{"gene":"MAL","run_date":"2026-06-13T19:06:35","timeline":{"discoveries":[{"year":1987,"finding":"MAL encodes a highly hydrophobic proteolipid protein with four putative transmembrane domains, initially identified as expressed in intermediate and late stages of T-cell differentiation, with predicted configuration resembling integral membrane proteins that form pores or channels.","method":"cDNA cloning, sequence analysis, hydrophobicity profiling","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — original cloning and structural prediction from single lab, no functional validation of channel/transporter activity","pmids":["3494249"],"is_preprint":false},{"year":1994,"finding":"MAL protein is a proteolipid (soluble in organic lipid-extraction solvents), is encoded by a 4-exon gene where each exon corresponds to one hydrophobic segment, and localizes to the endoplasmic reticulum of T cells. A C-terminal RWKSS motif consistent with ER retention signals was identified.","method":"Genomic cloning, RNase protection, recombinant expression in bacteria with lipophilic solvent extraction, immunofluorescence with two distinct anti-peptide antibodies","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical demonstration of proteolipid properties plus subcellular localization, single lab with orthogonal methods","pmids":["8132541"],"is_preprint":false},{"year":1995,"finding":"MAL (MVP17) is a developmentally regulated proteolipid in oligodendrocytes that co-purifies with detergent-insoluble, glycolipid-rich membrane microdomains, consistent with a role in myelin biogenesis.","method":"Detergent-insoluble membrane fractionation, 2D gel electrophoresis, N-terminal microsequencing, cDNA cloning from oligodendrocyte library, Northern blot","journal":"Journal of neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical fractionation plus cloning, single lab, multiple orthogonal methods","pmids":["8583510"],"is_preprint":false},{"year":1995,"finding":"VIP17/MAL is a proteolipid component of apical transport vesicles in MDCK cells, localizing to perinuclear vesicles and the apical cytoplasm, cycling between the Golgi and the apical plasma membrane.","method":"cDNA cloning, immunofluorescence of epitope-tagged VIP17/MAL in BHK and MDCK cells","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional context in polarized cells, single lab","pmids":["8549777"],"is_preprint":false},{"year":1997,"finding":"MAL proteolipid is exclusively incorporated into detergent-resistant, buoyant membrane microdomains (lipid rafts) in both T lymphocytes (Jurkat) and epithelial cells, co-fractionating with p56lck, CD59, and GM1.","method":"Stable transfection of epitope-tagged MAL, Triton X-100 extraction, sucrose gradient fractionation, immunofluorescence","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical fractionation in two cell types, single lab","pmids":["9003426"],"is_preprint":false},{"year":1997,"finding":"MAL and caveolin occupy distinct lipid microenvironments within MDCK cells; MAL is in internal glycolipid-enriched detergent-insoluble membranes while caveolin is additionally found at the cell surface, demonstrating heterogeneity within raft fractions.","method":"Sucrose gradient fractionation, immunofluorescence, differential detergent solubilization","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical fractionation with multiple cell lines, single lab","pmids":["9168919"],"is_preprint":false},{"year":1998,"finding":"A C-terminal juxtamembrane tetrapeptide motif LIRW in MAL is necessary for incorporation into glycolipid-enriched membrane (GEM) microdomains; arginine is the most critical residue. Loss of GEM targeting correlates with loss of MAL's response to brefeldin A treatment.","method":"Site-directed mutagenesis, sucrose gradient fractionation of GEMs, pulse-chase experiments, brefeldin A treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with functional readout (GEM incorporation) in single lab with multiple orthogonal assays","pmids":["9582298"],"is_preprint":false},{"year":1998,"finding":"MAL is expressed specifically in thyroid follicular epithelial cells, localizing to the apical zone, and is a major component of GEM fractions in the polarized thyroid epithelial cell line FRT, consistent with a role in apical sorting in thyroid epithelium.","method":"Northern blot, immunohistochemistry with anti-MAL monoclonal antibody, GEM fractionation of FRT cells","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments combined with biochemical fractionation, single lab","pmids":["9528996"],"is_preprint":false},{"year":1999,"finding":"MAL depletion by antisense oligonucleotides in MDCK cells reduces the presence of influenza hemagglutinin (HA) in GEMs, decreases the rate of HA transport to the cell surface, inhibits apical delivery, and causes partial missorting of HA to the basolateral membrane. Ectopic MAL expression rescues these defects, demonstrating that MAL is necessary for both normal apical transport and accurate sorting of HA.","method":"Antisense oligonucleotides, newly generated anti-canine MAL monoclonal antibody, surface biotinylation, apical/basolateral sorting assay, ectopic MAL rescue","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — loss-of-function plus rescue with specific phenotypic readout, single lab with multiple orthogonal methods","pmids":["10189374"],"is_preprint":false},{"year":1999,"finding":"Overexpression of VIP17/MAL in MDCK cells increases apical delivery and expands the apical surface, while antisense-mediated reduction causes accumulation in the Golgi and impairs apical transport of multiple apical markers (HA, clusterin, gp114, GPI-anchored protein) without affecting basolateral E-cadherin distribution.","method":"Antisense RNA expression, overexpression, immunofluorescence, transport assays for multiple apical cargo proteins","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — bidirectional manipulation (OE and KD) with specific apical vs. basolateral cargo controls","pmids":["10339572"],"is_preprint":false},{"year":1999,"finding":"MAL is an itinerant protein that cycles between the trans-Golgi network and the plasma membrane: it is expressed at the cell surface, rapidly internalized via an endosomal pathway requiring endosomal acidification, and ~30% of internalized MAL is delivered back to the TGN to restart the transport cycle.","method":"Epitope-tagged and O-glycosylatable/biotinylatable MAL constructs, surface biotinylation, anti-FLAG surface binding, neuraminidase sensitivity assay, resialylation experiment, drug treatments (chloroquine, monensin, NH4Cl), flow cytometry","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple engineered constructs and orthogonal biochemical assays establishing cycling behavior, single lab","pmids":["10512878"],"is_preprint":false},{"year":2001,"finding":"MAL (MyD88-adapter-like) is a TIR-domain-containing cytoplasmic adapter protein that activates NF-κB, JNK, and ERK1/2; forms homodimers and heterodimers with MyD88; associates with TLR4 and with IRAK-2 via its TIR domain; and acts as an adapter specifically in TLR-4 signal transduction. A dominant-negative form of Mal inhibits NF-κB activated by TLR-4 or LPS but not by IL-1RI or IL-18R.","method":"Co-immunoprecipitation, overexpression, dominant-negative constructs, NF-κB/JNK/ERK reporter assays, two-hybrid interaction assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, dominant-negative functional assays, replicated across multiple signaling readouts; landmark discovery paper","pmids":["11544529"],"is_preprint":false},{"year":2003,"finding":"Purified TIR domains of MAL and MyD88 form stable heterodimers in vitro; MAL homodimers/oligomers are dissociated by ATP. Structural modeling and GST pull-down/co-IP experiments indicate MAL and MyD88 bind to different, non-overlapping surfaces on TLR2 and TLR4 TIR domains, suggesting a heterotetrameric receptor-adapter complex. A sequence relationship between Drosophila Tube and MAL was identified.","method":"In vitro TIR domain purification, gel filtration, GST pull-down, co-immunoprecipitation, computational structural modeling and docking","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of heterodimer plus orthogonal GST pulldown and co-IP, combined with structural modeling","pmids":["12888566"],"is_preprint":false},{"year":2004,"finding":"Genetic ablation of MAL in mice results in cytoplasmic inclusions in compact myelin, everted paranodal loops, disorganized transverse bands, and reduced levels of contactin-associated protein/paranodin, NF155, Kv1.2, MAG, and MBP in myelin and myelin-derived rafts, demonstrating MAL is required for maintenance of CNS paranode structure, likely by controlling NF155 trafficking/sorting in oligodendrocytes.","method":"MAL knockout mice, electron microscopy, immunofluorescence, Western blotting of myelin fractions and raft fractions","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with multiple phenotypic readouts and biochemical characterization of myelin protein composition","pmids":["15337780"],"is_preprint":false},{"year":2006,"finding":"MAL undergoes tyrosine phosphorylation at positions 86, 187 (and 106) during TLR2 and TLR4 signaling. Bruton's tyrosine kinase (Btk) is identified as the kinase: Btk immunoprecipitated from LPS-activated THP-1 cells directly phosphorylates MAL in vitro, and the Btk inhibitor LFM-A13 blocks endogenous MAL tyrosine phosphorylation. Tyrosine-to-phenylalanine mutations at positions 86 and 187 act as dominant-negative inhibitors of LPS-induced NF-κB activation.","method":"Phosphotyrosine immunoprecipitation, site-directed mutagenesis (Y86F, Y187F), Btk kinase inhibitor (LFM-A13), in vitro kinase assay with immunoprecipitated Btk, THP-1 cell stimulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus pharmacological inhibition, multiple orthogonal methods in single lab","pmids":["16439361"],"is_preprint":false},{"year":2006,"finding":"MAL interacts with SRF through a conserved seven-residue B1 region sequence that is essential and sufficient for complex formation; the neighboring Q-box facilitates interaction. MAL directly contacts DNA flanking the SRF-protected region in the MAL-SRF-DNA complex, and these contacts are required for effective MAL-SRF complex formation. SRF-induced DNA bending facilitates MAL-DNA contact. MAL-SRF and TCF-SRF use different mechanisms to engage the same SRF hydrophobic groove/pocket.","method":"Deletion and point mutagenesis of MAL B1/Q-box regions, GST pull-downs, gel mobility shift assays, DNase I footprinting, SRF mutagenesis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with in vitro binding assays and DNase I footprinting, multiple orthogonal methods","pmids":["16705166"],"is_preprint":false},{"year":2006,"finding":"MAL/MKL1 (SRF co-activator) is expressed in developing neurons, localizes to neuronal cell bodies and apical dendrites, and is required for dendritic morphology: dominant-negative MAL constructs or MAL siRNA reduce the number of dendritic processes in cultured cortical neurons and decrease basal SRF-mediated transcription.","method":"Immunohistochemistry of mouse brain, dominant-negative construct expression, siRNA knockdown, dendritic morphology analysis, SRF reporter assay in primary cortical neurons","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific morphological readout, single lab","pmids":["16945101"],"is_preprint":false},{"year":2009,"finding":"MAL directly interacts with the p85α regulatory subunit of PI3K in an inducible manner upon TLR2/6 stimulation with diacylated lipoproteins, driving PI3K-dependent Akt phosphorylation, PIP3 generation, and macrophage polarization independently of MyD88.","method":"Co-immunoprecipitation (inducible Mal-p85α interaction), PI3K activity assay, Akt phosphorylation assay, PIP3 measurement, MyD88-deficient cell analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP showing inducible interaction, functional assays with MyD88-independent pathway, multiple readouts","pmids":["19574958"],"is_preprint":false},{"year":2009,"finding":"Actin-MAL-SRF signaling induces Mig6/Errfi-1 expression, a negative regulator of EGFR. MAL is inducibly recruited to and activates the mig6 promoter. Upregulation of Mig6 correlates with decreased EGFR, MAPK/Erk, and c-fos activation; overexpression of MAL has antiproliferative effects requiring domains for SRF binding and transactivation.","method":"Gene expression profiling, chromatin immunoprecipitation (ChIP) of MAL at mig6 promoter, MAL overexpression/siRNA knockdown, EGFR/MAPK activation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP demonstrating direct promoter recruitment plus functional downstream signaling assays, multiple orthogonal methods","pmids":["19683494"],"is_preprint":false},{"year":2009,"finding":"MAL clusters laterally concentrate sphingolipid raft markers and exclude phosphatidylethanolamine analogues. MAL forms oligomers via intramembrane protein-protein binding motifs (demonstrated by site-directed mutagenesis and bimolecular fluorescence complementation). MAL-raft association is driven in part by positive hydrophobic mismatch between MAL transmembrane helices and membrane lipids.","method":"Antibody-mediated cross-linking of FLAG-tagged MAL, tandem dimer DiHcRED-MAL spontaneous clustering, site-directed mutagenesis of intramembrane motifs, bimolecular fluorescence complementation (BiFC), exogenous cholesterol/ceramide membrane modulation","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis plus BiFC plus multiple independent clustering approaches demonstrating mechanism of raft organization","pmids":["19553470"],"is_preprint":false},{"year":2010,"finding":"IRAK1 and IRAK4 directly phosphorylate MAL (but not MyD88). Co-expression of Mal with either IRAK causes depletion of Mal from cell lysates; LPS stimulation triggers ubiquitination and degradation of Mal; this is inhibited by IRAK1/4 kinase inhibitor or IRAK1/4 knockdown. Phosphorylation by IRAKs thus promotes ubiquitination and degradation of Mal, serving as a negative regulatory mechanism for TLR2/TLR4 signaling.","method":"In vitro kinase assay (direct phosphorylation of Mal by IRAK1/4), kinase-inactive IRAK mutants, co-expression degradation assay, ubiquitination assay, IRAK1/4 siRNA knockdown, IRAK inhibitor treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus mutagenesis (kinase-dead controls) plus ubiquitination and degradation assays, multiple orthogonal methods","pmids":["20400509"],"is_preprint":false},{"year":2010,"finding":"MAL interacts with Inverted Formin 2 (INF2) in Jurkat T cells, where they colocalize at the cell periphery, pericentriolar endosomes, and along microtubules. MAL+ vesicles transport Lck to the plasma membrane along microtubule tracks. INF2 knockdown greatly reduces MAL+ transport vesicle formation and Lck plasma membrane levels and impairs immunological synapse formation. Both actin polymerization and depolymerization activities of INF2 are required. Cdc42 and Rac1 regulate this Lck transport process.","method":"Co-immunoprecipitation, videomicroscopy (live imaging of MAL+ vesicle movement), INF2 siRNA knockdown, INF2 activity mutants, Cdc42/Rac1 manipulation in Jurkat and primary T cells","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging, co-IP, loss-of-function with specific phenotypic readout, validated in primary T cells","pmids":["20881207"],"is_preprint":false},{"year":2010,"finding":"MAL/VIP17 coimmunoprecipitates with NKCC2 (Na+-K+-2Cl- cotransporter) in LLC-PK1 cells and rat kidney medullae; a 150-amino-acid stretch of the NKCC2 C-terminal tail mediates the interaction. MAL/VIP17 overexpression increases NKCC2 cell surface retention by attenuating internalization and coincides with increased cotransporter phosphorylation. Transgenic mice overexpressing MAL/VIP17 show cyst formation in distal nephron with highly glycosylated and phosphorylated NKCC2.","method":"Co-immunoprecipitation, truncation mutagenesis of NKCC2, surface biotinylation internalization assay, Western blotting for phosphorylation, transgenic mouse model","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP with domain mapping, surface retention assay, and in vivo transgenic validation, multiple orthogonal methods","pmids":["20861303"],"is_preprint":false},{"year":2011,"finding":"MAL localizes to the central SMAC (cSMAC) of the immunological synapse (IS) in T cells, where it colocalizes with condensed membranes. Mislocalization of MAL to the peripheral SMAC (pSMAC) reduces membrane condensation at the cSMAC, redistributes microtubule/vesicle docking machinery, and causes missorting of raft-associated Lck and LAT (but not TCR) to the pSMAC. MAL thus regulates membrane order and protein sorting at the IS.","method":"Live imaging, Laurdan fluorescent probe for membrane condensation, MAL mislocalization constructs, co-localization analysis in Jurkat and primary T cells","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging combined with functional membrane order measurement and specific cargo missorting phenotype, validated in primary T cells","pmids":["21508261"],"is_preprint":false},{"year":2011,"finding":"Mal is required for TLR2- and TLR4-induced CREB activation in macrophages. Mal-deficient macrophages fail to express CREB-responsive genes IL-10 and COX-2 in response to TLR2/TLR4 ligands. This pathway requires Pellino3, TRAF6, p38 MAPK, and MAPK-activated protein kinase 2 (MK2), which is directly activated by Mal.","method":"Mal-deficient murine macrophages (BMDM), CREB reporter/phosphorylation assays, Pellino3 siRNA knockdown, TRAF6-deficient cells, p38 MAPK inhibitor, MK2 inhibition, overexpression assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout plus pharmacological inhibition with specific transcription factor readout, single lab","pmids":["21398611"],"is_preprint":false},{"year":2012,"finding":"MAL/MRTF-A upregulates cytoskeleton-associated genes including integrin α5, plakophilin 2 (Pkp2), and FHL1 via direct SRF recruitment to their cis-regulatory elements (shown by ChIP). Elevated MAL expression impairs migration of fibroblasts and epithelial cells, while dominant-negative MAL or partial knockdown enhances motility. Knockdown of Pkp2 and FHL1 partially reverses MAL-induced migration inhibition.","method":"ChIP of MAL and SRF at integrin α5 and Pkp2 gene regulatory elements, active MAL and dominant-negative constructs, MAL knockdown, siRNA of target genes, cell migration assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP plus loss-of-function rescue experiment, multiple target genes validated","pmids":["22223881"],"is_preprint":false},{"year":2013,"finding":"Mal interacts with multiple protein kinase C (PKC) isoforms in intestinal epithelial cells (Caco-2). Inhibition of Mal or PKC increases paracellular permeability and bacterial invasion. Mal-deficient mice show altered expression of tight junction proteins (occludin, ZO-1, claudin-3) and increased susceptibility to oral Salmonella infection; bone marrow chimeras indicate the role is in non-hematopoietic (epithelial) cells.","method":"Co-immunoprecipitation (Mal-PKC), transepithelial resistance measurement, Mal−/− mice, bone marrow chimeras, oral vs. intraperitoneal infection comparison, tight junction protein Western blot/IF","journal":"Mucosal immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus genetic knockout with functional permeability and infection readouts, single lab","pmids":["23612054"],"is_preprint":false},{"year":2014,"finding":"Genetic evidence in both Drosophila and human cellular models establishes that actin is the key MAL/SRF target gene required for invasive cell migration. Actin protein feeds back on actin mRNA production via MAL/SRF, constituting a dedicated homeostatic feedback loop ensuring sufficient actin supply.","method":"Genetic epistasis in Drosophila (MAL-D mutants rescued by actin overexpression), human cell models with specific actin manipulation, gene expression analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis across two organisms plus human cellular validation, key regulatory circuit established","pmids":["24831700"],"is_preprint":false},{"year":2015,"finding":"MAL (myelin and lymphocyte protein) is required for binding of Clostridium perfringens ε-toxin (ETX) to mammalian cells and for ETX-mediated cytotoxicity. Native CHO cells are resistant to ETX; exogenous MAL expression confers ETX binding and cell death susceptibility. FLAG insertion into the second extracellular loop of MAL abolishes ETX binding. MAL-knockout mice show complete absence of ETX binding to tissues and complete resistance to ETX at doses >1000x the symptomatic dose for wild-type mice.","method":"Exogenous MAL expression in CHO cells, FLAG epitope insertion mutagenesis, ETX binding assay, cytotoxicity assay, MAL knockout mice, immunofluorescence tissue binding","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — gain-of-function (CHO expression), mutagenesis, and complete in vivo knockout validation with quantitative resistance phenotype","pmids":["25993478"],"is_preprint":false},{"year":2016,"finding":"Mal has a TLR-independent role in IFN-γ receptor (IFNGR) signaling: Mal-dependent IFNGR signaling leads to p38 MAPK phosphorylation and autophagy, is required for phagosome maturation and killing of intracellular M. tuberculosis. The common S180L Mal polymorphism (S200L in mice) reduces Mal's affinity for the IFNGR, thereby compromising IFNGR signaling in macrophages.","method":"Mal-deficient macrophages, IFNGR co-immunoprecipitation with Mal, p38 phosphorylation assays, autophagy assays, phagosome maturation assay, Mtb killing assay, S200L knock-in mice","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP demonstrating direct Mal-IFNGR interaction plus genetic and functional validation in macrophages and knock-in mice","pmids":["26885859"],"is_preprint":false},{"year":2017,"finding":"MAL TIR domains spontaneously and reversibly form filaments in vitro; they form co-filaments with TLR4 TIR domains and induce MyD88 assembly. A 7-Å cryo-EM structure reveals a stable MAL protofilament of two parallel TIR-domain strands in a BB-loop-mediated head-to-tail arrangement. Structure-guided mutagenesis of interface residues combined with in vivo interaction assays shows these MAL interactions represent a conserved mode of TIR-domain interaction for TLR and IL-1R signaling.","method":"Cryo-EM (7-Å resolution), in vitro TIR domain filament reconstitution, site-directed mutagenesis of interface residues, in vivo interaction assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure combined with in vitro reconstitution and structure-guided mutagenesis validated in vivo","pmids":["28759049"],"is_preprint":false},{"year":2017,"finding":"Src family kinase (SFK) activation induces tyrosine phosphorylation of TLR4, which dissociates both MyD88 and Mal/TIRAP from TLR4 and suppresses LPS-induced NFκB and JNK1/2 activation, constituting a negative feedback loop. Kinase-dead SFK-Lyn inhibits TLR4 tyrosine phosphorylation and has reduced binding to TLR4, whereas constitutively active SFK-Lyn strongly promotes TLR4 phosphorylation.","method":"Chemical rescuing approach for temporal SFK activation, TLR4 co-immunoprecipitation with MyD88/Mal, kinase-dead and constitutively active SFK-Lyn mutants, NFκB/JNK reporter assays","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP showing dissociation plus kinase-dead mutant controls, single lab","pmids":["29175418"],"is_preprint":false},{"year":2019,"finding":"MAL is required for ETX-induced blood-brain barrier permeability through caveolae-dependent transcytosis in brain endothelial cells. ETX binding to CNS microvasculature is MAL-dependent (absent in MAL-/- mice). ETX-induced BBB permeability additionally requires caveolin-1: MAL-/- or caveolin-1-/- mice do not show ETX-induced BBB permeability. ETX treatment increases caveolae in brain endothelial cells and causes loss of EEA1+ early endosomes with accumulation of RAB7+ late endosomes/MVBs.","method":"MAL-/- and caveolin-1-/- mice, intravascular ETX injection, molecular tracer extravasation (fluorescein, albumin, dextran, IgG), primary murine brain endothelial cell analysis, EEA1/RAB7 immunofluorescence, electron microscopy of caveolae","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout validation in two mouse lines with mechanistic endocytic pathway characterization","pmids":["31703116"],"is_preprint":false},{"year":2024,"finding":"Upon LPS stimulation, lysine acetyltransferase CBP is recruited to the TLR4 signalosome, leading to acetylation of the TIR domains of TLR4, MAL, and MyD88. This TIR domain acetylation enhances NF-κB (but not IRF3) pathway activation and M1 macrophage polarization. HDAC1 deacetylates the TIR domains; pharmacological modulation of CBP or HDAC1 plays opposite roles in sepsis.","method":"LPS stimulation of macrophages, CBP co-immunoprecipitation with TLR4 signalosome, acetylation mass spectrometry, NF-κB/IRF3 reporter assays, HDAC1 inhibitor, CBP inhibitor, patient monocyte analysis","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrating CBP recruitment plus acetylation identification, functional pathway assays, single lab","pmids":["39294473"],"is_preprint":false},{"year":2006,"finding":"Protein kinase Cδ (PKCδ) binds to TIRAP/Mal via the TIR domain of TIRAP/Mal. TLR2- and TLR4-mediated phosphorylation of p38 MAPK, IKK, and IκB in RAW264.7 cells is abolished by depletion of PKCδ.","method":"GST-fusion pull-down, co-immunoprecipitation from macrophage and THP1 lysates, TIRAP/Mal truncation mutants, PKCδ depletion (siRNA), p38/IKK/IκB phosphorylation assays","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GST pulldown and co-IP with domain mapping, functional knockdown validation, single lab","pmids":["17161867"],"is_preprint":false},{"year":2011,"finding":"Poxvirus A46 protein binds to MAL/TIRAP via a dimeric α-helical C-terminal domain. Biophysical analysis shows this A46 segment adopts a dimeric α-helical structure and interacts with monomeric Mal in vitro. The A46-derived peptide VIPER does not interact with Mal in vitro.","method":"Biophysical binding assays (in vitro), A46 C-terminal domain expression and dimerization characterization, Mal binding assay","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biophysical analysis, single lab, no mutagenesis or cellular validation","pmids":["21831443"],"is_preprint":false},{"year":2009,"finding":"In epithelial cells, dissociation of E-cadherin-dependent cell-cell junctions activates SRF-mediated transcription via monomeric actin and MAL. This pathway requires Rac1 (not RhoA) and actomyosin contractility as a prerequisite. Rac1 inhibition blocks MAL/SRF activation during junction disassembly in contrast to the Rho-ROCK pathway used in serum-stimulated fibroblasts.","method":"Ca2+-dependent junction dissociation, clostridial cytotoxin inhibition of Rac vs. RhoA, MAL reporter assays, direct evidence of actin-MAL signaling","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic dissection of pathway, single lab","pmids":["18334560"],"is_preprint":false},{"year":2004,"finding":"Border cell migration in Drosophila oogenesis requires SRF and its cofactor MAL-D; nuclear accumulation of MAL-D is induced by cell stretching/tension. Border cells that cannot migrate lack nuclear MAL-D but accumulate it when pulled by other cells. MAL-D responds to activated Diaphanous (affecting actin dynamics). MAL-D/SRF activity builds a robust actin cytoskeleton in migrating cells; mutant cells break apart during migration initiation.","method":"Drosophila genetics (MAL-D and SRF mutants), live imaging of border cell migration, nuclear localization assay under tension, activated Diaphanous expression","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function in vivo with mechanistic live imaging and tension manipulation showing nuclear accumulation","pmids":["15239956"],"is_preprint":false},{"year":2009,"finding":"MAL/SRF complex directly regulates MYL9 (MLC2) and MMP9 transcription in megakaryocytes, as demonstrated by luciferase assays and ChIP in primary megakaryocytes. MAL knockdown reduces filopodia/lamellipodia/stress fiber formation, proplatelet formation, and megakaryocyte migration (via MMP9). MAL expression increases during late megakaryopoiesis and localizes to the nucleus after Rho GTPase activation by adhesion.","method":"ChIP in primary megakaryocytes, luciferase assays (HEK293T), MAL siRNA knockdown, gene expression profiling, migration assay through Matrigel, MYL9 shRNA knockdown","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP demonstrating direct promoter binding plus target gene knockdown rescue experiments, validated in primary cells","pmids":["19724058"],"is_preprint":false},{"year":2016,"finding":"In urothelial umbrella cells, MAL facilitates apical fusion of uroplakin-delivering fusiform vesicles (FVs). Sequential action is established: Rab8/11 and Rab27b/Slac2-a mediate apical transport along actin; Rab27b/Slp2-a mediates membrane anchorage; then SNARE-mediated and MAL-facilitated apical fusion occurs. Rab27b acts upstream of MAL in this pathway.","method":"Immunomicroscopy of normal and mutant mouse urothelia, Rab27b knockout mice, epistasis analysis placing Rab27b upstream of MAL","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in mouse model with pathway order established, single lab","pmids":["27009205"],"is_preprint":false}],"current_model":"MAL (also known as VIP17/MVP17) is a four-transmembrane proteolipid that selectively partitions into ordered/raft membrane domains via a C-terminal LIRW motif and intramembrane oligomerization, where it organizes condensed membrane platforms to drive apical protein sorting (cycling between TGN and plasma membrane) in polarized epithelial cells, facilitates Lck and LAT targeting to the immunological synapse cSMAC in T cells, maintains CNS paranodal structure in oligodendrocytes, and serves as the obligatory receptor for Clostridium perfringens ε-toxin; in innate immunity, a distinct gene product sharing the symbol (MyD88-adapter-like/TIRAP) functions as a TIR-domain adapter that recruits MyD88 to TLR2 and TLR4, forms BB-loop-mediated TIR filaments structurally defined by cryo-EM, is phosphorylated by Btk (promoting signaling) and by IRAK1/4 (promoting ubiquitination and degradation), activates NF-κB/JNK/ERK and CREB pathways, connects TLR2/6 to PI3K-Akt via p85α interaction, and mediates IFN-γ receptor signaling for phagosome maturation and Mycobacterium tuberculosis killing; the proteolipid MAL additionally acts as a transcriptional coactivator context—distinct from TIRAP—through SRF binding via its B1 domain to regulate actin homeostasis, dendritic morphology, megakaryocyte migration, and invasive cell migration."},"narrative":{"mechanistic_narrative":"Parse failed — see logs","teleology":[],"mechanism_profile":null},"prefetch_data":{"uniprot":{"accession":"P21145","full_name":"Myelin and lymphocyte protein","aliases":["T-lymphocyte maturation-associated protein"],"length_aa":153,"mass_kda":16.7,"function":"May be involved in vesicular trafficking from the Golgi apparatus to the cell membrane. Plays a role in the maintenance of the myelin sheath, and in axon-glia and glia-glia interactions","subcellular_location":"Membrane; Cell membrane","url":"https://www.uniprot.org/uniprotkb/P21145/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAL","classification":"Not Classified","n_dependent_lines":18,"n_total_lines":1208,"dependency_fraction":0.014900662251655629},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAL","total_profiled":1310},"omim":[{"mim_id":"620978","title":"LEUKODYSTROPHY, HYPOMYELINATING, 28; HLD28","url":"https://www.omim.org/entry/620978"},{"mim_id":"619222","title":"SUPPRESSOR OF CANCER CELL INVASION; SCAI","url":"https://www.omim.org/entry/619222"},{"mim_id":"617924","title":"EPILEPSY, JUVENILE MYOCLONIC, SUSCEPTIBILITY TO, 10; EJM10","url":"https://www.omim.org/entry/617924"},{"mim_id":"617831","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL DOMINANT 55, WITH SEIZURES; MRD55","url":"https://www.omim.org/entry/617831"},{"mim_id":"616409","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 33; DEE33","url":"https://www.omim.org/entry/616409"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Centrosome","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"cervix","ntpm":687.1},{"tissue":"esophagus","ntpm":2851.1},{"tissue":"vagina","ntpm":835.9}],"url":"https://www.proteinatlas.org/search/MAL"},"hgnc":{"alias_symbol":["MVP17","VIP17"],"prev_symbol":[]},"alphafold":{"accession":"P21145","domains":[{"cath_id":"1.20.58","chopping":"36-151","consensus_level":"high","plddt":94.0599,"start":36,"end":151}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P21145","model_url":"https://alphafold.ebi.ac.uk/files/AF-P21145-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P21145-F1-predicted_aligned_error_v6.png","plddt_mean":88.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAL","jax_strain_url":"https://www.jax.org/strain/search?query=MAL"},"sequence":{"accession":"P21145","fasta_url":"https://rest.uniprot.org/uniprotkb/P21145.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P21145/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P21145"}},"corpus_meta":[{"pmid":"11544529","id":"PMC_11544529","title":"Mal 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Signaling.","date":"2016","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/26885859","citation_count":28,"is_preprint":false},{"pmid":"10447589","id":"PMC_10447589","title":"Functional domain analysis of the Saccharomyces MAL-activator.","date":"1999","source":"Current genetics","url":"https://pubmed.ncbi.nlm.nih.gov/10447589","citation_count":28,"is_preprint":false},{"pmid":"28240513","id":"PMC_28240513","title":"Human Papillomavirus Genotypes and Methylation of CADM1, PAX1, MAL and ADCYAP1 Genes in Epithelial Ovarian Cancer Patients.","date":"2017","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/28240513","citation_count":28,"is_preprint":false},{"pmid":"23612054","id":"PMC_23612054","title":"MyD88 adaptor-like (Mal) functions in the epithelial barrier and contributes to intestinal integrity via protein kinase C.","date":"2013","source":"Mucosal immunology","url":"https://pubmed.ncbi.nlm.nih.gov/23612054","citation_count":28,"is_preprint":false},{"pmid":"16911365","id":"PMC_16911365","title":"Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy.","date":"2006","source":"Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology","url":"https://pubmed.ncbi.nlm.nih.gov/16911365","citation_count":28,"is_preprint":false},{"pmid":"38062068","id":"PMC_38062068","title":"Cancer CD39 drives metabolic adaption and mal-differentiation of CD4+ T cells in patients with non-small-cell lung cancer.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38062068","citation_count":27,"is_preprint":false},{"pmid":"31258734","id":"PMC_31258734","title":"On The Role of Myelin and Lymphocyte Protein (MAL) In Cancer: A Puzzle With Two Faces.","date":"2019","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31258734","citation_count":26,"is_preprint":false},{"pmid":"27329150","id":"PMC_27329150","title":"MAL and TMEM220 are novel DNA methylation markers in human gastric cancer.","date":"2016","source":"Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals","url":"https://pubmed.ncbi.nlm.nih.gov/27329150","citation_count":26,"is_preprint":false},{"pmid":"9582298","id":"PMC_9582298","title":"A short peptide motif at the carboxyl terminus is required for incorporation of the integral membrane MAL protein to glycolipid-enriched membranes.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9582298","citation_count":26,"is_preprint":false},{"pmid":"39294473","id":"PMC_39294473","title":"Acetylation of TIR domains in the TLR4-Mal-MyD88 complex regulates immune responses in sepsis.","date":"2024","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/39294473","citation_count":25,"is_preprint":false},{"pmid":"31703116","id":"PMC_31703116","title":"Clostridium perfringens epsilon toxin induces blood brain barrier permeability via caveolae-dependent transcytosis and requires expression of MAL.","date":"2019","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/31703116","citation_count":25,"is_preprint":false},{"pmid":"32436385","id":"PMC_32436385","title":"Myelin-Associated MAL and PLP Are Unusual among Multipass Transmembrane Proteins in Preferring Ordered Membrane Domains.","date":"2020","source":"The journal of physical chemistry. B","url":"https://pubmed.ncbi.nlm.nih.gov/32436385","citation_count":24,"is_preprint":false},{"pmid":"27009205","id":"PMC_27009205","title":"Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells.","date":"2016","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/27009205","citation_count":24,"is_preprint":false},{"pmid":"23932646","id":"PMC_23932646","title":"Synthesis and preclinical characterization of [64Cu]NODAGA-MAL-exendin-4 with a Nε-maleoyl-L-lysyl-glycine linkage.","date":"2013","source":"Nuclear medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/23932646","citation_count":24,"is_preprint":false},{"pmid":"33946345","id":"PMC_33946345","title":"The MAL Protein, an Integral Component of Specialized Membranes, in Normal Cells and Cancer.","date":"2021","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/33946345","citation_count":23,"is_preprint":false},{"pmid":"37106795","id":"PMC_37106795","title":"From Non-Alcoholic Fatty Liver to Hepatocellular Carcinoma: A Story of (Mal)Adapted Mitochondria.","date":"2023","source":"Biology","url":"https://pubmed.ncbi.nlm.nih.gov/37106795","citation_count":23,"is_preprint":false},{"pmid":"29895215","id":"PMC_29895215","title":"RAC2 promotes abnormal proliferation of quiescent cells by enhanced JUNB expression via the MAL-SRF pathway.","date":"2018","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/29895215","citation_count":22,"is_preprint":false},{"pmid":"15514050","id":"PMC_15514050","title":"Mutations in SIN4 and RGR1 cause constitutive expression of MAL structural genes in Saccharomyces cerevisiae.","date":"2004","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15514050","citation_count":21,"is_preprint":false},{"pmid":"24603458","id":"PMC_24603458","title":"MyD88 adaptor-like (Mal) regulates intestinal homeostasis and colitis-associated colorectal cancer in mice.","date":"2014","source":"American journal of physiology. Gastrointestinal and liver physiology","url":"https://pubmed.ncbi.nlm.nih.gov/24603458","citation_count":21,"is_preprint":false},{"pmid":"21276161","id":"PMC_21276161","title":"MAL-PDT for difficult to treat nonmelanoma skin cancer.","date":"2011","source":"Dermatologic therapy","url":"https://pubmed.ncbi.nlm.nih.gov/21276161","citation_count":20,"is_preprint":false},{"pmid":"36010947","id":"PMC_36010947","title":"Combined Liquid Biopsy Methylation Analysis of CADM1 and MAL in Cervical Cancer Patients.","date":"2022","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/36010947","citation_count":20,"is_preprint":false},{"pmid":"35806185","id":"PMC_35806185","title":"The Role of Microglia in the (Mal)adaptive Response to Traumatic Experience in an Animal Model of PTSD.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35806185","citation_count":20,"is_preprint":false},{"pmid":"35241698","id":"PMC_35241698","title":"Gene methylation of CADM1 and MAL identified as a biomarker of high grade anal intraepithelial neoplasia.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/35241698","citation_count":19,"is_preprint":false},{"pmid":"21831443","id":"PMC_21831443","title":"Poxvirus A46 protein binds to TIR domain-containing Mal/TIRAP via an α-helical sub-domain.","date":"2011","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/21831443","citation_count":19,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49440,"output_tokens":10775,"usd":0.154973,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":21501,"output_tokens":8,"usd":0.053852,"stage2_stop_reason":"refusal"},"total_usd":0.208825,"stage1_batch_id":"msgbatch_0147LFpL5iCPFZF66xp23BqC","stage2_batch_id":"msgbatch_01Crtwc6eCsZh1CrnfJhY7J7","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1987,\n      \"finding\": \"MAL encodes a highly hydrophobic proteolipid protein with four putative transmembrane domains, initially identified as expressed in intermediate and late stages of T-cell differentiation, with predicted configuration resembling integral membrane proteins that form pores or channels.\",\n      \"method\": \"cDNA cloning, sequence analysis, hydrophobicity profiling\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — original cloning and structural prediction from single lab, no functional validation of channel/transporter activity\",\n      \"pmids\": [\"3494249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"MAL protein is a proteolipid (soluble in organic lipid-extraction solvents), is encoded by a 4-exon gene where each exon corresponds to one hydrophobic segment, and localizes to the endoplasmic reticulum of T cells. A C-terminal RWKSS motif consistent with ER retention signals was identified.\",\n      \"method\": \"Genomic cloning, RNase protection, recombinant expression in bacteria with lipophilic solvent extraction, immunofluorescence with two distinct anti-peptide antibodies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical demonstration of proteolipid properties plus subcellular localization, single lab with orthogonal methods\",\n      \"pmids\": [\"8132541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"MAL (MVP17) is a developmentally regulated proteolipid in oligodendrocytes that co-purifies with detergent-insoluble, glycolipid-rich membrane microdomains, consistent with a role in myelin biogenesis.\",\n      \"method\": \"Detergent-insoluble membrane fractionation, 2D gel electrophoresis, N-terminal microsequencing, cDNA cloning from oligodendrocyte library, Northern blot\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical fractionation plus cloning, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"8583510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"VIP17/MAL is a proteolipid component of apical transport vesicles in MDCK cells, localizing to perinuclear vesicles and the apical cytoplasm, cycling between the Golgi and the apical plasma membrane.\",\n      \"method\": \"cDNA cloning, immunofluorescence of epitope-tagged VIP17/MAL in BHK and MDCK cells\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional context in polarized cells, single lab\",\n      \"pmids\": [\"8549777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"MAL proteolipid is exclusively incorporated into detergent-resistant, buoyant membrane microdomains (lipid rafts) in both T lymphocytes (Jurkat) and epithelial cells, co-fractionating with p56lck, CD59, and GM1.\",\n      \"method\": \"Stable transfection of epitope-tagged MAL, Triton X-100 extraction, sucrose gradient fractionation, immunofluorescence\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical fractionation in two cell types, single lab\",\n      \"pmids\": [\"9003426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"MAL and caveolin occupy distinct lipid microenvironments within MDCK cells; MAL is in internal glycolipid-enriched detergent-insoluble membranes while caveolin is additionally found at the cell surface, demonstrating heterogeneity within raft fractions.\",\n      \"method\": \"Sucrose gradient fractionation, immunofluorescence, differential detergent solubilization\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical fractionation with multiple cell lines, single lab\",\n      \"pmids\": [\"9168919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"A C-terminal juxtamembrane tetrapeptide motif LIRW in MAL is necessary for incorporation into glycolipid-enriched membrane (GEM) microdomains; arginine is the most critical residue. Loss of GEM targeting correlates with loss of MAL's response to brefeldin A treatment.\",\n      \"method\": \"Site-directed mutagenesis, sucrose gradient fractionation of GEMs, pulse-chase experiments, brefeldin A treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with functional readout (GEM incorporation) in single lab with multiple orthogonal assays\",\n      \"pmids\": [\"9582298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"MAL is expressed specifically in thyroid follicular epithelial cells, localizing to the apical zone, and is a major component of GEM fractions in the polarized thyroid epithelial cell line FRT, consistent with a role in apical sorting in thyroid epithelium.\",\n      \"method\": \"Northern blot, immunohistochemistry with anti-MAL monoclonal antibody, GEM fractionation of FRT cells\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments combined with biochemical fractionation, single lab\",\n      \"pmids\": [\"9528996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MAL depletion by antisense oligonucleotides in MDCK cells reduces the presence of influenza hemagglutinin (HA) in GEMs, decreases the rate of HA transport to the cell surface, inhibits apical delivery, and causes partial missorting of HA to the basolateral membrane. Ectopic MAL expression rescues these defects, demonstrating that MAL is necessary for both normal apical transport and accurate sorting of HA.\",\n      \"method\": \"Antisense oligonucleotides, newly generated anti-canine MAL monoclonal antibody, surface biotinylation, apical/basolateral sorting assay, ectopic MAL rescue\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — loss-of-function plus rescue with specific phenotypic readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"10189374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Overexpression of VIP17/MAL in MDCK cells increases apical delivery and expands the apical surface, while antisense-mediated reduction causes accumulation in the Golgi and impairs apical transport of multiple apical markers (HA, clusterin, gp114, GPI-anchored protein) without affecting basolateral E-cadherin distribution.\",\n      \"method\": \"Antisense RNA expression, overexpression, immunofluorescence, transport assays for multiple apical cargo proteins\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — bidirectional manipulation (OE and KD) with specific apical vs. basolateral cargo controls\",\n      \"pmids\": [\"10339572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MAL is an itinerant protein that cycles between the trans-Golgi network and the plasma membrane: it is expressed at the cell surface, rapidly internalized via an endosomal pathway requiring endosomal acidification, and ~30% of internalized MAL is delivered back to the TGN to restart the transport cycle.\",\n      \"method\": \"Epitope-tagged and O-glycosylatable/biotinylatable MAL constructs, surface biotinylation, anti-FLAG surface binding, neuraminidase sensitivity assay, resialylation experiment, drug treatments (chloroquine, monensin, NH4Cl), flow cytometry\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple engineered constructs and orthogonal biochemical assays establishing cycling behavior, single lab\",\n      \"pmids\": [\"10512878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"MAL (MyD88-adapter-like) is a TIR-domain-containing cytoplasmic adapter protein that activates NF-κB, JNK, and ERK1/2; forms homodimers and heterodimers with MyD88; associates with TLR4 and with IRAK-2 via its TIR domain; and acts as an adapter specifically in TLR-4 signal transduction. A dominant-negative form of Mal inhibits NF-κB activated by TLR-4 or LPS but not by IL-1RI or IL-18R.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, dominant-negative constructs, NF-κB/JNK/ERK reporter assays, two-hybrid interaction assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, dominant-negative functional assays, replicated across multiple signaling readouts; landmark discovery paper\",\n      \"pmids\": [\"11544529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Purified TIR domains of MAL and MyD88 form stable heterodimers in vitro; MAL homodimers/oligomers are dissociated by ATP. Structural modeling and GST pull-down/co-IP experiments indicate MAL and MyD88 bind to different, non-overlapping surfaces on TLR2 and TLR4 TIR domains, suggesting a heterotetrameric receptor-adapter complex. A sequence relationship between Drosophila Tube and MAL was identified.\",\n      \"method\": \"In vitro TIR domain purification, gel filtration, GST pull-down, co-immunoprecipitation, computational structural modeling and docking\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of heterodimer plus orthogonal GST pulldown and co-IP, combined with structural modeling\",\n      \"pmids\": [\"12888566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Genetic ablation of MAL in mice results in cytoplasmic inclusions in compact myelin, everted paranodal loops, disorganized transverse bands, and reduced levels of contactin-associated protein/paranodin, NF155, Kv1.2, MAG, and MBP in myelin and myelin-derived rafts, demonstrating MAL is required for maintenance of CNS paranode structure, likely by controlling NF155 trafficking/sorting in oligodendrocytes.\",\n      \"method\": \"MAL knockout mice, electron microscopy, immunofluorescence, Western blotting of myelin fractions and raft fractions\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with multiple phenotypic readouts and biochemical characterization of myelin protein composition\",\n      \"pmids\": [\"15337780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MAL undergoes tyrosine phosphorylation at positions 86, 187 (and 106) during TLR2 and TLR4 signaling. Bruton's tyrosine kinase (Btk) is identified as the kinase: Btk immunoprecipitated from LPS-activated THP-1 cells directly phosphorylates MAL in vitro, and the Btk inhibitor LFM-A13 blocks endogenous MAL tyrosine phosphorylation. Tyrosine-to-phenylalanine mutations at positions 86 and 187 act as dominant-negative inhibitors of LPS-induced NF-κB activation.\",\n      \"method\": \"Phosphotyrosine immunoprecipitation, site-directed mutagenesis (Y86F, Y187F), Btk kinase inhibitor (LFM-A13), in vitro kinase assay with immunoprecipitated Btk, THP-1 cell stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus pharmacological inhibition, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"16439361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MAL interacts with SRF through a conserved seven-residue B1 region sequence that is essential and sufficient for complex formation; the neighboring Q-box facilitates interaction. MAL directly contacts DNA flanking the SRF-protected region in the MAL-SRF-DNA complex, and these contacts are required for effective MAL-SRF complex formation. SRF-induced DNA bending facilitates MAL-DNA contact. MAL-SRF and TCF-SRF use different mechanisms to engage the same SRF hydrophobic groove/pocket.\",\n      \"method\": \"Deletion and point mutagenesis of MAL B1/Q-box regions, GST pull-downs, gel mobility shift assays, DNase I footprinting, SRF mutagenesis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with in vitro binding assays and DNase I footprinting, multiple orthogonal methods\",\n      \"pmids\": [\"16705166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MAL/MKL1 (SRF co-activator) is expressed in developing neurons, localizes to neuronal cell bodies and apical dendrites, and is required for dendritic morphology: dominant-negative MAL constructs or MAL siRNA reduce the number of dendritic processes in cultured cortical neurons and decrease basal SRF-mediated transcription.\",\n      \"method\": \"Immunohistochemistry of mouse brain, dominant-negative construct expression, siRNA knockdown, dendritic morphology analysis, SRF reporter assay in primary cortical neurons\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific morphological readout, single lab\",\n      \"pmids\": [\"16945101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MAL directly interacts with the p85α regulatory subunit of PI3K in an inducible manner upon TLR2/6 stimulation with diacylated lipoproteins, driving PI3K-dependent Akt phosphorylation, PIP3 generation, and macrophage polarization independently of MyD88.\",\n      \"method\": \"Co-immunoprecipitation (inducible Mal-p85α interaction), PI3K activity assay, Akt phosphorylation assay, PIP3 measurement, MyD88-deficient cell analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP showing inducible interaction, functional assays with MyD88-independent pathway, multiple readouts\",\n      \"pmids\": [\"19574958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Actin-MAL-SRF signaling induces Mig6/Errfi-1 expression, a negative regulator of EGFR. MAL is inducibly recruited to and activates the mig6 promoter. Upregulation of Mig6 correlates with decreased EGFR, MAPK/Erk, and c-fos activation; overexpression of MAL has antiproliferative effects requiring domains for SRF binding and transactivation.\",\n      \"method\": \"Gene expression profiling, chromatin immunoprecipitation (ChIP) of MAL at mig6 promoter, MAL overexpression/siRNA knockdown, EGFR/MAPK activation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP demonstrating direct promoter recruitment plus functional downstream signaling assays, multiple orthogonal methods\",\n      \"pmids\": [\"19683494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MAL clusters laterally concentrate sphingolipid raft markers and exclude phosphatidylethanolamine analogues. MAL forms oligomers via intramembrane protein-protein binding motifs (demonstrated by site-directed mutagenesis and bimolecular fluorescence complementation). MAL-raft association is driven in part by positive hydrophobic mismatch between MAL transmembrane helices and membrane lipids.\",\n      \"method\": \"Antibody-mediated cross-linking of FLAG-tagged MAL, tandem dimer DiHcRED-MAL spontaneous clustering, site-directed mutagenesis of intramembrane motifs, bimolecular fluorescence complementation (BiFC), exogenous cholesterol/ceramide membrane modulation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis plus BiFC plus multiple independent clustering approaches demonstrating mechanism of raft organization\",\n      \"pmids\": [\"19553470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"IRAK1 and IRAK4 directly phosphorylate MAL (but not MyD88). Co-expression of Mal with either IRAK causes depletion of Mal from cell lysates; LPS stimulation triggers ubiquitination and degradation of Mal; this is inhibited by IRAK1/4 kinase inhibitor or IRAK1/4 knockdown. Phosphorylation by IRAKs thus promotes ubiquitination and degradation of Mal, serving as a negative regulatory mechanism for TLR2/TLR4 signaling.\",\n      \"method\": \"In vitro kinase assay (direct phosphorylation of Mal by IRAK1/4), kinase-inactive IRAK mutants, co-expression degradation assay, ubiquitination assay, IRAK1/4 siRNA knockdown, IRAK inhibitor treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus mutagenesis (kinase-dead controls) plus ubiquitination and degradation assays, multiple orthogonal methods\",\n      \"pmids\": [\"20400509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MAL interacts with Inverted Formin 2 (INF2) in Jurkat T cells, where they colocalize at the cell periphery, pericentriolar endosomes, and along microtubules. MAL+ vesicles transport Lck to the plasma membrane along microtubule tracks. INF2 knockdown greatly reduces MAL+ transport vesicle formation and Lck plasma membrane levels and impairs immunological synapse formation. Both actin polymerization and depolymerization activities of INF2 are required. Cdc42 and Rac1 regulate this Lck transport process.\",\n      \"method\": \"Co-immunoprecipitation, videomicroscopy (live imaging of MAL+ vesicle movement), INF2 siRNA knockdown, INF2 activity mutants, Cdc42/Rac1 manipulation in Jurkat and primary T cells\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging, co-IP, loss-of-function with specific phenotypic readout, validated in primary T cells\",\n      \"pmids\": [\"20881207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MAL/VIP17 coimmunoprecipitates with NKCC2 (Na+-K+-2Cl- cotransporter) in LLC-PK1 cells and rat kidney medullae; a 150-amino-acid stretch of the NKCC2 C-terminal tail mediates the interaction. MAL/VIP17 overexpression increases NKCC2 cell surface retention by attenuating internalization and coincides with increased cotransporter phosphorylation. Transgenic mice overexpressing MAL/VIP17 show cyst formation in distal nephron with highly glycosylated and phosphorylated NKCC2.\",\n      \"method\": \"Co-immunoprecipitation, truncation mutagenesis of NKCC2, surface biotinylation internalization assay, Western blotting for phosphorylation, transgenic mouse model\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP with domain mapping, surface retention assay, and in vivo transgenic validation, multiple orthogonal methods\",\n      \"pmids\": [\"20861303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MAL localizes to the central SMAC (cSMAC) of the immunological synapse (IS) in T cells, where it colocalizes with condensed membranes. Mislocalization of MAL to the peripheral SMAC (pSMAC) reduces membrane condensation at the cSMAC, redistributes microtubule/vesicle docking machinery, and causes missorting of raft-associated Lck and LAT (but not TCR) to the pSMAC. MAL thus regulates membrane order and protein sorting at the IS.\",\n      \"method\": \"Live imaging, Laurdan fluorescent probe for membrane condensation, MAL mislocalization constructs, co-localization analysis in Jurkat and primary T cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging combined with functional membrane order measurement and specific cargo missorting phenotype, validated in primary T cells\",\n      \"pmids\": [\"21508261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Mal is required for TLR2- and TLR4-induced CREB activation in macrophages. Mal-deficient macrophages fail to express CREB-responsive genes IL-10 and COX-2 in response to TLR2/TLR4 ligands. This pathway requires Pellino3, TRAF6, p38 MAPK, and MAPK-activated protein kinase 2 (MK2), which is directly activated by Mal.\",\n      \"method\": \"Mal-deficient murine macrophages (BMDM), CREB reporter/phosphorylation assays, Pellino3 siRNA knockdown, TRAF6-deficient cells, p38 MAPK inhibitor, MK2 inhibition, overexpression assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout plus pharmacological inhibition with specific transcription factor readout, single lab\",\n      \"pmids\": [\"21398611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MAL/MRTF-A upregulates cytoskeleton-associated genes including integrin α5, plakophilin 2 (Pkp2), and FHL1 via direct SRF recruitment to their cis-regulatory elements (shown by ChIP). Elevated MAL expression impairs migration of fibroblasts and epithelial cells, while dominant-negative MAL or partial knockdown enhances motility. Knockdown of Pkp2 and FHL1 partially reverses MAL-induced migration inhibition.\",\n      \"method\": \"ChIP of MAL and SRF at integrin α5 and Pkp2 gene regulatory elements, active MAL and dominant-negative constructs, MAL knockdown, siRNA of target genes, cell migration assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP plus loss-of-function rescue experiment, multiple target genes validated\",\n      \"pmids\": [\"22223881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mal interacts with multiple protein kinase C (PKC) isoforms in intestinal epithelial cells (Caco-2). Inhibition of Mal or PKC increases paracellular permeability and bacterial invasion. Mal-deficient mice show altered expression of tight junction proteins (occludin, ZO-1, claudin-3) and increased susceptibility to oral Salmonella infection; bone marrow chimeras indicate the role is in non-hematopoietic (epithelial) cells.\",\n      \"method\": \"Co-immunoprecipitation (Mal-PKC), transepithelial resistance measurement, Mal−/− mice, bone marrow chimeras, oral vs. intraperitoneal infection comparison, tight junction protein Western blot/IF\",\n      \"journal\": \"Mucosal immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus genetic knockout with functional permeability and infection readouts, single lab\",\n      \"pmids\": [\"23612054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Genetic evidence in both Drosophila and human cellular models establishes that actin is the key MAL/SRF target gene required for invasive cell migration. Actin protein feeds back on actin mRNA production via MAL/SRF, constituting a dedicated homeostatic feedback loop ensuring sufficient actin supply.\",\n      \"method\": \"Genetic epistasis in Drosophila (MAL-D mutants rescued by actin overexpression), human cell models with specific actin manipulation, gene expression analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis across two organisms plus human cellular validation, key regulatory circuit established\",\n      \"pmids\": [\"24831700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MAL (myelin and lymphocyte protein) is required for binding of Clostridium perfringens ε-toxin (ETX) to mammalian cells and for ETX-mediated cytotoxicity. Native CHO cells are resistant to ETX; exogenous MAL expression confers ETX binding and cell death susceptibility. FLAG insertion into the second extracellular loop of MAL abolishes ETX binding. MAL-knockout mice show complete absence of ETX binding to tissues and complete resistance to ETX at doses >1000x the symptomatic dose for wild-type mice.\",\n      \"method\": \"Exogenous MAL expression in CHO cells, FLAG epitope insertion mutagenesis, ETX binding assay, cytotoxicity assay, MAL knockout mice, immunofluorescence tissue binding\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — gain-of-function (CHO expression), mutagenesis, and complete in vivo knockout validation with quantitative resistance phenotype\",\n      \"pmids\": [\"25993478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mal has a TLR-independent role in IFN-γ receptor (IFNGR) signaling: Mal-dependent IFNGR signaling leads to p38 MAPK phosphorylation and autophagy, is required for phagosome maturation and killing of intracellular M. tuberculosis. The common S180L Mal polymorphism (S200L in mice) reduces Mal's affinity for the IFNGR, thereby compromising IFNGR signaling in macrophages.\",\n      \"method\": \"Mal-deficient macrophages, IFNGR co-immunoprecipitation with Mal, p38 phosphorylation assays, autophagy assays, phagosome maturation assay, Mtb killing assay, S200L knock-in mice\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP demonstrating direct Mal-IFNGR interaction plus genetic and functional validation in macrophages and knock-in mice\",\n      \"pmids\": [\"26885859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MAL TIR domains spontaneously and reversibly form filaments in vitro; they form co-filaments with TLR4 TIR domains and induce MyD88 assembly. A 7-Å cryo-EM structure reveals a stable MAL protofilament of two parallel TIR-domain strands in a BB-loop-mediated head-to-tail arrangement. Structure-guided mutagenesis of interface residues combined with in vivo interaction assays shows these MAL interactions represent a conserved mode of TIR-domain interaction for TLR and IL-1R signaling.\",\n      \"method\": \"Cryo-EM (7-Å resolution), in vitro TIR domain filament reconstitution, site-directed mutagenesis of interface residues, in vivo interaction assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure combined with in vitro reconstitution and structure-guided mutagenesis validated in vivo\",\n      \"pmids\": [\"28759049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Src family kinase (SFK) activation induces tyrosine phosphorylation of TLR4, which dissociates both MyD88 and Mal/TIRAP from TLR4 and suppresses LPS-induced NFκB and JNK1/2 activation, constituting a negative feedback loop. Kinase-dead SFK-Lyn inhibits TLR4 tyrosine phosphorylation and has reduced binding to TLR4, whereas constitutively active SFK-Lyn strongly promotes TLR4 phosphorylation.\",\n      \"method\": \"Chemical rescuing approach for temporal SFK activation, TLR4 co-immunoprecipitation with MyD88/Mal, kinase-dead and constitutively active SFK-Lyn mutants, NFκB/JNK reporter assays\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP showing dissociation plus kinase-dead mutant controls, single lab\",\n      \"pmids\": [\"29175418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MAL is required for ETX-induced blood-brain barrier permeability through caveolae-dependent transcytosis in brain endothelial cells. ETX binding to CNS microvasculature is MAL-dependent (absent in MAL-/- mice). ETX-induced BBB permeability additionally requires caveolin-1: MAL-/- or caveolin-1-/- mice do not show ETX-induced BBB permeability. ETX treatment increases caveolae in brain endothelial cells and causes loss of EEA1+ early endosomes with accumulation of RAB7+ late endosomes/MVBs.\",\n      \"method\": \"MAL-/- and caveolin-1-/- mice, intravascular ETX injection, molecular tracer extravasation (fluorescein, albumin, dextran, IgG), primary murine brain endothelial cell analysis, EEA1/RAB7 immunofluorescence, electron microscopy of caveolae\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout validation in two mouse lines with mechanistic endocytic pathway characterization\",\n      \"pmids\": [\"31703116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Upon LPS stimulation, lysine acetyltransferase CBP is recruited to the TLR4 signalosome, leading to acetylation of the TIR domains of TLR4, MAL, and MyD88. This TIR domain acetylation enhances NF-κB (but not IRF3) pathway activation and M1 macrophage polarization. HDAC1 deacetylates the TIR domains; pharmacological modulation of CBP or HDAC1 plays opposite roles in sepsis.\",\n      \"method\": \"LPS stimulation of macrophages, CBP co-immunoprecipitation with TLR4 signalosome, acetylation mass spectrometry, NF-κB/IRF3 reporter assays, HDAC1 inhibitor, CBP inhibitor, patient monocyte analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrating CBP recruitment plus acetylation identification, functional pathway assays, single lab\",\n      \"pmids\": [\"39294473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Protein kinase Cδ (PKCδ) binds to TIRAP/Mal via the TIR domain of TIRAP/Mal. TLR2- and TLR4-mediated phosphorylation of p38 MAPK, IKK, and IκB in RAW264.7 cells is abolished by depletion of PKCδ.\",\n      \"method\": \"GST-fusion pull-down, co-immunoprecipitation from macrophage and THP1 lysates, TIRAP/Mal truncation mutants, PKCδ depletion (siRNA), p38/IKK/IκB phosphorylation assays\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GST pulldown and co-IP with domain mapping, functional knockdown validation, single lab\",\n      \"pmids\": [\"17161867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Poxvirus A46 protein binds to MAL/TIRAP via a dimeric α-helical C-terminal domain. Biophysical analysis shows this A46 segment adopts a dimeric α-helical structure and interacts with monomeric Mal in vitro. The A46-derived peptide VIPER does not interact with Mal in vitro.\",\n      \"method\": \"Biophysical binding assays (in vitro), A46 C-terminal domain expression and dimerization characterization, Mal binding assay\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biophysical analysis, single lab, no mutagenesis or cellular validation\",\n      \"pmids\": [\"21831443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In epithelial cells, dissociation of E-cadherin-dependent cell-cell junctions activates SRF-mediated transcription via monomeric actin and MAL. This pathway requires Rac1 (not RhoA) and actomyosin contractility as a prerequisite. Rac1 inhibition blocks MAL/SRF activation during junction disassembly in contrast to the Rho-ROCK pathway used in serum-stimulated fibroblasts.\",\n      \"method\": \"Ca2+-dependent junction dissociation, clostridial cytotoxin inhibition of Rac vs. RhoA, MAL reporter assays, direct evidence of actin-MAL signaling\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic dissection of pathway, single lab\",\n      \"pmids\": [\"18334560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Border cell migration in Drosophila oogenesis requires SRF and its cofactor MAL-D; nuclear accumulation of MAL-D is induced by cell stretching/tension. Border cells that cannot migrate lack nuclear MAL-D but accumulate it when pulled by other cells. MAL-D responds to activated Diaphanous (affecting actin dynamics). MAL-D/SRF activity builds a robust actin cytoskeleton in migrating cells; mutant cells break apart during migration initiation.\",\n      \"method\": \"Drosophila genetics (MAL-D and SRF mutants), live imaging of border cell migration, nuclear localization assay under tension, activated Diaphanous expression\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function in vivo with mechanistic live imaging and tension manipulation showing nuclear accumulation\",\n      \"pmids\": [\"15239956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MAL/SRF complex directly regulates MYL9 (MLC2) and MMP9 transcription in megakaryocytes, as demonstrated by luciferase assays and ChIP in primary megakaryocytes. MAL knockdown reduces filopodia/lamellipodia/stress fiber formation, proplatelet formation, and megakaryocyte migration (via MMP9). MAL expression increases during late megakaryopoiesis and localizes to the nucleus after Rho GTPase activation by adhesion.\",\n      \"method\": \"ChIP in primary megakaryocytes, luciferase assays (HEK293T), MAL siRNA knockdown, gene expression profiling, migration assay through Matrigel, MYL9 shRNA knockdown\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP demonstrating direct promoter binding plus target gene knockdown rescue experiments, validated in primary cells\",\n      \"pmids\": [\"19724058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In urothelial umbrella cells, MAL facilitates apical fusion of uroplakin-delivering fusiform vesicles (FVs). Sequential action is established: Rab8/11 and Rab27b/Slac2-a mediate apical transport along actin; Rab27b/Slp2-a mediates membrane anchorage; then SNARE-mediated and MAL-facilitated apical fusion occurs. Rab27b acts upstream of MAL in this pathway.\",\n      \"method\": \"Immunomicroscopy of normal and mutant mouse urothelia, Rab27b knockout mice, epistasis analysis placing Rab27b upstream of MAL\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in mouse model with pathway order established, single lab\",\n      \"pmids\": [\"27009205\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAL (also known as VIP17/MVP17) is a four-transmembrane proteolipid that selectively partitions into ordered/raft membrane domains via a C-terminal LIRW motif and intramembrane oligomerization, where it organizes condensed membrane platforms to drive apical protein sorting (cycling between TGN and plasma membrane) in polarized epithelial cells, facilitates Lck and LAT targeting to the immunological synapse cSMAC in T cells, maintains CNS paranodal structure in oligodendrocytes, and serves as the obligatory receptor for Clostridium perfringens ε-toxin; in innate immunity, a distinct gene product sharing the symbol (MyD88-adapter-like/TIRAP) functions as a TIR-domain adapter that recruits MyD88 to TLR2 and TLR4, forms BB-loop-mediated TIR filaments structurally defined by cryo-EM, is phosphorylated by Btk (promoting signaling) and by IRAK1/4 (promoting ubiquitination and degradation), activates NF-κB/JNK/ERK and CREB pathways, connects TLR2/6 to PI3K-Akt via p85α interaction, and mediates IFN-γ receptor signaling for phagosome maturation and Mycobacterium tuberculosis killing; the proteolipid MAL additionally acts as a transcriptional coactivator context—distinct from TIRAP—through SRF binding via its B1 domain to regulate actin homeostasis, dendritic morphology, megakaryocyte migration, and invasive cell migration.\"\n}\n```","stage2_raw":"","audit_flag":{"gene":"MAL","tier":"BEHAVIOR","verdict":"Model-behavior concern","subtype":"model_safety_refusal","uniprot_band":"sparse","rules_fired":"R10","issue":"R10: API stop_reason='refusal' at stage2"},"evaluation":null}