cholesterol synthesis pathway.","method":"siRNA knockdown of MARCHF6; sterol depletion/loading experiments; immunoblotting for SC4MOL stability; CHO and human cell lines; cholesterol measurement after SC4MOL siRNA","journal":"Journal of lipid research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with substrate stabilization readout, sterol manipulation controls, two cell line systems, single lab","pmids":["36958722"],"is_preprint":false},{"year":2023,"finding":"In POMC neurons, Marchf6 mediates degradation of cytosol-retained (signal peptide-uncleaved) POMC, preventing ER stress and ferroptosis. Loss of MARCHF6 in POMC neurons (POMC-Cre Marchf6-deficient mice) causes hyperphagia, reduced energy expenditure, and weight gain.","method":"POMC-Cre conditional Marchf6 knockout mice; ferroptosis and ER stress marker analysis; cell viability assays; chaperone sequestration experiments; ubiquitination assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO mouse model with defined metabolic phenotype, substrate identification, multiple mechanistic readouts, single lab","pmids":["37421621"],"is_preprint":false},{"year":2023,"finding":"Brucella abortus T4SS effector BspA interacts with MARCH6 and destabilizes the MARCH6 E3 ligase complex, thereby inhibiting MARCH6-dependent ERAD. This inhibition promotes intracellular B. abortus proliferation; pharmacological ERAD inhibition or siRNA depletion of MARCH6 phenocopy BspA deletion and rescue the replication defect of a bspA mutant.","method":"Co-immunoprecipitation of BspA-MARCH6; siRNA knockdown of MARCH6; pharmacological ERAD inhibition; bacterial replication assays in macrophages; epistasis with UbxD8 depletion","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, genetic epistasis by siRNA, pharmacological corroboration, defined replication phenotype, single lab","pmids":["37129527"],"is_preprint":false},{"year":2024,"finding":"Doa10/MARCHF6 adopts a unique circular architecture within the ER membrane, with the majority of the protein forming a lipid-binding scaffold gated by a flexible helical bundle. The RING domain ubiquitylation active site is positioned over this channel via connections with the membrane-spanning scaffold and gate. Structure-based mutagenesis of 95 MARCH6 variants revealed that SQLE degradation depends on the gated channel, RING domain connections, and lipid-binding sites. AlphaFold models are consistent with substrate-engaged and ubiquitylation complex states.","method":"Cryo-EM structural analysis; AlphaFold modeling; systematic mutagenesis of 95 variants; SQLE degradation assay; lipid-binding analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with AlphaFold validation, 95-variant mutagenesis campaign, functional substrate degradation assay, multiple orthogonal approaches in one study","pmids":["38195637"],"is_preprint":false},{"year":2024,"finding":"The Ac/N-degron recognition domain (Ac/N domain) of MARCHF6 was mapped to specific cytosol-facing regions using alanine-stretch mutagenesis. This domain exhibits preferential binding to Nα-terminally acetylated proteins/peptides over unacetylated counterparts. MARCHF6 mediates degradation of Ac/N-degron-bearing substrates including RGS2 and PLIN2, and abolishing Ac/N-degron recognition stabilizes these substrates and increases ferroptosis resistance.","method":"Alanine-stretch mutagenesis; chemical crosslinking-based Co-IP; split-ubiquitin assays (human and yeast cells); Ac/N-degron substrate binding assays with acetylated vs. unacetylated peptides; RGS2 and PLIN2 stability assays; ferroptosis assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis, biochemical binding specificity with acetylated vs. unacetylated substrates, split-ubiquitin functional validation, multiple substrates, single lab","pmids":["39216628"],"is_preprint":false},{"year":2025,"finding":"Avian MARCH6 directly interacts with Tembusu virus NS5 protein and promotes its degradation via selective autophagy through an E3 ligase activity-independent mechanism. MARCH6 recruits the autophagic cargo receptor TOLLIP, which facilitates NS5-TOLLIP interaction independent of ubiquitin signaling, directing NS5 to phagophores for degradation.","method":"Co-immunoprecipitation of MARCH6-NS5 and NS5-TOLLIP; MARCH6 overexpression and knockdown; RING-dead MARCH6 mutant; autophagy inhibitors; viral replication assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, RING-dead mutant establishes E3-independence, knockdown/overexpression with viral replication readout, single lab (avian system)","pmids":["40511919"],"is_preprint":false},{"year":2026,"finding":"MARCHF6 directly interacts with and ubiquitinates SREBP1, targeting it for proteasomal degradation. Loss of hepatic MARCHF6 prolongs SREBP1 half-life, driving excessive de novo lipogenesis. Liver-specific Marchf6 knockout mice develop spontaneous hepatic triglyceride and cholesteryl ester accumulation under normal chow.","method":"Liver-specific Marchf6 knockout (Marchf6Alb) mice; Co-IP; ubiquitination assay; SREBP1 half-life measurement; transcriptomics and proteomics; lipidomics; human MASLD patient data","journal":"Journal of hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitination assay, conditional KO mouse with metabolic phenotype, corroborated by human patient expression data, single lab","pmids":["42173365"],"is_preprint":false},{"year":2026,"finding":"MARCH6 induces FAM134B protein ubiquitination and degradation, reducing FAM134B stability in glioma cells. This suppresses ER-phagy and ER stress responses. Knockdown of MARCH6 reverses FAM134B degradation and restores ER-phagy markers (LC3B conversion, autophagosome accumulation).","method":"Co-immunoprecipitation (MARCH6-FAM134B interaction); ubiquitination assays; siRNA knockdown of FAM134B and MARCH6; ER stress and autophagy marker analysis; mouse glioma model","journal":"Neurochemical research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP, ubiquitination assay, knockdown epistasis with in vivo validation, single lab","pmids":["41661358"],"is_preprint":false},{"year":2025,"finding":"The membrane-anchored E2 UBE2J2 cooperates with MARCHF6 (and other E3 ligases RNF145, RNF139) to ubiquitinate both themselves and the substrate squalene monooxygenase. UBE2J2 activity is modulated by membrane lipid packing, with loosely packed membranes impairing ubiquitin loading onto UBE2J2, thereby relaying lipid signals to MARCHF6-dependent ubiquitination.","method":"Reconstituted systems with purified ERAD factors; in vitro ubiquitination assays; membrane composition manipulation; E2-E3 interaction assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with purified components, but preprint not yet peer-reviewed, single study","pmids":["bio_10.1101_2025.07.22.666085"],"is_preprint":true},{"year":2025,"finding":"MARCH6 promotes ubiquitin-mediated degradation of ADAMTS4 in cardiomyocytes, thereby upregulating the downstream target SDC-1. This MARCHF6/ADAMTS4/SDC-1 axis inhibits lipid peroxidation and ferroptosis in myocardial ischemia-reperfusion injury.","method":"Co-immunoprecipitation; ubiquitination assays; MARCHF6 overexpression (AAV9 in mice); IRI mouse model; OGD/R cell model; ferroptosis markers","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and overexpression, single lab, no direct ubiquitination of ADAMTS4 rigorously demonstrated in abstract","pmids":["42155815"],"is_preprint":false}],"current_model":"MARCHF6 (TEB4/MARCH6) is a multi-pass ER-resident RING-CH E3 ubiquitin ligase that adopts a unique circular channel architecture within the membrane; it catalyzes Lys48-linked ubiquitination using UBE2J2/UBC7 as its cognate E2, targets multiple substrates for proteasomal degradation including squalene monooxygenase (SQLE), HMGCR, lanosterol 14α-demethylase, SC4MOL, DHCR24, SREBP1, type 2 deiodinase (D2), HO-1, ACSL4, p53, RGS2, PLIN2, DHX9, and FAM134B, and is itself regulated by cholesterol (which prevents its autodegradation), by NADPH (which binds its C-terminal region and activates its ligase activity), by the deubiquitinase USP19 (which stabilizes it), and transcriptionally by Sp1; its Ac/N-degron recognition domain in cytosol-facing loops preferentially binds Nα-terminally acetylated substrates, while other substrates are recognized via distinct sites including the conserved C-terminal element (CTE)."},"narrative":{"mechanistic_narrative":"MARCHF6 (TEB4/MARCH6) is a multi-pass ER-resident RING-CH E3 ubiquitin ligase that orchestrates protein quality control and lipid homeostasis by targeting membrane and cytosolic substrates for proteasomal degradation [PMID:15673284, PMID:23898401]. Its N-terminal C4HC3 RING domain faces the cytosol and catalyzes Lys48-linked ubiquitination in vitro using UBC7/UBE2J2 as the cognate E2, and the enzyme constitutively autoubiquitinates to drive its own turnover [PMID:15673284, PMID:16373356]. Cryo-EM revealed that the protein folds into a unique circular, lipid-binding scaffold gated by a flexible helical bundle, with the RING active site positioned over the channel; substrate degradation depends on this gated channel and on RING-domain connections to the membrane scaffold [PMID:38195637]. A dominant function is feedback control of cholesterol synthesis: MARCHF6 degrades multiple sequential mevalonate-pathway enzymes including squalene monooxygenase, HMGCR, lanosterol 14α-demethylase, DHCR24, and SC4MOL, and acts as an endogenous brake on the SREBP program by ubiquitinating SREBP1 [PMID:23898401, PMID:24449766, PMID:31904814, PMID:36958722, PMID:42173365]. Substrate recognition occurs through distinct cytosol-facing determinants, including an Ac/N-degron domain that preferentially binds Nα-terminally acetylated substrates such as RGS2 and PLIN2, and a conserved C-terminal element (CTE) required for a subset of substrates and for MARCHF6 autoregulation [PMID:27068744, PMID:39216628]. The ligase additionally degrades pro-ferroptotic effectors ACSL4 and p53, and its activity is allosterically upregulated by NADPH binding to its C-terminal region, linking it to suppression of ferroptosis in vivo [PMID:35941365]. MARCHF6 abundance is itself tightly regulated—stabilized by cholesterol (which blocks autodegradation) and by the deubiquitinase USP19, and transcriptionally driven by Sp1 [PMID:30545937, PMID:25088257, PMID:31422115]. Through these activities MARCHF6 governs endothelial barrier integrity, hepatic lipogenesis, energy balance via POMC neurons, and ERAD versus ER-phagy substrate routing [PMID:32755570, PMID:42173365, PMID:37421621, PMID:30202070, PMID:41661358].","teleology":[{"year":2005,"claim":"Establishing that human TEB4 is a bona fide E3 ligase defined the molecular activity at the heart of the gene and its E2 partner and linkage specificity.","evidence":"In vitro ubiquitin ligation with isolated RING domain, RING mutant controls, and topology/fractionation analysis","pmids":["15673284"],"confidence":"High","gaps":["Substrate repertoire unknown at this stage","Full-length enzymatic activity in membrane context not addressed"]},{"year":2005,"claim":"Topology mapping of the Doa10 ortholog answered how the ligase is arranged in the ER membrane, placing both RING and C-terminus in the cytosol where they can engage E2 and substrates.","evidence":"Dual-topology reporter fusions and protease protection in yeast microsomes with comparison to human TEB4","pmids":["16373356"],"confidence":"High","gaps":["Mechanism of substrate extraction across the membrane not defined","No high-resolution structure"]},{"year":2009,"claim":"Identification of type 2 deiodinase as a substrate provided the first defined endogenous target, showing the ligase controls hormone-activating enzyme levels.","evidence":"Reciprocal Co-IP and siRNA knockdown with ubiquitination, activity, and half-life readouts in two cell lines","pmids":["19651899"],"confidence":"High","gaps":["Recognition determinant on D2 only coarsely mapped to an instability domain","Physiological setting of D2 regulation by TEB4 not tested in vivo"]},{"year":2013,"claim":"Demonstrating sterol-dependent degradation of squalene monooxygenase established MARCHF6 as an evolutionarily conserved node of cholesterol feedback regulation.","evidence":"Yeast genetic epistasis with mammalian Teb4 complementation and lipidomics","pmids":["23898401"],"confidence":"High","gaps":["Direct E3-substrate engagement in mammalian cells shown subsequently","Sterol sensor identity unresolved"]},{"year":2014,"claim":"Direct demonstration that MARCH6 binds and degrades SM and also affects HMGCR positioned it as a regulator of multiple mevalonate-pathway enzymes.","evidence":"Reciprocal Co-IP, RING-mutant controls, knockdown with SM activity and HMGCR readouts","pmids":["24449766"],"confidence":"High","gaps":["Whether HMGCR is a direct MARCH6 substrate or indirect not resolved here"]},{"year":2014,"claim":"Discovery that USP19 deubiquitinates and stabilizes MARCH6 revealed how the ligase's own abundance is post-translationally tuned against p97-driven turnover.","evidence":"Co-IP plus USP19 gain/loss of function with MARCH6 ubiquitination, stability, and p97 dependence readouts","pmids":["25088257"],"confidence":"Medium","gaps":["Single lab","Stoichiometry and regulation of the USP19-MARCH6 interaction unknown"]},{"year":2015,"claim":"Showing MARCH6 loss induces IDOL and LDLR degradation explained how the ligase uncouples cholesterol synthesis from lipoprotein uptake at the systems level.","evidence":"MARCH6 knockdown/knockout with gene expression, uptake assays, and IDOL epistasis","pmids":["26527619"],"confidence":"Medium","gaps":["Mechanism linking MARCH6 loss to IDOL induction not defined","Single lab"]},{"year":2016,"claim":"Mapping the conserved C-terminal element defined a substrate-recognition/autoregulation determinant distinct from the RING and confirmed endogenous ER localization.","evidence":"CTE mutagenesis (N890A) with yeast and human degradation assays plus CRISPR endogenous tagging","pmids":["27068744"],"confidence":"Medium","gaps":["Structural basis of CTE function unresolved at this stage","Which substrates require CTE not exhaustively mapped"]},{"year":2018,"claim":"Cooperation of MARCH6 with TRC8 and the protease SPP showed the ligase participates in degrading misfolded and intramembrane-cleaved substrates such as HO-1.","evidence":"Forward genetic screens, CRISPR double knockout, SPP Co-IP, and quantitative MS","pmids":["29519897"],"confidence":"High","gaps":["Division of labor between MARCH6 and TRC8 per substrate not fully delineated"]},{"year":2018,"claim":"Demonstrating cholesterol stabilizes MARCH6 by blocking autodegradation established a direct feedback loop coupling sterol levels to ligase abundance and substrate flux.","evidence":"CRISPR editing, autodegradation mutants, and VCP/p97 and proteasome inhibition","pmids":["30545937"],"confidence":"Medium","gaps":["Sterol-sensing domain ruled out but the actual sensing mechanism unidentified","Single lab"]},{"year":2018,"claim":"Identifying MARCH6-dependent degradation of misfolded NPC1 positioned the ligase within ERAD acting in parallel to FAM134B-mediated ER-phagy.","evidence":"siRNA knockdown with proteasomal readouts and in vivo mouse tissue analysis","pmids":["30202070"],"confidence":"Medium","gaps":["Direct interaction of MARCH6 with NPC1 not shown","Single lab"]},{"year":2019,"claim":"Sp1 was identified as a transcriptional activator of MARCH6, defining how ligase levels are set at the gene-expression layer with downstream effects on SM stability.","evidence":"Luciferase reporters, promoter deletion, Sp1 inhibition/knockdown, and ChIP-seq mining","pmids":["31422115"],"confidence":"Medium","gaps":["Whether Sp1 regulation responds to metabolic cues not addressed","Single lab"]},{"year":2020,"claim":"Establishing LDM and DHCR24 as substrates, in a sterol-independent manner, showed MARCH6 controls multiple cholesterol-synthesis steps beyond SM.","evidence":"siRNA knockdown with substrate stability and sterol-manipulation controls","pmids":["31904814"],"confidence":"Medium","gaps":["Direct ubiquitination of these substrates not shown","Recognition determinants undefined"]},{"year":2020,"claim":"Linking MARCH6-mediated SQLE degradation to endothelial membrane order and barrier function extended the ligase's lipid role to tissue physiology.","evidence":"MARCHF6 ablation and SQLE overexpression with membrane-order, barrier, and angiogenesis assays","pmids":["32755570"],"confidence":"Medium","gaps":["In vivo vascular phenotype not tested","Single lab"]},{"year":2021,"claim":"Degradation of DHX9 connected MARCH6 to AKT/mTOR signaling and proliferation in thyroid cancer, broadening substrates beyond lipid metabolism.","evidence":"Co-IP, MARCH6 overexpression/knockdown, DHX9 knockdown epistasis, and pathway/phenotype readouts","pmids":["34512155"],"confidence":"Medium","gaps":["Direct ubiquitination of DHX9 not rigorously shown","Single lab/cancer context"]},{"year":2022,"claim":"Discovery that NADPH binds and activates MARCHF6 and that the ligase degrades ACSL4 and p53 revealed an allosteric metabolic sensor controlling ferroptosis suppression.","evidence":"NADPH binding and ligase-activity assays, ubiquitination of ACSL4/p53, and knockout mice with ferroptosis-rescue","pmids":["35941365"],"confidence":"High","gaps":["Structural basis of NADPH binding/activation not resolved","Relative contribution of each substrate to ferroptosis phenotype unclear"]},{"year":2023,"claim":"Identification of SC4MOL as a substrate brought the count of MARCH6-controlled cholesterol-synthesis enzymes to at least five, cementing it as a multi-enzyme pathway regulator.","evidence":"siRNA knockdown with substrate stability and sterol-manipulation controls in two cell systems","pmids":["36958722"],"confidence":"Medium","gaps":["Direct ubiquitination of SC4MOL not shown","Single lab"]},{"year":2023,"claim":"Degradation of cytosol-retained POMC by Marchf6 in hypothalamic neurons tied the ligase to ER-stress/ferroptosis protection and systemic energy balance.","evidence":"POMC-Cre conditional knockout mice with ER-stress, ferroptosis, and metabolic phenotyping","pmids":["37421621"],"confidence":"Medium","gaps":["Generalizability to other mislocalized substrates unknown","Single lab"]},{"year":2023,"claim":"The Brucella effector BspA was shown to destabilize MARCH6 and inhibit ERAD, identifying the ligase as a target hijacked during bacterial infection.","evidence":"Co-IP, MARCH6 siRNA, pharmacological ERAD inhibition, and bacterial replication assays in macrophages","pmids":["37129527"],"confidence":"Medium","gaps":["Mechanism by which BspA destabilizes the complex not detailed","Single lab"]},{"year":2024,"claim":"Cryo-EM solved the unique circular channel architecture and, with 95-variant mutagenesis, defined how the gated channel, RING connections, and lipid-binding sites drive substrate degradation.","evidence":"Cryo-EM, AlphaFold modeling, systematic mutagenesis, and SQLE degradation assays","pmids":["38195637"],"confidence":"High","gaps":["Substrate-engaged and ubiquitylation-complex states only modeled, not directly resolved","Mechanism of membrane substrate retrotranslocation not visualized"]},{"year":2024,"claim":"Mapping the Ac/N-degron recognition domain explained how MARCHF6 selectively binds Nα-terminally acetylated substrates like RGS2 and PLIN2, defining a discrete degron-reading module.","evidence":"Alanine-stretch mutagenesis, crosslinking Co-IP, split-ubiquitin assays, and acetylated vs unacetylated binding assays","pmids":["39216628"],"confidence":"High","gaps":["Structural view of the Ac/N domain bound to degron not available","Full set of Ac/N-degron substrates undefined"]},{"year":2025,"claim":"An E3-activity-independent role degrading Tembusu virus NS5 via TOLLIP-mediated selective autophagy revealed a non-canonical, ubiquitin-independent function for MARCH6 as an autophagic adaptor.","evidence":"Co-IP, RING-dead mutant, autophagy inhibitors, and viral replication assays in avian system","pmids":["40511919"],"confidence":"Medium","gaps":["Generalization to mammalian MARCHF6 untested","How a ligase scaffolds autophagy receptors mechanistically unclear"]},{"year":2026,"claim":"Direct ubiquitination of SREBP1 and the hepatic-knockout lipogenesis phenotype established MARCHF6 as a proteostatic brake on de novo lipogenesis relevant to fatty liver disease.","evidence":"Liver-specific knockout mice, Co-IP, ubiquitination and half-life assays, multi-omics, and human MASLD data","pmids":["42173365"],"confidence":"Medium","gaps":["Recognition determinant on SREBP1 undefined","Single lab"]},{"year":2026,"claim":"Ubiquitination-driven degradation of FAM134B in glioma showed MARCH6 actively suppresses ER-phagy, complementing its ERAD role in setting ER homeostasis balance.","evidence":"Co-IP, ubiquitination assays, knockdown epistasis, ER-stress/autophagy markers, and mouse glioma model","pmids":["41661358"],"confidence":"Medium","gaps":["Recognition of FAM134B not mapped","Single lab"]},{"year":null,"claim":"How lipid composition, NADPH, cholesterol, and degron type are integrated by the channel architecture to select among the diverse substrate repertoire, and the structural basis of substrate retrotranslocation, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No substrate-bound structure","Rules governing substrate selection across degron classes undefined","In vivo hierarchy among substrates and tissues unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,15]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,15,22]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[19,24]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[15,9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[20,21]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,1,7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,8,10]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,4,12,16,22]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[15,17,20]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[10,23,21]}],"complexes":[],"partners":["UBE2J2","USP19","SQLE","SREBP1","DHX9","FAM134B","SPP","TRC8"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O60337","full_name":"E3 ubiquitin-protein ligase MARCHF6","aliases":["Doa10 homolog","Membrane-associated RING finger protein 6","Membrane-associated RING-CH protein VI","MARCH-VI","Protein TEB-4","RING finger protein 176","RING-type E3 ubiquitin transferase MARCHF6"],"length_aa":910,"mass_kda":102.5,"function":"Endoplasmic reticulum membrane-associated E3 ubiquitin ligase that plays a critical role in mitigating endoplasmic reticulum stress, the regulation of cholesterol and lipid homeostasis, and ferroptosis (PubMed:25088257, PubMed:35941365, PubMed:39216628). Acts as a pivotal component of both the Ac/N-degron pathway (targeting the N-terminal acetyl group of substrates) and the ER-associated protein degradation-cytosol (ERAD-C) pathway (targeting misfolded substrates) (PubMed:30425097, PubMed:35941365). For instance, mediates the degradation of Ac/N-degron-bearing proteins such as the G-protein regulator RGS2 and the lipid droplet protein PLIN2 (PubMed:39216628). Suppresses endoplasmic reticulum stress and ferroptosis through cytosolic POMC degradation (By similarity). Prevents ferroptosis by acting as a NADPH sensor during lipid peroxidation through its C-terminal regulatory region (PubMed:35941365). Facilitates also the degradation of selected endoplasmic reticulum proteins by associating with signal peptide peptidase for the turnover of endogenous tail-anchored proteins (PubMed:29519897). Promotes ubiquitination of DIO2, leading to its degradation (PubMed:19651899). By ubiquitinating and thereby modulating the stability of many proteins of the cholesterol pathway including SQLE, CYP51A1, CYP11A1 and HMGCR, acts as a crucial post-translational regulator of cholesterol synthesis (PubMed:24449766, PubMed:31904814, PubMed:36958722)","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/O60337/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MARCHF6","classification":"Not Classified","n_dependent_lines":104,"n_total_lines":1090,"dependency_fraction":0.09541284403669725},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CCDC47","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MARCHF6","total_profiled":1310},"omim":[{"mim_id":"620096","title":"RING FINGER PROTEIN 185; RNF185","url":"https://www.omim.org/entry/620096"},{"mim_id":"613608","title":"EPILEPSY, FAMILIAL ADULT MYOCLONIC, 3; FAME3","url":"https://www.omim.org/entry/613608"},{"mim_id":"613297","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 6; MARCHF6","url":"https://www.omim.org/entry/613297"},{"mim_id":"601068","title":"EPILEPSY, FAMILIAL ADULT MYOCLONIC, 1; FAME1","url":"https://www.omim.org/entry/601068"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MARCHF6"},"hgnc":{"alias_symbol":["TEB4","MARCH-VI","RNF176"],"prev_symbol":["MARCH6"]},"alphafold":{"accession":"O60337","domains":[{"cath_id":"-","chopping":"87-174_276-291","consensus_level":"high","plddt":84.3042,"start":87,"end":291},{"cath_id":"-","chopping":"377-504","consensus_level":"high","plddt":88.9866,"start":377,"end":504},{"cath_id":"-","chopping":"512-575_622-700","consensus_level":"medium","plddt":81.3673,"start":512,"end":700},{"cath_id":"-","chopping":"704-851","consensus_level":"high","plddt":89.7783,"start":704,"end":851}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60337","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60337-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60337-F1-predicted_aligned_error_v6.png","plddt_mean":77.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MARCHF6","jax_strain_url":"https://www.jax.org/strain/search?query=MARCHF6"},"sequence":{"accession":"O60337","fasta_url":"https://rest.uniprot.org/uniprotkb/O60337.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60337/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60337"}},"corpus_meta":[{"pmid":"23898401","id":"PMC_23898401","title":"Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4.","date":"2013","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/23898401","citation_count":167,"is_preprint":false},{"pmid":"15673284","id":"PMC_15673284","title":"TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum.","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15673284","citation_count":149,"is_preprint":false},{"pmid":"24449766","id":"PMC_24449766","title":"The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.","date":"2014","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24449766","citation_count":145,"is_preprint":false},{"pmid":"31664039","id":"PMC_31664039","title":"Unstable TTTTA/TTTCA expansions in MARCH6 are associated with Familial Adult Myoclonic 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MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes.","date":"2015","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/26527619","citation_count":38,"is_preprint":false},{"pmid":"27068744","id":"PMC_27068744","title":"A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27068744","citation_count":37,"is_preprint":false},{"pmid":"33049405","id":"PMC_33049405","title":"The E3 ubiquitin ligase MARCHF6 as a metabolic integrator in cholesterol synthesis and beyond.","date":"2020","source":"Biochimica et biophysica acta. Molecular and cell biology of lipids","url":"https://pubmed.ncbi.nlm.nih.gov/33049405","citation_count":28,"is_preprint":false},{"pmid":"31904814","id":"PMC_31904814","title":"The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.","date":"2020","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/31904814","citation_count":27,"is_preprint":false},{"pmid":"25088257","id":"PMC_25088257","title":"Ubiquitin-specific protease 19 regulates the stability of the E3 ubiquitin ligase MARCH6.","date":"2014","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/25088257","citation_count":27,"is_preprint":false},{"pmid":"32755570","id":"PMC_32755570","title":"The MARCH6-SQLE Axis Controls Endothelial Cholesterol Homeostasis and Angiogenic Sprouting.","date":"2020","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32755570","citation_count":23,"is_preprint":false},{"pmid":"34512155","id":"PMC_34512155","title":"MARCH6 promotes Papillary Thyroid Cancer development by destabilizing DHX9.","date":"2021","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34512155","citation_count":21,"is_preprint":false},{"pmid":"37421621","id":"PMC_37421621","title":"Marchf6 E3 ubiquitin ligase critically regulates endoplasmic reticulum stress, ferroptosis, and metabolic homeostasis in POMC neurons.","date":"2023","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/37421621","citation_count":20,"is_preprint":false},{"pmid":"36958722","id":"PMC_36958722","title":"Cholesterol synthesis enzyme SC4MOL is fine-tuned by sterols and targeted for degradation by the E3 ligase MARCHF6.","date":"2023","source":"Journal of lipid research","url":"https://pubmed.ncbi.nlm.nih.gov/36958722","citation_count":18,"is_preprint":false},{"pmid":"38195637","id":"PMC_38195637","title":"Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38195637","citation_count":14,"is_preprint":false},{"pmid":"34273954","id":"PMC_34273954","title":"MARCH6 promotes hepatocellular carcinoma development through up-regulation of ATF2.","date":"2021","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/34273954","citation_count":14,"is_preprint":false},{"pmid":"31422115","id":"PMC_31422115","title":"Consulting prostate cancer cohort data uncovers transcriptional control: Regulation of the MARCH6 gene.","date":"2019","source":"Biochimica et biophysica acta. Molecular and cell biology of lipids","url":"https://pubmed.ncbi.nlm.nih.gov/31422115","citation_count":10,"is_preprint":false},{"pmid":"37129527","id":"PMC_37129527","title":"The Brucella abortus Type IV Effector BspA Inhibits MARCH6-Dependent ERAD To Promote Intracellular Growth.","date":"2023","source":"Infection and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/37129527","citation_count":9,"is_preprint":false},{"pmid":"39216628","id":"PMC_39216628","title":"Delineation of the substrate recognition domain of MARCHF6 E3 ubiquitin ligase in the Ac/N-degron pathway and its regulatory role in ferroptosis.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39216628","citation_count":8,"is_preprint":false},{"pmid":"40511919","id":"PMC_40511919","title":"MARCH6 suppresses Tembusu virus replication by targeting viral NS5 protein for TOLLIP-mediated selective autophagic degradation.","date":"2025","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/40511919","citation_count":4,"is_preprint":false},{"pmid":"38760877","id":"PMC_38760877","title":"A paREDOX in the control of cholesterol biosynthesis: Does the NADPH sensor and E3 ubiquitin ligase MARCHF6 protect mammalian cells during oxidative stress by controlling sterol biosynthesis?","date":"2024","source":"BioEssays : news and reviews in molecular, cellular and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/38760877","citation_count":4,"is_preprint":false},{"pmid":"40445517","id":"PMC_40445517","title":"March6 Protects Against Acute Kidney Injury by Suppressing Renal Tubular Epithelial Cell Ferroptosis Through the Destabilization of P53 and ACSL4 Proteins.","date":"2025","source":"Inflammation","url":"https://pubmed.ncbi.nlm.nih.gov/40445517","citation_count":3,"is_preprint":false},{"pmid":"39306038","id":"PMC_39306038","title":"Development of a monoclonal antibody to study MARCH6, an E3 ligase that regulates proteins that control lipid homeostasis.","date":"2024","source":"Journal of lipid research","url":"https://pubmed.ncbi.nlm.nih.gov/39306038","citation_count":2,"is_preprint":false},{"pmid":"42107511","id":"PMC_42107511","title":"Unraveling MARCH6's role in cancer progression and metabolism from protein homeostasis to oncogenesis.","date":"2026","source":"Pharmacological research","url":"https://pubmed.ncbi.nlm.nih.gov/42107511","citation_count":0,"is_preprint":false},{"pmid":"40643475","id":"PMC_40643475","title":"The Ac/N-Degron Domain of MARCHF6 E3 Ubiquitin Ligase and Its Role in Regulating Ferroptosis.","date":"2025","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/40643475","citation_count":0,"is_preprint":false},{"pmid":"42173365","id":"PMC_42173365","title":"MARCHF6 orchestrates hepatic lipid homeostasis by targeting SREBP1 for ER-associated degradation.","date":"2026","source":"Journal of hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/42173365","citation_count":0,"is_preprint":false},{"pmid":"41268177","id":"PMC_41268177","title":"Novel, complex configurations of the MARCHF6 repeat expansion associated with progressive myoclonic epilepsy and familial adult myoclonic epilepsy.","date":"2025","source":"Brain communications","url":"https://pubmed.ncbi.nlm.nih.gov/41268177","citation_count":0,"is_preprint":false},{"pmid":"41661358","id":"PMC_41661358","title":"MARCH6 Confers Protection Against Endoplasmic Reticulum Autophagy in Gliomas by Destabilizing FAM134B.","date":"2026","source":"Neurochemical research","url":"https://pubmed.ncbi.nlm.nih.gov/41661358","citation_count":0,"is_preprint":false},{"pmid":"40788430","id":"PMC_40788430","title":"First clinical diagnosis of FAME3 via commercial Long-Read sequencing reveals mosaic repeat expansion in MARCHF6 gene.","date":"2025","source":"Neurogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/40788430","citation_count":0,"is_preprint":false},{"pmid":"42155815","id":"PMC_42155815","title":"MARCHF6 regulates ferroptosis in myocardial ischemia-reperfusion injury via the ADAMTS4/SDC-1 pathway.","date":"2026","source":"Biochimica et biophysica acta. Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/42155815","citation_count":0,"is_preprint":false},{"pmid":"40992841","id":"PMC_40992841","title":"Identification of the Ac/N-degron recognition domain in the MARCHF6 E3 ubiquitin ligase.","date":"2025","source":"Methods in enzymology","url":"https://pubmed.ncbi.nlm.nih.gov/40992841","citation_count":0,"is_preprint":false},{"pmid":"42082151","id":"PMC_42082151","title":"Loss of the E3 ubiquitin ligase MARCHF6 alters hepatic lipid metabolism and drives spontaneous hepatosteatosis.","date":"2026","source":"Molecular metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/42082151","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.22.666085","title":"UBE2J2 sensitizes the ERAD ubiquitination cascade to changes in membrane lipid saturation","date":"2025-07-25","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.22.666085","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20626,"output_tokens":7378,"usd":0.086274,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16680,"output_tokens":6464,"usd":0.1225,"stage2_stop_reason":"end_turn"},"total_usd":0.208774,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"Human TEB4 (MARCHF6) is an ER-resident ubiquitin ligase with a C4HC3 RING finger at its N-terminus located in the cytosol. The isolated RING domain catalyzes ubiquitin ligation in vitro in a Lys48-specific manner involving UBC7 as the E2. TEB4 promotes its own proteasomal degradation in a RING finger-dependent manner (autoubiquitination).\",\n      \"method\": \"In vitro ubiquitin ligation assay with isolated RING domain; mutational analysis of RING finger; proteasomal inhibitor experiments; subcellular fractionation/topology analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of E3 activity with defined E2 (UBC7), Lys48 specificity shown, RING mutant controls, multiple orthogonal methods in single study\",\n      \"pmids\": [\"15673284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Yeast Doa10 (ortholog of human TEB4/MARCHF6) contains 14 transmembrane helices with both its N-terminal RING-CH domain and C-terminus facing the cytosol. Biochemical evidence supports a similar topology for human TEB4 (MARCHF6). The yeast Derlins are not required for degradation of Doa10 membrane substrates.\",\n      \"method\": \"Dual-topology reporter fusions at 16 positions in Doa10; protease digestion of yeast microsomes; bioinformatic topology prediction; in silico mutagenesis; topology comparison with human TEB4\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic topology mapping with 16 reporter fusions plus protease protection, replicated across yeast and comparative analysis with human ortholog\",\n      \"pmids\": [\"16373356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TEB4 (MARCHF6) interacts with and mediates ubiquitination and degradation of type 2 iodothyronine deiodinase (D2). TEB4 knockdown decreases D2 ubiquitination and increases D2 activity and protein levels ~4-fold, prolonging D2 half-life. The effect is specific to D2 and requires a critical instability domain in D2; the other deiodinase D1 and a truncated D2 lacking the instability domain are unaffected.\",\n      \"method\": \"Co-immunoprecipitation; TEB4 overexpression (activity assay); siRNA knockdown of TEB4 (>90% reduction); measurement of D2 activity, ubiquitination, and protein levels; lentivirus-based knockdown in D2-expressing MSTO-211 cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, knockdown with multiple readouts (activity, ubiquitination, half-life), substrate specificity tested with controls, replicated in two cell lines\",\n      \"pmids\": [\"19651899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The yeast Doa10 (and its mammalian ortholog Teb4/MARCHF6) mediates sterol-dependent ubiquitination and degradation of squalene monooxygenase (SM), a sterol-specific step in the mevalonate pathway. This constitutes an evolutionarily conserved feedback system for sterol homeostasis, distinct from and complementary to HMGR (Hrd1 branch) regulation.\",\n      \"method\": \"Genetic epistasis in yeast (doa10 deletion); sterol-dependent degradation assays; lipidomics; complementation with mammalian Teb4\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in yeast plus complementation with mammalian ortholog, multiple orthogonal methods, independently corroborated by subsequent mammalian studies\",\n      \"pmids\": [\"23898401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MARCH6 (TEB4) physically interacts with squalene monooxygenase (SM) and acts as the E3 ligase controlling its cholesterol-dependent proteasomal degradation. MARCH6 overexpression reduces SM abundance in a RING-dependent manner; MARCH6 knockdown increases SM protein and activity and prevents its cholesterol-regulated degradation. MARCH6 knockdown also increases HMGCR levels in hepatocytes, establishing MARCH6 as a regulator of both SM and HMGCR.\",\n      \"method\": \"Co-immunoprecipitation; MARCH6 overexpression with RING mutant control; siRNA knockdown; immunoblotting; SM activity assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, RING-domain mutant control, knockdown with multiple substrates tested, independently replicated across multiple labs\",\n      \"pmids\": [\"24449766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP19, an ER-anchored deubiquitinating enzyme, interacts with MARCH6 and stabilizes it by deubiquitination, protecting MARCH6 from p97-dependent proteasomal degradation. USP19 overexpression delays MARCH6 degradation and reduces its ubiquitination; USP19 knockdown decreases MARCH6 levels and increases ubiquitination of MARCH6. Loss of USP19 also increases levels of the ERAD substrate ABCB11, consistent with MARCH6 being the mediating ligase.\",\n      \"method\": \"Co-immunoprecipitation; USP19 overexpression and siRNA knockdown; immunoblotting for MARCH6 ubiquitination and stability; p97 inhibition experiments\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction, gain- and loss-of-function with mechanistic readout (deubiquitination, p97 dependence), single lab\",\n      \"pmids\": [\"25088257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MARCH6 acts as an endogenous inhibitor of the SREBP transcriptional program. Loss of MARCH6 increases SREBP-regulated gene expression (cholesterol biosynthesis and lipoprotein uptake genes) but paradoxically decreases cellular lipoprotein uptake due to enhanced lysosomal LDLR degradation. This is mediated by MARCH6-loss-induced upregulation of the E3 ligase IDOL, which drives LDLR degradation. Thus, MARCH6 uncouples cholesterol synthesis from lipoprotein uptake.\",\n      \"method\": \"Genetic knockdown/knockout of MARCH6; gene expression analysis; lipoprotein uptake assays; IDOL knockdown epistasis experiments\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (MARCH6 loss → IDOL induction → LDLR degradation), multiple pathway readouts, single lab\",\n      \"pmids\": [\"26527619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A conserved C-terminal element (CTE), a 16-residue cytosol-facing motif after the final TM helix of Doa10/MARCH6, is required for degradation of a subset of substrates. Mutation of the conserved asparagine in the MARCH6 CTE (N890A) stabilizes MARCH6 itself to the same degree as a catalytically inactivating RING mutation (C9A), indicating the CTE is required for MARCH6 autoregulation. CRISPR/Cas9 endogenous tagging confirmed MARCH6 localizes to the ER.\",\n      \"method\": \"Alanine/asparagine mutagenesis of CTE; yeast ubiquitylation and degradation assays; MARCH6 autoregulation assays in human cells; CRISPR/Cas9 endogenous epitope tagging for ER localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis at defined residue with multiple substrate readouts, endogenous localization by CRISPR tagging, single lab\",\n      \"pmids\": [\"27068744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MARCH6 and TRC8 are both required for proteasomal degradation of misfolded cytosolic/ER-membrane substrates (mCherry-CL1 reporter); complete stabilization requires double knockout of both E3 ligases. MARCH6 and TRC8 both associate with the intramembrane protease SPP and cooperate to degrade tail-anchored heme oxygenase-1 (HO-1) following intramembrane proteolysis. The two ligases act independently of each other on these substrates.\",\n      \"method\": \"Forward genetic screens in human cells; CRISPR double knockout; quantitative mass spectrometry for protein turnover; Co-immunoprecipitation of SPP association; HO-1 degradation assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — forward genetic screen, double KO, reciprocal Co-IP with SPP, quantitative MS, multiple substrates tested, rigorous controls\",\n      \"pmids\": [\"29519897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cholesterol stabilizes MARCH6 protein by preventing its autodegradation, likely through a conformational change. This stabilization requires functional VCP/p97-dependent membrane extraction and proteasomal degradation, is absent in MARCH6 autodegradation-deficient mutants, and leads to increased degradation of at least three known MARCH6 substrates (SM and others). A putative sterol-sensing domain in MARCH6 is not required for this cholesterol-mediated stabilization.\",\n      \"method\": \"CRISPR/Cas9 gene editing; MARCH6 overexpression; immunoblotting; VCP/p97 inhibition; proteasome inhibition; MARCH6 autodegradation mutants; chemical chaperone treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO, multiple mutant controls, pharmacological dissection of pathway, single lab with orthogonal approaches\",\n      \"pmids\": [\"30545937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MARCH6-dependent ERAD mediates proteasomal degradation of misfolded I1061T NPC1. This pathway acts complementarily with FAM134B-dependent selective ER autophagy (ER-phagy) to regulate I1061T NPC1 turnover.\",\n      \"method\": \"siRNA knockdown of MARCH6; proteasome inhibitor treatment; subcellular fractionation; in vivo mouse tissue analysis; identification of FAM134B-dependent ER-phagy pathway as parallel route\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MARCH6 knockdown with proteasomal readout, complemented by in vivo mouse model, single lab\",\n      \"pmids\": [\"30202070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The transcription factor Sp1 binds to three Sp1 binding sites located ~100 bp downstream of the MARCH6 transcriptional start site and upregulates MARCH6 gene expression. Pharmacological and genetic inhibition of Sp1 reduces MARCH6 expression, which in turn affects stability of its substrate squalene monooxygenase.\",\n      \"method\": \"Luciferase reporter assays; qRT-PCR; pharmacological Sp1 inhibition; siRNA knockdown of Sp1; ChIP-seq data mining; promoter deletion analysis\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular and cell biology of lipids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase reporter, genetic and pharmacological inhibition of Sp1, functional downstream readout (SM stability), single lab\",\n      \"pmids\": [\"31422115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MARCH6 controls levels of lanosterol 14α-demethylase (LDM) and 24-dehydrocholesterol reductase (DHCR24) by promoting their degradation; this degradation is not triggered by sterols. MARCH6 thereby targets multiple steps in the cholesterol synthesis pathway, representing the first E3 ligase known to control multiple enzymes in a single biochemical pathway.\",\n      \"method\": \"siRNA knockdown of MARCH6; immunoblotting for LDM and DHCR24 stability; sterol manipulation experiments; cell-based degradation assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with multiple substrate readouts and sterol treatment controls, single lab\",\n      \"pmids\": [\"31904814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Ablation of MARCHF6 in endothelial cells increases SQLE protein and cholesterol load, leading to altered membrane order, disorganized VE-cadherin-based adherens junctions, decreased endothelial barrier function, and impaired SQLE-dependent sprouting angiogenesis. This positions MARCH6-mediated SQLE degradation as a determinant of endothelial integrity.\",\n      \"method\": \"siRNA/CRISPR-mediated MARCHF6 ablation in endothelial cells; SQLE overexpression; cholesterol measurement; membrane order assays; barrier function assays; sprouting angiogenesis assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with defined cellular phenotypes, SQLE overexpression phenocopy, single lab\",\n      \"pmids\": [\"32755570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MARCH6 interacts with and promotes degradation (destabilization) of DHX9 in thyroid cancer cells. Mechanistically, MARCH6-mediated DHX9 destabilization activates the AKT/mTOR signaling pathway. DHX9 knockdown phenocopies MARCH6 overexpression in promoting proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation; MARCH6 overexpression and knockdown; DHX9 knockdown; AKT/mTOR pathway immunoblotting; cell proliferation and migration assays\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction, epistasis via DHX9 knockdown rescue, downstream pathway readout, single lab\",\n      \"pmids\": [\"34512155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCHF6 recognizes NADPH through its C-terminal regulatory region; this NADPH binding upregulates MARCHF6 E3 ligase activity. MARCHF6 mediates ubiquitin-dependent degradation of the pro-ferroptotic effectors ACSL4 and p53. Loss of MARCHF6 increases ferroptosis sensitivity; inhibiting ferroptosis rescues growth of MARCHF6-deficient tumors and perinatal lethality of Marchf6-/- mice.\",\n      \"method\": \"NADPH binding assays; MARCHF6 ligase activity assays; ubiquitination assays for ACSL4 and p53; MARCHF6 knockout mice; tumor growth rescue experiments; ferroptosis inhibitor treatment\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — biochemical NADPH binding, in vitro/in vivo E3 activity, knockout mouse model with rescue, multiple substrates identified with orthogonal methods\",\n      \"pmids\": [\"35941365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SC4MOL (the first acting enzyme of the C4-demethylation complex in cholesterol synthesis) is a substrate of MARCHF6; SC4MOL is rapidly turned over and sensitive to sterols. MARCHF6 thereby controls at least five enzymes in the cholesterol synthesis pathway.\",\n      \"method\": \"siRNA knockdown of MARCHF6; sterol depletion/loading experiments; immunoblotting for SC4MOL stability; CHO and human cell lines; cholesterol measurement after SC4MOL siRNA\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with substrate stabilization readout, sterol manipulation controls, two cell line systems, single lab\",\n      \"pmids\": [\"36958722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In POMC neurons, Marchf6 mediates degradation of cytosol-retained (signal peptide-uncleaved) POMC, preventing ER stress and ferroptosis. Loss of MARCHF6 in POMC neurons (POMC-Cre Marchf6-deficient mice) causes hyperphagia, reduced energy expenditure, and weight gain.\",\n      \"method\": \"POMC-Cre conditional Marchf6 knockout mice; ferroptosis and ER stress marker analysis; cell viability assays; chaperone sequestration experiments; ubiquitination assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO mouse model with defined metabolic phenotype, substrate identification, multiple mechanistic readouts, single lab\",\n      \"pmids\": [\"37421621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Brucella abortus T4SS effector BspA interacts with MARCH6 and destabilizes the MARCH6 E3 ligase complex, thereby inhibiting MARCH6-dependent ERAD. This inhibition promotes intracellular B. abortus proliferation; pharmacological ERAD inhibition or siRNA depletion of MARCH6 phenocopy BspA deletion and rescue the replication defect of a bspA mutant.\",\n      \"method\": \"Co-immunoprecipitation of BspA-MARCH6; siRNA knockdown of MARCH6; pharmacological ERAD inhibition; bacterial replication assays in macrophages; epistasis with UbxD8 depletion\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, genetic epistasis by siRNA, pharmacological corroboration, defined replication phenotype, single lab\",\n      \"pmids\": [\"37129527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Doa10/MARCHF6 adopts a unique circular architecture within the ER membrane, with the majority of the protein forming a lipid-binding scaffold gated by a flexible helical bundle. The RING domain ubiquitylation active site is positioned over this channel via connections with the membrane-spanning scaffold and gate. Structure-based mutagenesis of 95 MARCH6 variants revealed that SQLE degradation depends on the gated channel, RING domain connections, and lipid-binding sites. AlphaFold models are consistent with substrate-engaged and ubiquitylation complex states.\",\n      \"method\": \"Cryo-EM structural analysis; AlphaFold modeling; systematic mutagenesis of 95 variants; SQLE degradation assay; lipid-binding analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with AlphaFold validation, 95-variant mutagenesis campaign, functional substrate degradation assay, multiple orthogonal approaches in one study\",\n      \"pmids\": [\"38195637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The Ac/N-degron recognition domain (Ac/N domain) of MARCHF6 was mapped to specific cytosol-facing regions using alanine-stretch mutagenesis. This domain exhibits preferential binding to Nα-terminally acetylated proteins/peptides over unacetylated counterparts. MARCHF6 mediates degradation of Ac/N-degron-bearing substrates including RGS2 and PLIN2, and abolishing Ac/N-degron recognition stabilizes these substrates and increases ferroptosis resistance.\",\n      \"method\": \"Alanine-stretch mutagenesis; chemical crosslinking-based Co-IP; split-ubiquitin assays (human and yeast cells); Ac/N-degron substrate binding assays with acetylated vs. unacetylated peptides; RGS2 and PLIN2 stability assays; ferroptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis, biochemical binding specificity with acetylated vs. unacetylated substrates, split-ubiquitin functional validation, multiple substrates, single lab\",\n      \"pmids\": [\"39216628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Avian MARCH6 directly interacts with Tembusu virus NS5 protein and promotes its degradation via selective autophagy through an E3 ligase activity-independent mechanism. MARCH6 recruits the autophagic cargo receptor TOLLIP, which facilitates NS5-TOLLIP interaction independent of ubiquitin signaling, directing NS5 to phagophores for degradation.\",\n      \"method\": \"Co-immunoprecipitation of MARCH6-NS5 and NS5-TOLLIP; MARCH6 overexpression and knockdown; RING-dead MARCH6 mutant; autophagy inhibitors; viral replication assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, RING-dead mutant establishes E3-independence, knockdown/overexpression with viral replication readout, single lab (avian system)\",\n      \"pmids\": [\"40511919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MARCHF6 directly interacts with and ubiquitinates SREBP1, targeting it for proteasomal degradation. Loss of hepatic MARCHF6 prolongs SREBP1 half-life, driving excessive de novo lipogenesis. Liver-specific Marchf6 knockout mice develop spontaneous hepatic triglyceride and cholesteryl ester accumulation under normal chow.\",\n      \"method\": \"Liver-specific Marchf6 knockout (Marchf6Alb) mice; Co-IP; ubiquitination assay; SREBP1 half-life measurement; transcriptomics and proteomics; lipidomics; human MASLD patient data\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitination assay, conditional KO mouse with metabolic phenotype, corroborated by human patient expression data, single lab\",\n      \"pmids\": [\"42173365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MARCH6 induces FAM134B protein ubiquitination and degradation, reducing FAM134B stability in glioma cells. This suppresses ER-phagy and ER stress responses. Knockdown of MARCH6 reverses FAM134B degradation and restores ER-phagy markers (LC3B conversion, autophagosome accumulation).\",\n      \"method\": \"Co-immunoprecipitation (MARCH6-FAM134B interaction); ubiquitination assays; siRNA knockdown of FAM134B and MARCH6; ER stress and autophagy marker analysis; mouse glioma model\",\n      \"journal\": \"Neurochemical research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP, ubiquitination assay, knockdown epistasis with in vivo validation, single lab\",\n      \"pmids\": [\"41661358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The membrane-anchored E2 UBE2J2 cooperates with MARCHF6 (and other E3 ligases RNF145, RNF139) to ubiquitinate both themselves and the substrate squalene monooxygenase. UBE2J2 activity is modulated by membrane lipid packing, with loosely packed membranes impairing ubiquitin loading onto UBE2J2, thereby relaying lipid signals to MARCHF6-dependent ubiquitination.\",\n      \"method\": \"Reconstituted systems with purified ERAD factors; in vitro ubiquitination assays; membrane composition manipulation; E2-E3 interaction assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with purified components, but preprint not yet peer-reviewed, single study\",\n      \"pmids\": [\"bio_10.1101_2025.07.22.666085\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCH6 promotes ubiquitin-mediated degradation of ADAMTS4 in cardiomyocytes, thereby upregulating the downstream target SDC-1. This MARCHF6/ADAMTS4/SDC-1 axis inhibits lipid peroxidation and ferroptosis in myocardial ischemia-reperfusion injury.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assays; MARCHF6 overexpression (AAV9 in mice); IRI mouse model; OGD/R cell model; ferroptosis markers\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and overexpression, single lab, no direct ubiquitination of ADAMTS4 rigorously demonstrated in abstract\",\n      \"pmids\": [\"42155815\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MARCHF6 (TEB4/MARCH6) is a multi-pass ER-resident RING-CH E3 ubiquitin ligase that adopts a unique circular channel architecture within the membrane; it catalyzes Lys48-linked ubiquitination using UBE2J2/UBC7 as its cognate E2, targets multiple substrates for proteasomal degradation including squalene monooxygenase (SQLE), HMGCR, lanosterol 14α-demethylase, SC4MOL, DHCR24, SREBP1, type 2 deiodinase (D2), HO-1, ACSL4, p53, RGS2, PLIN2, DHX9, and FAM134B, and is itself regulated by cholesterol (which prevents its autodegradation), by NADPH (which binds its C-terminal region and activates its ligase activity), by the deubiquitinase USP19 (which stabilizes it), and transcriptionally by Sp1; its Ac/N-degron recognition domain in cytosol-facing loops preferentially binds Nα-terminally acetylated substrates, while other substrates are recognized via distinct sites including the conserved C-terminal element (CTE).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MARCHF6 (TEB4/MARCH6) is a multi-pass ER-resident RING-CH E3 ubiquitin ligase that orchestrates protein quality control and lipid homeostasis by targeting membrane and cytosolic substrates for proteasomal degradation [#0, #3]. Its N-terminal C4HC3 RING domain faces the cytosol and catalyzes Lys48-linked ubiquitination in vitro using UBC7/UBE2J2 as the cognate E2, and the enzyme constitutively autoubiquitinates to drive its own turnover [#0, #1]. Cryo-EM revealed that the protein folds into a unique circular, lipid-binding scaffold gated by a flexible helical bundle, with the RING active site positioned over the channel; substrate degradation depends on this gated channel and on RING-domain connections to the membrane scaffold [#19]. A dominant function is feedback control of cholesterol synthesis: MARCHF6 degrades multiple sequential mevalonate-pathway enzymes including squalene monooxygenase, HMGCR, lanosterol 14\\u03b1-demethylase, DHCR24, and SC4MOL, and acts as an endogenous brake on the SREBP program by ubiquitinating SREBP1 [#3, #4, #12, #16, #22]. Substrate recognition occurs through distinct cytosol-facing determinants, including an Ac/N-degron domain that preferentially binds N\\u03b1-terminally acetylated substrates such as RGS2 and PLIN2, and a conserved C-terminal element (CTE) required for a subset of substrates and for MARCHF6 autoregulation [#7, #20]. The ligase additionally degrades pro-ferroptotic effectors ACSL4 and p53, and its activity is allosterically upregulated by NADPH binding to its C-terminal region, linking it to suppression of ferroptosis in vivo [#15]. MARCHF6 abundance is itself tightly regulated\\u2014stabilized by cholesterol (which blocks autodegradation) and by the deubiquitinase USP19, and transcriptionally driven by Sp1 [#9, #5, #11]. Through these activities MARCHF6 governs endothelial barrier integrity, hepatic lipogenesis, energy balance via POMC neurons, and ERAD versus ER-phagy substrate routing [#13, #22, #17, #10, #23].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing that human TEB4 is a bona fide E3 ligase defined the molecular activity at the heart of the gene and its E2 partner and linkage specificity.\",\n      \"evidence\": \"In vitro ubiquitin ligation with isolated RING domain, RING mutant controls, and topology/fractionation analysis\",\n      \"pmids\": [\"15673284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate repertoire unknown at this stage\", \"Full-length enzymatic activity in membrane context not addressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Topology mapping of the Doa10 ortholog answered how the ligase is arranged in the ER membrane, placing both RING and C-terminus in the cytosol where they can engage E2 and substrates.\",\n      \"evidence\": \"Dual-topology reporter fusions and protease protection in yeast microsomes with comparison to human TEB4\",\n      \"pmids\": [\"16373356\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of substrate extraction across the membrane not defined\", \"No high-resolution structure\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of type 2 deiodinase as a substrate provided the first defined endogenous target, showing the ligase controls hormone-activating enzyme levels.\",\n      \"evidence\": \"Reciprocal Co-IP and siRNA knockdown with ubiquitination, activity, and half-life readouts in two cell lines\",\n      \"pmids\": [\"19651899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recognition determinant on D2 only coarsely mapped to an instability domain\", \"Physiological setting of D2 regulation by TEB4 not tested in vivo\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrating sterol-dependent degradation of squalene monooxygenase established MARCHF6 as an evolutionarily conserved node of cholesterol feedback regulation.\",\n      \"evidence\": \"Yeast genetic epistasis with mammalian Teb4 complementation and lipidomics\",\n      \"pmids\": [\"23898401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct E3-substrate engagement in mammalian cells shown subsequently\", \"Sterol sensor identity unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Direct demonstration that MARCH6 binds and degrades SM and also affects HMGCR positioned it as a regulator of multiple mevalonate-pathway enzymes.\",\n      \"evidence\": \"Reciprocal Co-IP, RING-mutant controls, knockdown with SM activity and HMGCR readouts\",\n      \"pmids\": [\"24449766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HMGCR is a direct MARCH6 substrate or indirect not resolved here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that USP19 deubiquitinates and stabilizes MARCH6 revealed how the ligase's own abundance is post-translationally tuned against p97-driven turnover.\",\n      \"evidence\": \"Co-IP plus USP19 gain/loss of function with MARCH6 ubiquitination, stability, and p97 dependence readouts\",\n      \"pmids\": [\"25088257\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Stoichiometry and regulation of the USP19-MARCH6 interaction unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showing MARCH6 loss induces IDOL and LDLR degradation explained how the ligase uncouples cholesterol synthesis from lipoprotein uptake at the systems level.\",\n      \"evidence\": \"MARCH6 knockdown/knockout with gene expression, uptake assays, and IDOL epistasis\",\n      \"pmids\": [\"26527619\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking MARCH6 loss to IDOL induction not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mapping the conserved C-terminal element defined a substrate-recognition/autoregulation determinant distinct from the RING and confirmed endogenous ER localization.\",\n      \"evidence\": \"CTE mutagenesis (N890A) with yeast and human degradation assays plus CRISPR endogenous tagging\",\n      \"pmids\": [\"27068744\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of CTE function unresolved at this stage\", \"Which substrates require CTE not exhaustively mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Cooperation of MARCH6 with TRC8 and the protease SPP showed the ligase participates in degrading misfolded and intramembrane-cleaved substrates such as HO-1.\",\n      \"evidence\": \"Forward genetic screens, CRISPR double knockout, SPP Co-IP, and quantitative MS\",\n      \"pmids\": [\"29519897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Division of labor between MARCH6 and TRC8 per substrate not fully delineated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating cholesterol stabilizes MARCH6 by blocking autodegradation established a direct feedback loop coupling sterol levels to ligase abundance and substrate flux.\",\n      \"evidence\": \"CRISPR editing, autodegradation mutants, and VCP/p97 and proteasome inhibition\",\n      \"pmids\": [\"30545937\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Sterol-sensing domain ruled out but the actual sensing mechanism unidentified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identifying MARCH6-dependent degradation of misfolded NPC1 positioned the ligase within ERAD acting in parallel to FAM134B-mediated ER-phagy.\",\n      \"evidence\": \"siRNA knockdown with proteasomal readouts and in vivo mouse tissue analysis\",\n      \"pmids\": [\"30202070\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct interaction of MARCH6 with NPC1 not shown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Sp1 was identified as a transcriptional activator of MARCH6, defining how ligase levels are set at the gene-expression layer with downstream effects on SM stability.\",\n      \"evidence\": \"Luciferase reporters, promoter deletion, Sp1 inhibition/knockdown, and ChIP-seq mining\",\n      \"pmids\": [\"31422115\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Sp1 regulation responds to metabolic cues not addressed\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Establishing LDM and DHCR24 as substrates, in a sterol-independent manner, showed MARCH6 controls multiple cholesterol-synthesis steps beyond SM.\",\n      \"evidence\": \"siRNA knockdown with substrate stability and sterol-manipulation controls\",\n      \"pmids\": [\"31904814\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of these substrates not shown\", \"Recognition determinants undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linking MARCH6-mediated SQLE degradation to endothelial membrane order and barrier function extended the ligase's lipid role to tissue physiology.\",\n      \"evidence\": \"MARCHF6 ablation and SQLE overexpression with membrane-order, barrier, and angiogenesis assays\",\n      \"pmids\": [\"32755570\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo vascular phenotype not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Degradation of DHX9 connected MARCH6 to AKT/mTOR signaling and proliferation in thyroid cancer, broadening substrates beyond lipid metabolism.\",\n      \"evidence\": \"Co-IP, MARCH6 overexpression/knockdown, DHX9 knockdown epistasis, and pathway/phenotype readouts\",\n      \"pmids\": [\"34512155\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of DHX9 not rigorously shown\", \"Single lab/cancer context\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery that NADPH binds and activates MARCHF6 and that the ligase degrades ACSL4 and p53 revealed an allosteric metabolic sensor controlling ferroptosis suppression.\",\n      \"evidence\": \"NADPH binding and ligase-activity assays, ubiquitination of ACSL4/p53, and knockout mice with ferroptosis-rescue\",\n      \"pmids\": [\"35941365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of NADPH binding/activation not resolved\", \"Relative contribution of each substrate to ferroptosis phenotype unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of SC4MOL as a substrate brought the count of MARCH6-controlled cholesterol-synthesis enzymes to at least five, cementing it as a multi-enzyme pathway regulator.\",\n      \"evidence\": \"siRNA knockdown with substrate stability and sterol-manipulation controls in two cell systems\",\n      \"pmids\": [\"36958722\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of SC4MOL not shown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Degradation of cytosol-retained POMC by Marchf6 in hypothalamic neurons tied the ligase to ER-stress/ferroptosis protection and systemic energy balance.\",\n      \"evidence\": \"POMC-Cre conditional knockout mice with ER-stress, ferroptosis, and metabolic phenotyping\",\n      \"pmids\": [\"37421621\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generalizability to other mislocalized substrates unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The Brucella effector BspA was shown to destabilize MARCH6 and inhibit ERAD, identifying the ligase as a target hijacked during bacterial infection.\",\n      \"evidence\": \"Co-IP, MARCH6 siRNA, pharmacological ERAD inhibition, and bacterial replication assays in macrophages\",\n      \"pmids\": [\"37129527\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which BspA destabilizes the complex not detailed\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cryo-EM solved the unique circular channel architecture and, with 95-variant mutagenesis, defined how the gated channel, RING connections, and lipid-binding sites drive substrate degradation.\",\n      \"evidence\": \"Cryo-EM, AlphaFold modeling, systematic mutagenesis, and SQLE degradation assays\",\n      \"pmids\": [\"38195637\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate-engaged and ubiquitylation-complex states only modeled, not directly resolved\", \"Mechanism of membrane substrate retrotranslocation not visualized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mapping the Ac/N-degron recognition domain explained how MARCHF6 selectively binds N\\u03b1-terminally acetylated substrates like RGS2 and PLIN2, defining a discrete degron-reading module.\",\n      \"evidence\": \"Alanine-stretch mutagenesis, crosslinking Co-IP, split-ubiquitin assays, and acetylated vs unacetylated binding assays\",\n      \"pmids\": [\"39216628\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural view of the Ac/N domain bound to degron not available\", \"Full set of Ac/N-degron substrates undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"An E3-activity-independent role degrading Tembusu virus NS5 via TOLLIP-mediated selective autophagy revealed a non-canonical, ubiquitin-independent function for MARCH6 as an autophagic adaptor.\",\n      \"evidence\": \"Co-IP, RING-dead mutant, autophagy inhibitors, and viral replication assays in avian system\",\n      \"pmids\": [\"40511919\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generalization to mammalian MARCHF6 untested\", \"How a ligase scaffolds autophagy receptors mechanistically unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Direct ubiquitination of SREBP1 and the hepatic-knockout lipogenesis phenotype established MARCHF6 as a proteostatic brake on de novo lipogenesis relevant to fatty liver disease.\",\n      \"evidence\": \"Liver-specific knockout mice, Co-IP, ubiquitination and half-life assays, multi-omics, and human MASLD data\",\n      \"pmids\": [\"42173365\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Recognition determinant on SREBP1 undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Ubiquitination-driven degradation of FAM134B in glioma showed MARCH6 actively suppresses ER-phagy, complementing its ERAD role in setting ER homeostasis balance.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, knockdown epistasis, ER-stress/autophagy markers, and mouse glioma model\",\n      \"pmids\": [\"41661358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Recognition of FAM134B not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How lipid composition, NADPH, cholesterol, and degron type are integrated by the channel architecture to select among the diverse substrate repertoire, and the structural basis of substrate retrotranslocation, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrate-bound structure\", \"Rules governing substrate selection across degron classes undefined\", \"In vivo hierarchy among substrates and tissues unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 15]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 15, 22]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [19, 24]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [15, 9]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [20, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0005789\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 8, 10]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 4, 12, 16, 22]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [15, 17, 20]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10, 23, 21]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UBE2J2\", \"USP19\", \"SQLE\", \"SREBP1\", \"DHX9\", \"FAM134B\", \"SPP\", \"TRC8\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}