{"gene":"CLIC4","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1997,"finding":"CLIC4 (p64H1) localizes to the endoplasmic reticulum when expressed in HEK-293 and HT-4 cells; incorporation of HEK-293 ER vesicles into planar lipid bilayers produced intermediate-conductance, outwardly rectifying anion channels. Protein kinase C phosphorylation of p64H1 increased its apparent molecular weight from ~29 kDa to ~43 kDa.","method":"In vitro expression, immunolocalization, planar lipid bilayer electrophysiology, in vitro PKC phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct channel reconstitution in lipid bilayers plus biochemical phosphorylation assay; foundational characterization paper","pmids":["9295337"],"is_preprint":false},{"year":2001,"finding":"CLIC4 directly binds dynamin I and 14-3-3ζ (confirmed by gel overlay and reverse pull-down), and also associates with α-tubulin, β-actin, creatine kinase, and two 14-3-3 isoforms in rat brain (confirmed by affinity chromatography, mass spectrometry, and co-immunoprecipitation). CLIC4 partially co-localizes with caveolin and functional caveolae in HEK-293 cells, implicating it in caveolar endocytosis.","method":"Affinity chromatography, mass spectrometry, co-immunoprecipitation, gel overlay, reverse pull-down, immunofluorescence","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (pulldown, reciprocal Co-IP, gel overlay) identifying direct binding partners","pmids":["11563969"],"is_preprint":false},{"year":2002,"finding":"CLIC4 (mtCLIC) associates with the inner mitochondrial membrane. Overexpression reduces mitochondrial membrane potential, releases cytochrome c, activates caspases, and induces apoptosis. CLIC4 is transcriptionally regulated by p53 and TNF-α. CLIC4 antisense prevents p53-induced apoptosis but not Bax-induced apoptosis, placing CLIC4 in an independent proapoptotic pathway converging on mitochondria.","method":"Subcellular fractionation, transient transfection overexpression, mitochondrial membrane potential assay, cytochrome c release assay, caspase activation assay, antisense knockdown, genetic epistasis with Bax","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional assays with epistasis analysis in the same study","pmids":["11997498"],"is_preprint":false},{"year":2002,"finding":"Overexpressed CLIC4 in stably transfected HEK-293 cells forms novel low-conductance (~1 pS) plasma membrane anion channels with mild outward rectification, sensitive to IAA (IC50 ~100 µM). Anti-CLIC4 antibodies applied to the cytoplasmic face (but not external face) inhibit these channels, establishing that the C-terminus of the integral membrane form of CLIC4 faces the cytoplasm.","method":"Stable transfection, patch-clamp electrophysiology (whole-cell and single-channel), antibody inhibition from cytoplasmic vs. external face","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct channel recording with topology determination by sidedness of antibody block; single lab but rigorous electrophysiological approach","pmids":["12237120"],"is_preprint":false},{"year":2002,"finding":"TGF-β1 specifically upregulates CLIC4 (>16-fold) during fibroblast-to-myofibroblast conversion, an effect not shared by CLIC1, CLIC2, CLIC3, or CLIC5. Conditional expression of CLIC4 in MEF/3T3 fibroblasts inhibits cell motility by 27% in a migration assay.","method":"Differential display mRNA profiling, RT-PCR, tetracycline-regulated conditional expression, migration assay","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (expression profiling plus functional migration assay) in a single lab","pmids":["12163372"],"is_preprint":false},{"year":2003,"finding":"Multiple stress inducers (DNA damage, apoptotic stimuli) cause translocation of cytoplasmic CLIC4 to the nucleus. CLIC4 associates with Ran, NTF2, and Importin-α nuclear import complexes. Deletion or mutation of the C-terminal nuclear localization signal abolishes nuclear translocation; N-terminal deletion enhances it. Nuclear-targeted CLIC4 accelerates apoptosis and induces apoptosis even in Apaf-null fibroblasts or Bcl-2-overexpressing keratinocytes.","method":"Immunogold EM, confocal microscopy, co-immunoprecipitation, deletion/mutation analysis, adenoviral nuclear targeting, apoptosis assays in Apaf-null and Bcl-2-overexpressing cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (EM, IP, mutagenesis, genetic epistasis) characterizing nuclear import mechanism","pmids":["14610078"],"is_preprint":false},{"year":2003,"finding":"CLIC4 colocalizes with AKAP350 at the centrosome and midbody in cultured mammalian cells, and with AKAP350 and the tight junction protein ZO-1 in the apical region of polarized epithelial cells. CLIC4 is enriched in mitochondria, cortical actin-based structures, and the nuclear matrix, and associates with microtubule cytoskeletal proteins biochemically.","method":"Immunofluorescence microscopy, subcellular fractionation, biochemical co-sedimentation","journal":"Cell motility and the cytoskeleton","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by immunofluorescence and fractionation, but functional consequence not fully established in this study","pmids":["14569596"],"is_preprint":false},{"year":2005,"finding":"Crystal structure of CLIC4 resolved at 1.8 Å by X-ray crystallography. CLIC4 is monomeric and adopts a GST fold, similar to CLIC1 but with differences in helix 2 of the glutaredoxin-like N-terminal domain. Purified recombinant CLIC4 binds artificial lipid bilayers, induces chloride efflux when associated with liposomes, and forms a 30 pS ion channel in artificial bilayers. Oxidation enhances membrane binding; no channels were observed under reducing conditions.","method":"X-ray crystallography (1.8 Å), lipid bilayer reconstitution, chloride efflux assay, tip-dip electrophysiology, oxidation/reduction manipulation","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus reconstituted ion channel activity with redox regulation established in vitro","pmids":["16176272"],"is_preprint":false},{"year":2005,"finding":"CLIC4 protein levels are upregulated by c-Myc; Myc binds directly to the CLIC4 gene promoter and activates its transcription (by quantitative proteomics and ChIP). Suppression of CLIC4 by RNAi inhibits Myc-induced apoptosis under stress conditions and abolishes cooperative induction of apoptosis by Myc and Bax.","method":"Isotope-coded affinity tag quantitative proteomics, chromatin immunoprecipitation, RNAi knockdown, apoptosis assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP demonstrates direct promoter binding by Myc; functional RNAi epistasis with multiple stress conditions","pmids":["16316993"],"is_preprint":false},{"year":2005,"finding":"CLIC4 expression decreases during VEGF-A-induced endothelial cell tubular morphogenesis. siRNA- or antisense-mediated suppression of CLIC4 arrests tubular morphogenesis in vitro, establishing a required role for CLIC4 in endothelial tube/lumen formation.","method":"2D proteomics, antisense and siRNA knockdown, in vitro tubulogenesis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA and antisense knockdown with specific tubulogenesis phenotype in a single lab","pmids":["16239224"],"is_preprint":false},{"year":2007,"finding":"Reconstituted recombinant CLIC4 in planar lipid bilayers forms redox-regulated, poorly selective ion channels (maximum ~15 pS in KCl). A truncated version comprising only the N-terminal 61 residues (containing the predicted transmembrane domain) also forms non-selective channels with reduced conductance that retain trans-redox sensitivity and can be blocked by trans (not cis) thiol-reactive DTNB, suggesting the predicted TMD forms oligomeric pores and the trans cysteine is at the external pore entrance.","method":"Planar lipid bilayer reconstitution, site-specific truncation mutant analysis, redox manipulation, thiol-reactive DTNB block from trans/cis sides","journal":"Molecular membrane biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis/truncation approach probing pore topology, single lab","pmids":["17453412"],"is_preprint":false},{"year":2007,"finding":"CLIC1 and CLIC5 channel activity in planar bilayers is strongly and reversibly inhibited by F-actin; CLIC4 channels are NOT inhibited by F-actin under the same conditions, demonstrating differential actin regulation among CLIC family members.","method":"Planar lipid bilayer reconstitution with addition of F-actin; cytochalasin reversal experiment","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro reconstitution assay with pharmacological reversal; negative result for CLIC4 is itself mechanistically informative","pmids":["18028448"],"is_preprint":false},{"year":2007,"finding":"CLIC4 expression is reduced in multiple human epithelial cancers and excluded from the nucleus in cancer cells. In xenografts, adenoviral introduction of CLIC4 or nuclear-targeted CLIC4 into breast cancer cells inhibits tumor growth, whereas overexpression of CLIC4 in stromal cells enhances tumor growth.","method":"Tissue microarray, adenoviral transduction into xenografts, CLIC4 overexpression in stromal cells, in vivo tumor growth assay","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo functional data with nuclear targeting; single lab","pmids":["17200346"],"is_preprint":false},{"year":2008,"finding":"CLIC4 physically interacts with the C-terminus of the histamine H3 receptor, confirmed by in vitro pull-down, co-immunoprecipitation from rat brain lysate, and immunofluorescence co-localization in rat cerebellar neurons. CLIC4 enhances cell surface expression of H3R, but not a mutant H3R that cannot interact with CLIC4, as measured by flow cytometry, radioligand binding, and cell-based ELISA.","method":"In vitro pull-down, co-immunoprecipitation from rat brain, immunofluorescence, flow cytometry, radioligand binding assay, cell-based ELISA","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods confirming interaction and functional consequence (receptor surface expression) in the same study","pmids":["18302930"],"is_preprint":false},{"year":2009,"finding":"TGF-β promotes CLIC4 and Schnurri-2 expression, their cytoplasmic association, and their co-translocation to the nucleus. In the nucleus, CLIC4 associates with phospho-Smad2 and phospho-Smad3, protecting them from dephosphorylation by nuclear phosphatases, thereby sustaining TGF-β signaling. In the absence of CLIC4 or Schnurri-2, TGF-β signaling is abrogated; direct nuclear targeting of CLIC4 removes the requirement for Schnurri-2.","method":"Co-immunoprecipitation, nuclear fractionation, siRNA knockdown, adenoviral nuclear targeting, TGF-β signaling reporter assays, phospho-Smad stabilization assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, knockdown, nuclear targeting epistasis, signaling assays) in a high-impact journal","pmids":["19448624"],"is_preprint":false},{"year":2009,"finding":"Cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of Gα13-coupled, RhoA-activating receptors (LPA, thrombin, S1P). This translocation is strictly dependent on Gα13-mediated RhoA activation and F-actin integrity but not Rho kinase activity. Mutational analysis reveals dependence on at least six conserved residues including reactive Cys35. Membrane-targeted CLIC4 does not modulate transmembrane chloride currents.","method":"Live-cell imaging, pharmacological inhibitors (Y-27632, cytochalasin), dominant-negative/constitutively active RhoA constructs, site-directed mutagenesis, electrophysiology","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging with multiple genetic and pharmacological perturbations and mutagenesis in the same study","pmids":["19776349"],"is_preprint":false},{"year":2010,"finding":"CLIC4 is directly S-nitrosylated on a cysteine residue (detected by biotin switch assay), and this modification induces conformational unfolding (CD spectra, trypsinolysis) and enhanced association with importin-α and Ran, promoting nuclear translocation. TNF-α-induced nuclear translocation of CLIC4 depends on nitric oxide synthase activity, and NOS inhibition blocks TNF-α-induced CLIC4 nitrosylation and nuclear import. Cysteine mutants show altered nitrosylation, nuclear residence, and stability.","method":"Biotin switch assay (S-nitrosylation detection), CD spectroscopy, limited trypsinolysis, co-immunoprecipitation with importin-α/Ran, NOS inhibition, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical detection of PTM with multiple orthogonal methods (biotin switch, CD, IP) and mutagenesis","pmids":["20504765"],"is_preprint":false},{"year":2011,"finding":"CLIC4-null macrophages show reduced accumulation of phosphorylated IRF3 upon LPS stimulation, while CLIC4 overexpression enhances LPS-mediated IRF3 phosphorylation. CLIC4-null mice are protected from LPS-induced death with reduced serum inflammatory cytokines, and are impaired in Listeria monocytogenes clearance. Deletion of CLIC4 had little effect on MAPK and NF-κB activation, placing CLIC4 specifically in the IRF3 arm of LPS signaling.","method":"CLIC4-null mouse generation, LPS challenge in vivo, Western blot for phospho-IRF3/MAPK/NF-κB, stable CLIC4-overexpressing macrophage lines, Listeria infection assay","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with in vivo phenotype and mechanistic epistasis (IRF3 vs MAPK/NF-κB), replicated by overexpression","pmids":["21469130"],"is_preprint":false},{"year":2012,"finding":"CLIC4-null mice exhibit delayed wound reepithelialization and corneal wound healing, reduced β4 integrin and p21 expression in wounded skin, reduced TGF-β-induced phospho-Smad2 in CLIC4-null keratinocytes, slower keratinocyte migration, and failure to increase migration in response to TGF-β, placing CLIC4 upstream of TGF-β signaling in epidermal wound healing.","method":"CLIC4-null mouse (C57Bl/6 background), full-thickness skin wound and corneal wound assays, Western blot, keratinocyte migration assay, TGF-β stimulation of cultured keratinocytes","journal":"The American journal of pathology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple in vivo and in vitro phenotypic readouts establishing TGF-β pathway dependency","pmids":["22613027"],"is_preprint":false},{"year":2012,"finding":"In metabolically stressed keratinocytes, CLIC4 is S-nitrosylated and translocates to the nucleus, where it enhances TGF-β signaling by protecting phospho-Smad2/3 from dephosphorylation. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear CLIC4 translocation. Adenoviral nuclear targeting of CLIC4 in squamous cancer cells enhances TGF-β transcriptional activity and inhibits growth in vitro and in orthograft tumors.","method":"Adenoviral nuclear targeting, TGF-β reporter assay, S-nitrosylation assay, tumor orthograft model, transgenic epidermis overexpression model","journal":"Carcinogenesis","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo and in vitro experiments with mechanistic pathway dissection (S-nitrosylation → nuclear import → TGF-β/Smad signaling)","pmids":["22387366"],"is_preprint":false},{"year":2013,"finding":"CLIC4 is required for TGF-β-induced activation of p38 MAPK in stromal fibroblasts, and this requirement involves interaction of CLIC4 with PPM1a, the selective phosphatase of activated p38. Genetic ablation of CLIC4 in primary fibroblasts prevents TGF-β-induced expression of α-SMA and extracellular matrix components. Conditioned media from CLIC4-overexpressing fibroblasts increases tumor cell migration/invasion and promotes EMT in a TGF-β-dependent manner.","method":"CLIC4 knockout primary fibroblasts, co-immunoprecipitation (CLIC4–PPM1a), p38 MAPK phosphorylation assay, conditioned medium experiments, migration/invasion assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic ablation plus Co-IP identifying PPM1a as a CLIC4-binding phosphatase, multiple functional readouts","pmids":["23416981"],"is_preprint":false},{"year":2014,"finding":"CLIC4 knockdown in HeLa and MDA-MB-231 cells decreases cell-matrix adhesion, cell spreading, and integrin signaling, while increasing cell motility. LPA stimulates recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both internalization and serum/LPA-induced recycling of β1 integrin (but not EGFR). CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking.","method":"siRNA knockdown, live-cell imaging, co-immunoprecipitation, integrin internalization/recycling assays, Rab35 activity assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal assays (trafficking, signaling, GTPase activity) with receptor-specific controls in a single study","pmids":["25344254"],"is_preprint":false},{"year":2015,"finding":"Clic4 silencing or β-cell-specific knockout reduces cytokine-induced apoptosis, associated with increased expression and stability of Bcl-2, Bad, and phosphorylated Bad. Mass spectrometry of co-immunoprecipitated proteins found no direct association of CLIC4 with Bcl-2 family proteins, but CLIC4 co-purified with proteasome components, suggesting CLIC4 regulates Bcl-2/Bad stability via proteasomal degradation.","method":"siRNA knockdown, β-cell-specific knockout mice, co-immunoprecipitation with mass spectrometry, Bcl-2/Bad half-life measurement, cytokine-induced apoptosis assay","journal":"Molecular metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and Co-IP/MS; mechanism (proteasome association) is suggestive rather than fully demonstrated, single lab","pmids":["25830089"],"is_preprint":false},{"year":2016,"finding":"CLIC4 is required for apical exocytosis and lumen formation in renal tubulogenesis. CLIC4-null embryos have impaired renal tubulogenesis; in MDCK 3D cultures, CLIC4 localizes to early endosomes, recycling endosomes, and apical transport carriers before reaching apical membrane at steady state. CLIC4 suppression impairs apical vesicle coalescence and lumen formation, rescued by Rab8 and Cdc42. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating branched actin formation on early endosomes.","method":"CLIC4-null mouse embryos, MDCK 3D culture lumenogenesis assay, live confocal imaging, Rab8/Cdc42 rescue experiments, retromer knockdown, actin branching analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with in vivo phenotype, mechanistic dissection of retromer/actin pathway, rescue experiments in a high-impact journal","pmids":["26786190"],"is_preprint":false},{"year":2016,"finding":"Elevated CLIC4 induces expression of Smad7Δ, a novel truncated/alternatively spliced form of Smad7 missing 94 bp in exon 4 (predicted to lack the TGF-β inhibitory MH2 domain). Smad7Δ acts as a dominant-negative inhibitor of full-length Smad7, thereby further enhancing TGF-β signaling. TGF-β treatment also enhances Smad7Δ expression, amplified by CLIC4.","method":"CLIC4 overexpression in multiple cell types, RT-PCR splice variant identification, TGF-β reporter assay, proliferation assay, Smad phosphorylation analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — identification of splice variant with functional dominant-negative assay; single lab","pmids":["27536941"],"is_preprint":false},{"year":2016,"finding":"CLIC4 activates ERM proteins (ezrin, radixin, moesin) in glomerular endothelial cells; CLIC4 silencing reduces ERM phosphorylation and cytoskeletal association, and exogenous CLIC4 rescues ERM dephosphorylation. Mice lacking both CLIC4 and CLIC5 show profound reduction of glomerular EC ERM phosphorylation and develop glomerular capillary architectural defects, proteinuria, and glomerular cell proliferation.","method":"CLIC4/CLIC5 knockout mice (single and double), CLIC4 siRNA in cultured glomerular EC, exogenous CLIC4 expression rescue, ERM phosphorylation assay, histopathology","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — double knockout genetic model with in vitro rescue and specific molecular readout (ERM phosphorylation)","pmids":["27582103"],"is_preprint":false},{"year":2017,"finding":"Upon LPS stimulation of macrophages, CLIC4 translocates to the nucleus and cellular membrane (detected by confocal microscopy and cell fractionation). siRNA knockdown of CLIC4 in BMDMs impairs IL-1β transcription, ASC speck formation, and secretion of mature IL-1β in LPS/ATP-stimulated cells, showing CLIC4 participates in both the priming signal (IL-1β transcription) and the NLRP3 inflammasome activation step.","method":"Confocal microscopy, cell fractionation, siRNA knockdown, IL-1β transcription assay, ASC speck formation assay, ELISA for mature IL-1β","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal assays (fractionation, imaging, siRNA, inflammasome assembly readouts) in primary macrophages","pmids":["28576828"],"is_preprint":false},{"year":2018,"finding":"CLIC4 translocation to the plasma membrane upon LPA or EGF stimulation requires RhoA activation via the RhoA effector mDia2 and depends on F-actin polymerization. CLIC4 binds the G-actin-binding protein profilin-1 via residues required for CLIC4 trafficking. Profilin-1 silencing impairs agonist-induced CLIC4 trafficking and mDia2-dependent filopodium formation. CLIC4 knockdown increases filopodium formation (rescued by wild-type CLIC4 but not trafficking-incompetent CLIC4(C35A)), and CLIC4 accelerates LPA-induced filopodium retraction.","method":"Live-cell imaging, siRNA knockdown, co-immunoprecipitation, profilin-1 pull-down, mDia2 overexpression, filopodium quantification, CLIC4 mutant rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct profilin binding shown by pull-down, multiple genetic and imaging approaches, mutant rescue experiments","pmids":["30381396"],"is_preprint":false},{"year":2019,"finding":"CLIC4 localizes to the cytokinetic cleavage furrow and midbody in a RhoA-dependent manner. Mutations of GST activity-related residues (C35A, F37D) abolish cell-cycle-dependent CLIC4 localization. CLIC4 interacts with ezrin, anillin, and ALIX at the cleavage furrow and midbody. CLIC4 facilitates ezrin activation at the cleavage furrow; conversely, inhibition of ezrin activation reduces CLIC4 translocation. CLIC4 and CLIC1 double knockout causes abnormal polar cortex blebbing and cleavage furrow regression, resulting in multinucleated cells.","method":"Live-cell imaging, immunofluorescence, CLIC1/CLIC4 knockout, site-directed mutagenesis (C35A, F37D), co-immunoprecipitation identifying ezrin/anillin/ALIX, ezrin phosphorylation assay","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with specific cytokinesis phenotype, Co-IP partners, mutagenesis, and reciprocal functional dependency with ezrin","pmids":["31879279"],"is_preprint":false},{"year":2019,"finding":"Proteomic analysis of CLIC4-interacting proteins in pulmonary artery endothelial cells identified Arf6 GTPase-activating proteins and clathrin as binding partners. CLIC4 overexpression promotes Arf6-mediated reduction of gyrating clathrin and increased lysosomal targeting of BMPRII, reducing BMPRII expression and signaling. Arf6 siRNA, Arf inhibitor SecinH3, and clathrin-mediated endocytosis inhibitors restore BMPRII expression, but chloride channel inhibitor IAA-94 does not, placing CLIC4 in an Arf6-dependent trafficking pathway independent of its channel activity.","method":"Proteomic interactome (Co-IP/MS), Arf6 siRNA, pharmacological inhibitors (SecinH3, IAA-94, clathrin inhibitors), BMPRII expression and signaling assays, sugen/hypoxia and monocrotaline animal models","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — proteomic binding partners validated by siRNA epistasis and multiple inhibitors; in vivo disease models replicate findings","pmids":["30582444"],"is_preprint":false},{"year":2020,"finding":"CLIC4 localizes to the cytokinetic cleavage furrow and is required for successful completion of mitotic cell division. CLIC4 recruits MST4 kinase (STK26) to the cleavage furrow and regulates ezrin phosphorylation, thereby remodeling the sub-plasma-membrane actomyosin network during cytokinesis.","method":"Live-cell imaging, CLIC4 knockdown, MST4 kinase recruitment assay, ezrin phosphorylation assay, co-immunoprecipitation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with functional KD and molecular readout (MST4 recruitment, ezrin phosphorylation); single lab, limited orthogonal validation of direct interaction","pmids":["32184265"],"is_preprint":false},{"year":2020,"finding":"CLIC4 silencing enhances filopodium formation induced by constitutively active mDia2 mutants. CLIC4 binds the actin-regulatory region of mDia2 in vitro (pull-down assay), suggesting CLIC4 modulates the activity of the open conformation of mDia2 to inhibit filopodium formation.","method":"siRNA knockdown, in vitro pull-down (CLIC4 with mDia2 actin-regulatory region), filopodium quantification with constitutively active mDia2 mutants","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct binding shown by pull-down plus functional filopodium assay; single lab, limited mechanistic depth in abstract","pmids":["32145706"],"is_preprint":false},{"year":2021,"finding":"CLIC1 and CLIC4 in endothelial cells are required for S1P-induced activation of Rac1 downstream of S1PR1. CLIC1 and CLIC4 transiently translocate to the plasma membrane in response to S1P. Only CLIC1 (not CLIC4) was essential for S1P-induced RhoA activation downstream of S1PR2/S1PR3. CLIC1 and CLIC4 are not functionally interchangeable and are critical for S1P-induced endothelial barrier function.","method":"siRNA knockdown of CLIC1/CLIC4, live-cell imaging of membrane translocation, Rac1/RhoA GTPase activation assays, endothelial barrier assay, rescue experiments","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — specific GTPase activation assays with receptor-specific knockdown and rescue, establishing GPCR pathway specificity","pmids":["33879602"],"is_preprint":false},{"year":2022,"finding":"CLIC4 is present in mitochondrial-associated membranes (MAMs) of cardiomyocytes. CLIC4 loss increases myocardial infarction and reduces cardiac function after ischemia-reperfusion injury. CLIC4-null cardiomyocytes show increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation. CLIC4 modulates ER and mitochondrial calcium homeostasis under physiological and pathological conditions.","method":"MAM fractionation, CLIC4-null mouse IR injury model, echocardiography, cardiomyocyte hypoxia-reoxygenation assay, calcium imaging, apoptosis assay","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — subcellular fractionation demonstrating MAM localization combined with in vivo KO phenotype and in vitro calcium measurements","pmids":["36269835"],"is_preprint":false},{"year":2022,"finding":"FTO-mediated m6A demethylation stabilizes CLIC4 mRNA; FTO depletion increases m6A methylation on CLIC4 mRNA and reduces its stability, leading to decreased CLIC4 protein.","method":"MeRIP-RT-qPCR, mRNA stability assay, RNA-seq, FTO knockdown/overexpression","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct m6A mapping on CLIC4 mRNA with stability assay; single lab","pmids":["35397614"],"is_preprint":false},{"year":2022,"finding":"CLIC4 deletion from murine breast tumor cells using CRISPR enhances ROS accumulation, sensitizes cells to H2O2-induced apoptosis, and is associated with mitochondrial hyperactivity (increased membrane potential, organelle enlargement, increased superoxide). In the absence of CLIC4, H2O2-induced apoptosis involves degradation of Bcl2 and UCP2. CLIC4 therefore maintains redox homeostasis and mitochondrial function, consistent with a glutaredoxin-like enzymatic activity.","method":"CRISPR knockout, ROS measurement, mitochondrial membrane potential assay, apoptosis assay, transcriptomic profiling, Bcl2/UCP2 protein analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with multiple orthogonal mitochondrial/redox readouts; single lab","pmids":["35863434"],"is_preprint":false},{"year":2023,"finding":"CLIC4 is required for thrombin/PAR1-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Thrombin promotes CLIC4 (but not CLIC1) relocalization to HUVEC membranes. Endothelial-specific CLIC4 deletion in mice reduces lung edema and microvascular permeability induced by PAR1 activating peptide. CLIC1 was not required for thrombin-mediated barrier disruption but contributed to barrier recovery.","method":"CLIC4/CLIC1 siRNA knockdown, endothelial-specific conditional Clic4 knockout mice (PAR1 peptide-induced lung permeability), RhoA activation assay, ERM phosphorylation assay, endothelial barrier assay","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional in vivo KO with in vitro mechanistic dissection; receptor- and CLIC-specific functional differentiation","pmids":["37317855"],"is_preprint":false},{"year":2025,"finding":"CLIC4 is expressed on the plasma membrane of sperm cells and is required for IAA-94-sensitive chloride currents (genetic ablation of CLIC4 eliminates these currents). CLIC4 regulates cell volume during sperm maturation without altering membrane potential, motility, or acrosome reaction. CLIC4 channel activity in sperm is modulated by Protein Kinase C (PKC).","method":"Patch-clamp electrophysiology, CLIC4-null mouse sperm, IAA-94 pharmacology, PKC modulation assay, sperm morphology and functional assays","journal":"BMC biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct channel recording with genetic KO ablation of currents and PKC modulation; single lab","pmids":["41715063"],"is_preprint":false},{"year":2025,"finding":"CLIC4 C-terminus (not N-terminus) encodes CLIC4-specific functions required for S1P-induced Rac1 activation in endothelial cells. The CLIC4 N-terminus encodes determinants for S1P-induced plasma membrane relocalization, but is dispensable for Rac1 activation when the C-terminus is targeted to the membrane via a heterologous signal. The postulated ion channel and thiol-transferase (GST) activities of CLIC4 are NOT required for Rac1 activation.","method":"Structure-function mutagenesis (N/C-terminal domain swaps, heterologous membrane targeting), Rac1 activation assay, live-cell imaging of plasma membrane translocation, endothelial cell siRNA rescue experiments","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-function analysis with domain-specific rescue; preprint, not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"CLIC4 is a multifunctional, metamorphic protein that adopts a soluble GST-omega fold (crystal structure at 1.8 Å) and can auto-insert into membranes to form redox-regulated, poorly selective ion channels; it traffics constitutively between the cytoplasm, plasma membrane (recruited by RhoA/Gα13-coupled receptor activation via mDia2 and profilin-1 binding), mitochondria/MAMs, and nucleus (via S-nitrosylation of Cys35 and importin-α/Ran-mediated import), where nuclear CLIC4 stabilizes phospho-Smad2/3 against dephosphorylation to sustain TGF-β signaling, interacts with Schnurri-2, promotes cell cycle arrest and apoptosis (in part through mitochondrial membrane potential dissipation and cytochrome c release), and regulates cytokinesis by recruiting MST4 kinase and activating ezrin at the cleavage furrow; at the plasma membrane CLIC4 suppresses Rab35 activity to control β1 integrin trafficking, counteracts mDia2-driven filopodium formation, mediates GPCR (S1PR1, PAR1) coupling to Rac1 and RhoA via its C-terminus independently of ion channel or thiol-transferase activity, and promotes NLRP3 inflammasome priming; CLIC4 is also regulated post-translationally by PKC phosphorylation (in sperm) and at the mRNA level by FTO-dependent m6A demethylation."},"narrative":{"mechanistic_narrative":"CLIC4 is a metamorphic, multifunctional protein that interconverts between a soluble GST-omega/glutaredoxin-like fold (resolved by crystallography at 1.8 Å) and a membrane-inserted form, redistributing among the cytoplasm, plasma membrane, mitochondria, and nucleus to couple cellular redox state and small-GTPase signaling to cytoskeletal dynamics, membrane trafficking, apoptosis, and TGF-β signaling [PMID:16176272, PMID:19776349, PMID:20504765]. Reconstituted recombinant CLIC4 auto-inserts into lipid bilayers under oxidizing conditions to form poorly selective, redox-regulated ion channels whose pore is built by an N-terminal transmembrane segment with the C-terminus facing the cytoplasm [PMID:12237120, PMID:16176272, PMID:17453412]; CLIC4 supplies IAA-94-sensitive chloride currents in native sperm where it controls cell volume and is modulated by PKC [PMID:41715063]. A defining feature is stress- and agonist-triggered translocation: S-nitrosylation of a reactive cysteine unfolds CLIC4 and promotes importin-α/Ran-dependent nuclear import, where CLIC4 protects phospho-Smad2/3 from dephosphorylation—acting with Schnurri-2 and PPM1a—to sustain TGF-β/p38 signaling, drive myofibroblast conversion and wound reepithelialization, and execute proapoptotic programs converging on mitochondria [PMID:14610078, PMID:19448624, PMID:20504765, PMID:22387366, PMID:23416981, PMID:22613027, PMID:11997498]. At the plasma membrane, Gα13/RhoA-coupled receptor activation (LPA, thrombin/PAR1, S1P/S1PR1) recruits CLIC4 via mDia2- and profilin-1-dependent actin dynamics, and CLIC4 then transduces these signals to Rac1 and RhoA activation, ERM-protein phosphorylation, and endothelial barrier control through its C-terminus independently of ion-channel or thiol-transferase activity [PMID:19776349, PMID:30381396, PMID:33879602, PMID:37317855]. CLIC4 also governs endosomal trafficking—suppressing Rab35 to control β1-integrin internalization and recycling and modulating retromer/Arf6-dependent apical transport during lumenogenesis—and is required for cytokinesis, where it localizes to the cleavage furrow, recruits MST4 kinase, and activates ezrin to remodel the cortical actomyosin network [PMID:25344254, PMID:26786190, PMID:30582444, PMID:31879279, PMID:32184265]. CLIC4 expression is controlled transcriptionally by p53 and c-Myc and post-transcriptionally by FTO-dependent m6A demethylation [PMID:11997498, PMID:16316993, PMID:35397614].","teleology":[{"year":1997,"claim":"Established CLIC4 as a membrane-associating, channel-forming protein subject to PKC phosphorylation, framing the foundational question of whether it is a bona fide ion channel.","evidence":"ER localization in transfected cells plus planar lipid bilayer recordings and in vitro PKC assay","pmids":["9295337"],"confidence":"High","gaps":["Channel formation shown for ER-derived vesicles, not purified protein","Physiological ionic substrate and gating unresolved","Functional consequence of PKC phosphorylation unaddressed"]},{"year":2001,"claim":"Identified the first direct cytoskeletal and adaptor partners, placing CLIC4 at the interface of vesicle trafficking and the actin/tubulin cytoskeleton.","evidence":"Affinity chromatography, mass spectrometry, reciprocal Co-IP and gel overlay from rat brain identifying dynamin I, 14-3-3, tubulin, actin","pmids":["11563969"],"confidence":"High","gaps":["Functional roles of individual interactions not dissected","Caveolar endocytosis role inferred from colocalization only"]},{"year":2002,"claim":"Defined a mitochondrial, p53/TNF-α-inducible proapoptotic role distinct from Bax, and demonstrated low-conductance plasma membrane channels with cytoplasmic C-terminus topology.","evidence":"Mitochondrial fractionation, membrane-potential/cytochrome c/caspase assays with antisense epistasis; patch-clamp with sided antibody block","pmids":["11997498","12237120","12163372"],"confidence":"High","gaps":["Mechanism linking membrane insertion to mitochondrial depolarization unresolved","Whether channel activity drives apoptosis untested","TGF-β upregulation of CLIC4 mechanistically uncharacterized at this stage"]},{"year":2003,"claim":"Revealed regulated nuclear translocation via importin-α/Ran with a C-terminal NLS, establishing nuclear CLIC4 as a stress-responsive proapoptotic species.","evidence":"Immunogold EM, Co-IP with Ran/NTF2/importin-α, NLS deletion/mutation, adenoviral nuclear targeting in Apaf-null and Bcl-2 cells; plus centrosome/midbody/AKAP350 colocalization","pmids":["14610078","14569596"],"confidence":"High","gaps":["Signal triggering import not yet defined","Nuclear effectors downstream of CLIC4 unknown at this stage"]},{"year":2005,"claim":"Provided the atomic GST-fold structure and reconstituted redox-gated channel activity from purified protein, and placed CLIC4 under c-Myc transcriptional control with required roles in apoptosis and tubulogenesis.","evidence":"1.8 Å X-ray structure with bilayer reconstitution/chloride efflux under oxidation; quantitative proteomics and ChIP for Myc; siRNA/antisense tubulogenesis assays","pmids":["16176272","16316993","16239224"],"confidence":"High","gaps":["Structure is of the soluble form only; membrane conformation unresolved","How a soluble GST-fold monomer becomes a channel structurally unexplained","Enzymatic substrate of the glutaredoxin-like fold not identified"]},{"year":2007,"claim":"Mapped the N-terminal transmembrane segment as the pore-forming, redox-sensitive element and distinguished CLIC4 from CLIC1/CLIC5 by its insensitivity to F-actin inhibition.","evidence":"Bilayer reconstitution with N-terminal truncation, trans/cis DTNB block, and comparative F-actin addition experiments","pmids":["17453412","18028448"],"confidence":"High","gaps":["Oligomeric stoichiometry of the pore not determined","Physiological relevance of in vitro channel still open"]},{"year":2008,"claim":"Showed CLIC4 acts as a chaperone for GPCR surface delivery, expanding its role beyond channel/cytoskeletal functions.","evidence":"Pull-down, Co-IP from rat brain, immunofluorescence, and surface-expression assays with H3 receptor binding-deficient mutant control","pmids":["18302930"],"confidence":"High","gaps":["Trafficking step at which CLIC4 acts not pinpointed","Generality to other GPCRs untested here"]},{"year":2009,"claim":"Defined CLIC4 as a nuclear stabilizer of phospho-Smad2/3 acting with Schnurri-2 to sustain TGF-β signaling, and identified Gα13/RhoA-dependent recruitment to the plasma membrane.","evidence":"Co-IP, nuclear fractionation, siRNA, nuclear-targeting epistasis and reporter assays; live imaging with RhoA constructs and Cys35 mutagenesis","pmids":["19448624","19776349"],"confidence":"High","gaps":["Mechanism by which CLIC4 blocks Smad dephosphorylation unresolved","Identity of the nuclear phosphatase not defined","Function of membrane-targeted CLIC4 (non-channel) initially unclear"]},{"year":2010,"claim":"Connected nitric-oxide signaling to CLIC4 conformational change and nuclear import, defining S-nitrosylation as the molecular switch upstream of TGF-β stabilization.","evidence":"Biotin-switch assay, CD spectroscopy, limited trypsinolysis, importin-α/Ran Co-IP, and NOS inhibition with cysteine mutants","pmids":["20504765"],"confidence":"High","gaps":["Precise nitrosylated cysteine residue contribution among multiple cysteines","Reversal/denitrosylation mechanism unaddressed"]},{"year":2011,"claim":"Demonstrated in vivo immune function by placing CLIC4 selectively in the IRF3 arm of LPS/TLR signaling, separate from MAPK/NF-κB.","evidence":"CLIC4-null mice with LPS challenge, phospho-IRF3/MAPK/NF-κB blots, overexpressing macrophages, Listeria clearance","pmids":["21469130"],"confidence":"High","gaps":["Molecular target of CLIC4 in the IRF3 pathway unidentified","Whether channel or trafficking activity mediates the effect unknown"]},{"year":2012,"claim":"Showed CLIC4 is required in vivo for epidermal/corneal wound healing through TGF-β/Smad signaling, linking S-nitrosylation-driven nuclear CLIC4 to tissue repair and tumor suppression.","evidence":"CLIC4-null wound-healing models, keratinocyte migration and phospho-Smad2 assays; S-nitrosylation/nuclear-targeting orthograft tumor experiments","pmids":["22613027","22387366"],"confidence":"High","gaps":["Upstream NOS isoform in keratinocytes not specified","Stromal vs epithelial context-dependence of TGF-β output unresolved"]},{"year":2013,"claim":"Identified PPM1a as a CLIC4-binding phosphatase and extended TGF-β dependence to p38 MAPK and stromal myofibroblast/EMT-promoting functions.","evidence":"CLIC4-knockout primary fibroblasts, CLIC4–PPM1a Co-IP, p38 phosphorylation assays, conditioned-medium migration/invasion experiments","pmids":["23416981"],"confidence":"High","gaps":["Whether CLIC4 directly inhibits PPM1a catalytic activity not shown","Subcellular site of CLIC4–PPM1a interaction undefined"]},{"year":2014,"claim":"Established CLIC4 as a Rab35 suppressor controlling β1-integrin internalization and recycling, linking GPCR activation to adhesion and motility.","evidence":"siRNA, live imaging, Co-IP, integrin trafficking assays with EGFR control, and Rab35 activity assay","pmids":["25344254"],"confidence":"High","gaps":["Mechanism of Rab35 suppression (GAP/GEF vs sequestration) unknown","Direct vs indirect CLIC4–Rab35 relationship unresolved"]},{"year":2016,"claim":"Defined CLIC4 roles in retromer/actin-dependent apical lumenogenesis and in amplifying TGF-β via a dominant-negative Smad7 splice form, broadening its trafficking and signaling repertoire.","evidence":"CLIC4-null embryos, MDCK 3D lumen assays with Rab8/Cdc42 rescue and retromer knockdown; RT-PCR splice-variant identification with reporter assays","pmids":["26786190","27536941"],"confidence":"High","gaps":["How CLIC4 negatively regulates branched actin on endosomes mechanistically open","Trans-acting factor producing Smad7Δ splicing unidentified"]},{"year":2016,"claim":"Showed CLIC4 activates ERM proteins in glomerular endothelium, with CLIC4/CLIC5 redundancy required for glomerular capillary integrity.","evidence":"CLIC4/CLIC5 single and double knockout mice, glomerular EC siRNA with CLIC4 rescue, ERM phosphorylation and histopathology","pmids":["27582103"],"confidence":"High","gaps":["Kinase/phosphatase through which CLIC4 controls ERM phosphorylation undefined","Direct CLIC4–ERM interaction not established here"]},{"year":2018,"claim":"Mechanistically resolved agonist-induced membrane recruitment as mDia2/profilin-1/F-actin-dependent and assigned CLIC4 an antagonist role in filopodium dynamics.","evidence":"Live imaging, profilin-1 pull-down, mDia2 overexpression, filopodium quantification, and CLIC4(C35A) trafficking-mutant rescue","pmids":["30381396"],"confidence":"High","gaps":["How CLIC4 limits mDia2-driven actin elongation biochemically unclear at this point"]},{"year":2019,"claim":"Placed CLIC4 at the cytokinetic furrow with ezrin/anillin/ALIX partners and identified an Arf6/clathrin-dependent BMPRII-degradation pathway relevant to pulmonary hypertension, both independent of channel activity.","evidence":"CLIC1/CLIC4 knockout cytokinesis phenotyping with C35A/F37D mutants and Co-IP; interactome with Arf6-GAP/clathrin, Arf6 siRNA, SecinH3/IAA-94/clathrin inhibitors, in vivo PH models","pmids":["31879279","30582444"],"confidence":"High","gaps":["Direct vs scaffolded nature of ezrin/anillin/ALIX interactions not fully resolved","How CLIC4 modulates Arf6-GAP activity mechanistically open"]},{"year":2020,"claim":"Identified MST4 kinase recruitment as the means by which furrow CLIC4 controls ezrin phosphorylation and cytokinetic actomyosin remodeling, and clarified mDia2 inhibition via direct binding.","evidence":"Live imaging with CLIC4 knockdown, MST4 recruitment and ezrin phosphorylation assays, Co-IP; in vitro pull-down with mDia2 actin-regulatory region and constitutively active mDia2 filopodium assays","pmids":["32184265","32145706"],"confidence":"Medium","gaps":["Directness of CLIC4–MST4 interaction not orthogonally validated","Structural basis of CLIC4–mDia2 binding unresolved"]},{"year":2021,"claim":"Demonstrated non-interchangeable, receptor-specific roles for CLIC4 and CLIC1 in S1P-driven Rac1 versus RhoA activation and endothelial barrier function.","evidence":"CLIC1/CLIC4 siRNA, membrane translocation imaging, Rac1/RhoA activation assays, barrier assays and rescue","pmids":["33879602"],"confidence":"High","gaps":["Molecular basis of CLIC4 specificity for Rac1 not defined here","Effector linking CLIC4 to Rac1-GEF unknown"]},{"year":2022,"claim":"Extended CLIC4 to MAM/mitochondrial calcium and redox homeostasis with cardioprotective and ROS-buffering functions consistent with its glutaredoxin-like fold.","evidence":"MAM fractionation, CLIC4-null IR injury and hypoxia-reoxygenation models, calcium imaging; CRISPR knockout with ROS/membrane-potential/Bcl2/UCP2 readouts; plus FTO/m6A mRNA-stability mapping","pmids":["36269835","35863434","35397614"],"confidence":"High","gaps":["Whether CLIC4 directly catalyzes a redox reaction in vivo unproven","Mechanism of CLIC4 control over ER-mitochondrial calcium flux undefined","m6A reader linking FTO to CLIC4 stability not identified"]},{"year":2023,"claim":"Confirmed in vivo endothelial CLIC4 requirement for thrombin/PAR1-driven RhoA activation, ERM phosphorylation, and vascular permeability, with CLIC-isoform specificity.","evidence":"Endothelial-specific Clic4 knockout with PAR1-peptide lung permeability, CLIC4/CLIC1 siRNA, RhoA activation and barrier assays","pmids":["37317855"],"confidence":"High","gaps":["Direct CLIC4 effector coupling PAR1 to RhoA-GEF unknown","How membrane-recruited CLIC4 selectively engages RhoA vs Rac1 unresolved"]},{"year":2025,"claim":"Confirmed CLIC4 supplies a native IAA-94-sensitive chloride conductance regulating sperm volume under PKC control, and localized GPCR-to-Rac1 coupling to the CLIC4 C-terminus independent of channel and GST activities.","evidence":"Patch-clamp on CLIC4-null sperm with PKC modulation; domain-swap/heterologous-targeting structure-function with Rac1 activation assays (one preprint)","pmids":["41715063"],"confidence":"High","gaps":["How a soluble protein generates native sperm currents structurally unresolved","C-terminal effector(s) for Rac1 activation not identified","Domain-swap GPCR-coupling findings remain in preprint form"]},{"year":null,"claim":"It remains unknown how CLIC4 mechanistically converts between its soluble GST-fold and its membrane/signaling forms to select among its many outputs—channel activity, GTPase coupling, Smad stabilization, and trafficking—and which endogenous enzymatic substrate (if any) the glutaredoxin-like fold acts upon.","evidence":"No discovery in the corpus reconciles the membrane-inserted conformation with the soluble structure or identifies an in vivo catalytic substrate","pmids":[],"confidence":"Low","gaps":["No structure of the membrane-inserted conformation","No defined endogenous redox substrate","Logic determining which downstream function dominates in a given context unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,3,7,10,37]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[14,20,21,29,32]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[13,14,28,30]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[27,28,31]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5,15]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,15,21,36]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,14,16,19]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[2,33,35]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,33]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[21,23]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[6,28]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[14,15,32,36]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,5,8,35]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[17,26]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[21,23,29]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[28,30]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[36]}],"complexes":[],"partners":["SMAD2","SMAD3","PPM1A","EZR","MST4","PFN1","DIAPH3","RHOA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y696","full_name":"Chloride intracellular channel protein 4","aliases":["Glutaredoxin-like oxidoreductase CLIC4","Intracellular chloride ion channel protein p64H1"],"length_aa":253,"mass_kda":28.8,"function":"In the soluble state, catalyzes glutaredoxin-like thiol disulfide exchange reactions with reduced glutathione as electron donor (PubMed:25581026, PubMed:37759794). Can insert into membranes and form voltage-dependent multi-ion conductive channels. Membrane insertion seems to be redox-regulated and may occur only under oxidizing conditions (By similarity) (PubMed:16176272). Has alternate cellular functions like a potential role in angiogenesis or in maintaining apical-basolateral membrane polarity during mitosis and cytokinesis. Could also promote endothelial cell proliferation and regulate endothelial morphogenesis (tubulogenesis). Promotes cell-surface expression of HRH3","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasmic vesicle membrane; Nucleus; Cell membrane; Mitochondrion; Cell junction; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y696/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CLIC4","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SAR1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CLIC4","total_profiled":1310},"omim":[{"mim_id":"607293","title":"CHLORIDE INTRACELLULAR CHANNEL 5; CLIC5","url":"https://www.omim.org/entry/607293"},{"mim_id":"606536","title":"CHLORIDE INTRACELLULAR CHANNEL 4; CLIC4","url":"https://www.omim.org/entry/606536"},{"mim_id":"602872","title":"CHLORIDE INTRACELLULAR CHANNEL 1; CLIC1","url":"https://www.omim.org/entry/602872"},{"mim_id":"143054","title":"HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 ENHANCER-BINDING PROTEIN 2; HIVEP2","url":"https://www.omim.org/entry/143054"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Centrosome","reliability":"Supported"},{"location":"Calyx","reliability":"Additional"},{"location":"Connecting piece","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"blood vessel","ntpm":408.2}],"url":"https://www.proteinatlas.org/search/CLIC4"},"hgnc":{"alias_symbol":["DKFZP566G223","CLIC4L","P64H1","H1","huH1","p64H1"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y696","domains":[{"cath_id":"3.40.30.10","chopping":"18-97","consensus_level":"high","plddt":93.2343,"start":18,"end":97},{"cath_id":"1.20.1050.10","chopping":"111-243","consensus_level":"high","plddt":96.282,"start":111,"end":243}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y696","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y696-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y696-F1-predicted_aligned_error_v6.png","plddt_mean":91.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CLIC4","jax_strain_url":"https://www.jax.org/strain/search?query=CLIC4"},"sequence":{"accession":"Q9Y696","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y696.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y696/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y696"}},"corpus_meta":[{"pmid":"11997498","id":"PMC_11997498","title":"mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage and participates in the apoptotic response to p53.","date":"2002","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11997498","citation_count":163,"is_preprint":false},{"pmid":"28576828","id":"PMC_28576828","title":"The intracellular chloride channel proteins CLIC1 and CLIC4 induce IL-1β transcription and activate the NLRP3 inflammasome.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28576828","citation_count":146,"is_preprint":false},{"pmid":"14610078","id":"PMC_14610078","title":"The organellular chloride channel protein CLIC4/mtCLIC translocates to the nucleus in response to cellular stress and accelerates apoptosis.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14610078","citation_count":129,"is_preprint":false},{"pmid":"25546839","id":"PMC_25546839","title":"Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets.","date":"2014","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/25546839","citation_count":125,"is_preprint":false},{"pmid":"9295337","id":"PMC_9295337","title":"Rat brain p64H1, expression of a new member of the p64 chloride channel protein family in endoplasmic reticulum.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9295337","citation_count":107,"is_preprint":false},{"pmid":"16176272","id":"PMC_16176272","title":"Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4.","date":"2005","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/16176272","citation_count":107,"is_preprint":false},{"pmid":"11563969","id":"PMC_11563969","title":"Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms.","date":"2001","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/11563969","citation_count":93,"is_preprint":false},{"pmid":"18028448","id":"PMC_18028448","title":"Functional reconstitution of mammalian 'chloride intracellular channels' CLIC1, CLIC4 and CLIC5 reveals differential regulation by cytoskeletal actin.","date":"2007","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/18028448","citation_count":93,"is_preprint":false},{"pmid":"12163372","id":"PMC_12163372","title":"Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1-mediated conversion of fibroblasts to myofibroblasts.","date":"2002","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/12163372","citation_count":89,"is_preprint":false},{"pmid":"19448624","id":"PMC_19448624","title":"TGF-beta signalling is regulated by Schnurri-2-dependent nuclear translocation of CLIC4 and consequent stabilization of phospho-Smad2 and 3.","date":"2009","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/19448624","citation_count":88,"is_preprint":false},{"pmid":"16239224","id":"PMC_16239224","title":"Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for chloride intracellular channel 4 (CLIC4) in tubular morphogenesis.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16239224","citation_count":88,"is_preprint":false},{"pmid":"17453412","id":"PMC_17453412","title":"CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels.","date":"2007","source":"Molecular membrane biology","url":"https://pubmed.ncbi.nlm.nih.gov/17453412","citation_count":76,"is_preprint":false},{"pmid":"17200346","id":"PMC_17200346","title":"Reciprocal modifications of CLIC4 in tumor epithelium and stroma mark malignant progression of multiple human cancers.","date":"2007","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/17200346","citation_count":74,"is_preprint":false},{"pmid":"10191309","id":"PMC_10191309","title":"A 29 kDa intracellular chloride channel p64H1 is associated with large dense-core vesicles in rat hippocampal neurons.","date":"1999","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/10191309","citation_count":74,"is_preprint":false},{"pmid":"23416981","id":"PMC_23416981","title":"CLIC4 regulates TGF-β-dependent myofibroblast differentiation to produce a cancer stroma.","date":"2013","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/23416981","citation_count":70,"is_preprint":false},{"pmid":"19639201","id":"PMC_19639201","title":"CLIC4 mediates TGF-beta1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer.","date":"2009","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/19639201","citation_count":67,"is_preprint":false},{"pmid":"14569596","id":"PMC_14569596","title":"CLIC4 is enriched at cell-cell junctions and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells.","date":"2003","source":"Cell motility and the cytoskeleton","url":"https://pubmed.ncbi.nlm.nih.gov/14569596","citation_count":64,"is_preprint":false},{"pmid":"16316993","id":"PMC_16316993","title":"Quantitative proteomic analysis of myc-induced apoptosis: a direct role for Myc induction of the mitochondrial chloride ion channel, mtCLIC/CLIC4.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16316993","citation_count":61,"is_preprint":false},{"pmid":"25344254","id":"PMC_25344254","title":"CLIC4 regulates cell adhesion and β1 integrin trafficking.","date":"2014","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/25344254","citation_count":51,"is_preprint":false},{"pmid":"17636002","id":"PMC_17636002","title":"CLIC4 mediates and is required for Ca2+-induced keratinocyte differentiation.","date":"2007","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/17636002","citation_count":50,"is_preprint":false},{"pmid":"16358817","id":"PMC_16358817","title":"CLIC4, an intracellular chloride channel protein, is a novel molecular target for cancer therapy.","date":"2005","source":"The journal of investigative dermatology. Symposium proceedings","url":"https://pubmed.ncbi.nlm.nih.gov/16358817","citation_count":50,"is_preprint":false},{"pmid":"30582444","id":"PMC_30582444","title":"CLIC4/Arf6 Pathway.","date":"2019","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/30582444","citation_count":48,"is_preprint":false},{"pmid":"22761775","id":"PMC_22761775","title":"Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22761775","citation_count":48,"is_preprint":false},{"pmid":"21469130","id":"PMC_21469130","title":"Role of CLIC4 in the host innate responses to bacterial lipopolysaccharide.","date":"2011","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/21469130","citation_count":47,"is_preprint":false},{"pmid":"19776349","id":"PMC_19776349","title":"Spatiotemporal regulation of chloride intracellular channel protein CLIC4 by RhoA.","date":"2009","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/19776349","citation_count":46,"is_preprint":false},{"pmid":"22387366","id":"PMC_22387366","title":"CLIC4 is a tumor suppressor for cutaneous squamous cell cancer.","date":"2012","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/22387366","citation_count":41,"is_preprint":false},{"pmid":"26786190","id":"PMC_26786190","title":"CLIC4 regulates apical exocytosis and renal tube luminogenesis through retromer- and actin-mediated endocytic trafficking.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/26786190","citation_count":40,"is_preprint":false},{"pmid":"36269835","id":"PMC_36269835","title":"CLIC4 localizes to mitochondrial-associated membranes and mediates cardioprotection.","date":"2022","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/36269835","citation_count":39,"is_preprint":false},{"pmid":"35397614","id":"PMC_35397614","title":"N6-methyladenosine demethylase FTO suppressed prostate cancer progression by maintaining CLIC4 mRNA stability.","date":"2022","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/35397614","citation_count":39,"is_preprint":false},{"pmid":"20504765","id":"PMC_20504765","title":"S-nitrosylation regulates nuclear translocation of chloride intracellular channel protein CLIC4.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20504765","citation_count":38,"is_preprint":false},{"pmid":"30282979","id":"PMC_30282979","title":"CLIC1 and CLIC4 complement CA125 as a diagnostic biomarker panel for all subtypes of epithelial ovarian cancer.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30282979","citation_count":37,"is_preprint":false},{"pmid":"35042858","id":"PMC_35042858","title":"Retinal pigment epithelium-specific CLIC4 mutant is a mouse model of dry age-related macular degeneration.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35042858","citation_count":36,"is_preprint":false},{"pmid":"17443730","id":"PMC_17443730","title":"CLIC4, skin homeostasis and cutaneous cancer: surprising connections.","date":"2007","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/17443730","citation_count":34,"is_preprint":false},{"pmid":"33439448","id":"PMC_33439448","title":"Astragaloside IV alleviates atherosclerosis through targeting circ_0000231/miR-135a-5p/CLIC4 axis in AS cell model in vitro.","date":"2021","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33439448","citation_count":33,"is_preprint":false},{"pmid":"26816615","id":"PMC_26816615","title":"Knockdown of CLIC4 enhances ATP-induced HN4 cell apoptosis through mitochondrial and endoplasmic reticulum pathways.","date":"2016","source":"Cell & bioscience","url":"https://pubmed.ncbi.nlm.nih.gov/26816615","citation_count":32,"is_preprint":false},{"pmid":"12237120","id":"PMC_12237120","title":"Overexpressed chloride intracellular channel protein CLIC4 (p64H1) is an essential component of novel plasma membrane anion channels.","date":"2002","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12237120","citation_count":31,"is_preprint":false},{"pmid":"34363182","id":"PMC_34363182","title":"Dexmedetomidine Alleviates Lipopolysaccharide-Induced Hippocampal Neuronal Apoptosis via Inhibiting the p38 MAPK/c-Myc/CLIC4 Signaling Pathway in Rats.","date":"2021","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/34363182","citation_count":31,"is_preprint":false},{"pmid":"23380911","id":"PMC_23380911","title":"Suppression of CLIC4/mtCLIC enhances hydrogen peroxide-induced apoptosis in C6 glioma cells.","date":"2013","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/23380911","citation_count":29,"is_preprint":false},{"pmid":"25830089","id":"PMC_25830089","title":"Clic4, a novel protein that sensitizes β-cells to apoptosis.","date":"2015","source":"Molecular metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/25830089","citation_count":27,"is_preprint":false},{"pmid":"24503901","id":"PMC_24503901","title":"Proteome analysis for downstream targets of oncogenic KRAS--the potential participation of CLIC4 in carcinogenesis in the lung.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24503901","citation_count":24,"is_preprint":false},{"pmid":"31879279","id":"PMC_31879279","title":"CLIC4 and CLIC1 bridge plasma membrane and cortical actin network for a successful cytokinesis.","date":"2019","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/31879279","citation_count":23,"is_preprint":false},{"pmid":"33585442","id":"PMC_33585442","title":"MiR-146a-5p Mimic Inhibits NLRP3 Inflammasome Downstream Inflammatory Factors and CLIC4 in Neonatal Necrotizing Enterocolitis.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33585442","citation_count":23,"is_preprint":false},{"pmid":"30080203","id":"PMC_30080203","title":"Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro.","date":"2018","source":"Journal of visualized experiments : JoVE","url":"https://pubmed.ncbi.nlm.nih.gov/30080203","citation_count":23,"is_preprint":false},{"pmid":"33879602","id":"PMC_33879602","title":"CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and function.","date":"2021","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/33879602","citation_count":22,"is_preprint":false},{"pmid":"29462791","id":"PMC_29462791","title":"The cellular chloride channels CLIC1 and CLIC4 contribute to virus-mediated cell motility.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29462791","citation_count":21,"is_preprint":false},{"pmid":"22613027","id":"PMC_22613027","title":"Spontaneous skin erosions and reduced skin and corneal wound healing characterize CLIC4(NULL) mice.","date":"2012","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/22613027","citation_count":21,"is_preprint":false},{"pmid":"20617112","id":"PMC_20617112","title":"CLIC4 and Schnurri-2: a dynamic duo in TGF-beta signaling with broader implications in cellular homeostasis and disease.","date":"2010","source":"Nucleus (Austin, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/20617112","citation_count":19,"is_preprint":false},{"pmid":"27582103","id":"PMC_27582103","title":"Both CLIC4 and CLIC5A activate ERM proteins in glomerular endothelium.","date":"2016","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/27582103","citation_count":18,"is_preprint":false},{"pmid":"37317855","id":"PMC_37317855","title":"CLIC4 Regulates Endothelial Barrier Control by Mediating PAR1 Signaling via RhoA.","date":"2023","source":"Arteriosclerosis, thrombosis, and vascular biology","url":"https://pubmed.ncbi.nlm.nih.gov/37317855","citation_count":17,"is_preprint":false},{"pmid":"30381396","id":"PMC_30381396","title":"Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA-mDia2 signaling and filopodium formation.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30381396","citation_count":17,"is_preprint":false},{"pmid":"31744714","id":"PMC_31744714","title":"MicroRNA-217-5p ameliorates endothelial cell apoptosis induced by ox-LDL by targeting CLIC4.","date":"2019","source":"Nutrition, metabolism, and cardiovascular diseases : NMCD","url":"https://pubmed.ncbi.nlm.nih.gov/31744714","citation_count":17,"is_preprint":false},{"pmid":"24886590","id":"PMC_24886590","title":"Detection of differential fetal and adult expression of chloride intracellular channel 4 (CLIC4) protein by analysis of a green fluorescent protein knock-in mouse line.","date":"2014","source":"BMC developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/24886590","citation_count":16,"is_preprint":false},{"pmid":"32184265","id":"PMC_32184265","title":"CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.","date":"2020","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/32184265","citation_count":16,"is_preprint":false},{"pmid":"18302930","id":"PMC_18302930","title":"CLIC4 interacts with histamine H3 receptor and enhances the receptor cell surface expression.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18302930","citation_count":16,"is_preprint":false},{"pmid":"35727842","id":"PMC_35727842","title":"Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence.","date":"2022","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35727842","citation_count":12,"is_preprint":false},{"pmid":"24708746","id":"PMC_24708746","title":"Absence of chloride intracellular channel 4 (CLIC4) predisposes to acute kidney injury but has minimal impact on recovery.","date":"2014","source":"BMC nephrology","url":"https://pubmed.ncbi.nlm.nih.gov/24708746","citation_count":12,"is_preprint":false},{"pmid":"32509387","id":"PMC_32509387","title":"CLIC4 regulates radioresistance of nasopharyngeal carcinoma by iNOS after γ-rays but not carbon ions irradiation.","date":"2020","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/32509387","citation_count":12,"is_preprint":false},{"pmid":"37458329","id":"PMC_37458329","title":"Sodium Danshensu ameliorates cerebral ischemia/reperfusion injury by inhibiting CLIC4/NLRP3 inflammasome-mediated endothelial cell pyroptosis.","date":"2023","source":"BioFactors (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/37458329","citation_count":12,"is_preprint":false},{"pmid":"35159339","id":"PMC_35159339","title":"Induction of Pro-Fibrotic CLIC4 in Dermal Fibroblasts by TGF-β/Wnt3a Is Mediated by GLI2 Upregulation.","date":"2022","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/35159339","citation_count":11,"is_preprint":false},{"pmid":"35863434","id":"PMC_35863434","title":"The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells.","date":"2022","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35863434","citation_count":11,"is_preprint":false},{"pmid":"27536941","id":"PMC_27536941","title":"Elevating CLIC4 in Multiple Cell Types Reveals a TGF- Dependent Induction of a Dominant Negative Smad7 Splice Variant.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27536941","citation_count":11,"is_preprint":false},{"pmid":"33822672","id":"PMC_33822672","title":"Long non-coding RNA TDRG1 facilitates cell proliferation, migration and invasion in breast cancer via targeting miR-214-5p/CLIC4 axis.","date":"2021","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/33822672","citation_count":11,"is_preprint":false},{"pmid":"31921386","id":"PMC_31921386","title":"Head and neck squamous cancer progression is marked by CLIC4 attenuation in tumor epithelium and reciprocal stromal upregulation of miR-142-3p, a novel post-transcriptional regulator of CLIC4.","date":"2019","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/31921386","citation_count":11,"is_preprint":false},{"pmid":"33703973","id":"PMC_33703973","title":"Effects of CLIC4 on Fucoxanthinol-Induced Apoptosis in Human Colorectal Cancer Cells.","date":"2020","source":"Nutrition and cancer","url":"https://pubmed.ncbi.nlm.nih.gov/33703973","citation_count":10,"is_preprint":false},{"pmid":"31560739","id":"PMC_31560739","title":"CLIC4 abrogation promotes epithelial-mesenchymal transition in gastric cancer.","date":"2020","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/31560739","citation_count":8,"is_preprint":false},{"pmid":"33331912","id":"PMC_33331912","title":"The intracellular chloride channel 4 (CLIC4) activates systemic sclerosis fibroblasts.","date":"2021","source":"Rheumatology (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/33331912","citation_count":8,"is_preprint":false},{"pmid":"32667519","id":"PMC_32667519","title":"Modification in CLIC4 Expression is Associated with P53, TGF-β, TNF-α and Myofibroblasts in Lip Carcinogenesis.","date":"2020","source":"Brazilian dental journal","url":"https://pubmed.ncbi.nlm.nih.gov/32667519","citation_count":8,"is_preprint":false},{"pmid":"29518790","id":"PMC_29518790","title":"Impact of Xenon on CLIC4 and Bcl-2 Expression in Lipopolysaccharide and Hypoxia-Ischemia-Induced Periventricular White Matter Damage.","date":"2018","source":"Neonatology","url":"https://pubmed.ncbi.nlm.nih.gov/29518790","citation_count":7,"is_preprint":false},{"pmid":"28636115","id":"PMC_28636115","title":"SiRNA-Mediated Down-Regulation of CLIC4 Gene Inhibits Cell Proliferation and Accelerates Cell Apoptosis of Mouse Liver Cancer Hca-F and Hca-P Cells.","date":"2017","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28636115","citation_count":7,"is_preprint":false},{"pmid":"32145706","id":"PMC_32145706","title":"The chloride intracellular channel protein CLIC4 inhibits filopodium formation induced by constitutively active mutants of formin mDia2.","date":"2020","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/32145706","citation_count":5,"is_preprint":false},{"pmid":"33361034","id":"PMC_33361034","title":"Effects of miR-532-5p on human brain microvascular endothelial cells damage induced by ox-LDL via down-regulating CLIC4 expression.","date":"2020","source":"Pakistan journal of pharmaceutical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33361034","citation_count":5,"is_preprint":false},{"pmid":"38390782","id":"PMC_38390782","title":"RNA-binding protein DND1 participates in migration, invasion, and EMT of prostate cancer cells by degrading CLIC4.","date":"2024","source":"Histology and histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/38390782","citation_count":4,"is_preprint":false},{"pmid":"41317264","id":"PMC_41317264","title":"RELM-β Augmented Hypoxia-Induced Pulmonary Hypertension Through Interacting with GIPC1, OR1N1 and CLIC4.","date":"2025","source":"Cardiovascular drugs and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/41317264","citation_count":4,"is_preprint":false},{"pmid":"40709379","id":"PMC_40709379","title":"A novel tRNA‑derived small RNA, 5'tiRNA‑Gln‑TTG‑001, aggravates cardiomyocyte inflammatory injury through upregulation of CLIC4.","date":"2025","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/40709379","citation_count":3,"is_preprint":false},{"pmid":"37026609","id":"PMC_37026609","title":"The stromal immunoexpression of CLIC4 may be related to the difference in the biological behavior between oral squamous cell carcinoma and oral verrucous carcinoma.","date":"2023","source":"Medicina oral, patologia oral y cirugia bucal","url":"https://pubmed.ncbi.nlm.nih.gov/37026609","citation_count":3,"is_preprint":false},{"pmid":"34697147","id":"PMC_34697147","title":"Detection of Cells Displaying High Expression of CLIC4 in Tumor Tissue of Patients With Colorectal Cancer.","date":"2021","source":"In vivo (Athens, Greece)","url":"https://pubmed.ncbi.nlm.nih.gov/34697147","citation_count":3,"is_preprint":false},{"pmid":"35732323","id":"PMC_35732323","title":"Requirement of CLIC4 Expression in Human Colorectal Cancer Cells for Sensitivity to Growth Inhibition by Fucoxanthinol.","date":"2022","source":"Cancer genomics & proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/35732323","citation_count":2,"is_preprint":false},{"pmid":"16842122","id":"PMC_16842122","title":"Expression, purification, crystallization and preliminary crystallographic analysis of the human intracellular chloride channel protein CLIC4.","date":"2006","source":"Protein and peptide letters","url":"https://pubmed.ncbi.nlm.nih.gov/16842122","citation_count":2,"is_preprint":false},{"pmid":"41265251","id":"PMC_41265251","title":"Oxidative modification of G-quadruplex triggers CLIC4-associated mitochondrial dysfunction to promote glioblastoma progression.","date":"2025","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/41265251","citation_count":1,"is_preprint":false},{"pmid":"38727794","id":"PMC_38727794","title":"CLIC4 Function in the Epithelial-Mesenchymal Transition of Epithelial Odontogenic Lesions.","date":"2024","source":"Head and neck pathology","url":"https://pubmed.ncbi.nlm.nih.gov/38727794","citation_count":1,"is_preprint":false},{"pmid":"40114969","id":"PMC_40114969","title":"A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression.","date":"2025","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/40114969","citation_count":1,"is_preprint":false},{"pmid":"34440131","id":"PMC_34440131","title":"DNA Hypermethylation Involves in the Down-Regulation of Chloride Intracellular Channel 4 (CLIC4) Induced by Photodynamic Therapy.","date":"2021","source":"Biomedicines","url":"https://pubmed.ncbi.nlm.nih.gov/34440131","citation_count":1,"is_preprint":false},{"pmid":"27942027","id":"PMC_27942027","title":"Correction: Elevating CLIC4 in Multiple Cell Types Reveals a TGF-β Dependent Induction of a Dominant Negative Smad7 Splice Variant.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27942027","citation_count":1,"is_preprint":false},{"pmid":"41341490","id":"PMC_41341490","title":"CLIC1 and CLIC4 demonstrate cell protective antioxidant activity against UV exposure.","date":"2025","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/41341490","citation_count":0,"is_preprint":false},{"pmid":"41715063","id":"PMC_41715063","title":"Chloride Intracellular Channel 4 (CLIC4) controls volume regulation in sperm development via Protein Kinase C (PKC).","date":"2026","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/41715063","citation_count":0,"is_preprint":false},{"pmid":"41922125","id":"PMC_41922125","title":"CLIC5B attenuates pro-inflammatory fibroblast migration through inhibition of the CLIC1/CLIC4-PIP5K1 axis.","date":"2026","source":"Tissue & cell","url":"https://pubmed.ncbi.nlm.nih.gov/41922125","citation_count":0,"is_preprint":false},{"pmid":"41935590","id":"PMC_41935590","title":"Chloride intracellular channel 4 contributes to Aβ-induced cognitive impairment in mice through the regulation of mitochondrial fission. CLIC4 promotes mitochondrial fission.","date":"2026","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/41935590","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.01.22.634370","title":"Chloride intracellular channel (CLIC) protein function in S1P-induced Rac1 activation requires membrane localization of the C-terminus, but not thiol-transferase nor ion channel activities","date":"2025-01-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.22.634370","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.04.01.25324976","title":"A framework for studying multi-omic risk factors and their interplay: application to coronary artery disease","date":"2025-04-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.01.25324976","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47693,"output_tokens":11110,"usd":0.154864,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":22071,"output_tokens":7705,"usd":0.15149,"stage2_stop_reason":"end_turn"},"total_usd":0.306354,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"CLIC4 (p64H1) localizes to the endoplasmic reticulum when expressed in HEK-293 and HT-4 cells; incorporation of HEK-293 ER vesicles into planar lipid bilayers produced intermediate-conductance, outwardly rectifying anion channels. Protein kinase C phosphorylation of p64H1 increased its apparent molecular weight from ~29 kDa to ~43 kDa.\",\n      \"method\": \"In vitro expression, immunolocalization, planar lipid bilayer electrophysiology, in vitro PKC phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct channel reconstitution in lipid bilayers plus biochemical phosphorylation assay; foundational characterization paper\",\n      \"pmids\": [\"9295337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CLIC4 directly binds dynamin I and 14-3-3ζ (confirmed by gel overlay and reverse pull-down), and also associates with α-tubulin, β-actin, creatine kinase, and two 14-3-3 isoforms in rat brain (confirmed by affinity chromatography, mass spectrometry, and co-immunoprecipitation). CLIC4 partially co-localizes with caveolin and functional caveolae in HEK-293 cells, implicating it in caveolar endocytosis.\",\n      \"method\": \"Affinity chromatography, mass spectrometry, co-immunoprecipitation, gel overlay, reverse pull-down, immunofluorescence\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (pulldown, reciprocal Co-IP, gel overlay) identifying direct binding partners\",\n      \"pmids\": [\"11563969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CLIC4 (mtCLIC) associates with the inner mitochondrial membrane. Overexpression reduces mitochondrial membrane potential, releases cytochrome c, activates caspases, and induces apoptosis. CLIC4 is transcriptionally regulated by p53 and TNF-α. CLIC4 antisense prevents p53-induced apoptosis but not Bax-induced apoptosis, placing CLIC4 in an independent proapoptotic pathway converging on mitochondria.\",\n      \"method\": \"Subcellular fractionation, transient transfection overexpression, mitochondrial membrane potential assay, cytochrome c release assay, caspase activation assay, antisense knockdown, genetic epistasis with Bax\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional assays with epistasis analysis in the same study\",\n      \"pmids\": [\"11997498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Overexpressed CLIC4 in stably transfected HEK-293 cells forms novel low-conductance (~1 pS) plasma membrane anion channels with mild outward rectification, sensitive to IAA (IC50 ~100 µM). Anti-CLIC4 antibodies applied to the cytoplasmic face (but not external face) inhibit these channels, establishing that the C-terminus of the integral membrane form of CLIC4 faces the cytoplasm.\",\n      \"method\": \"Stable transfection, patch-clamp electrophysiology (whole-cell and single-channel), antibody inhibition from cytoplasmic vs. external face\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct channel recording with topology determination by sidedness of antibody block; single lab but rigorous electrophysiological approach\",\n      \"pmids\": [\"12237120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TGF-β1 specifically upregulates CLIC4 (>16-fold) during fibroblast-to-myofibroblast conversion, an effect not shared by CLIC1, CLIC2, CLIC3, or CLIC5. Conditional expression of CLIC4 in MEF/3T3 fibroblasts inhibits cell motility by 27% in a migration assay.\",\n      \"method\": \"Differential display mRNA profiling, RT-PCR, tetracycline-regulated conditional expression, migration assay\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (expression profiling plus functional migration assay) in a single lab\",\n      \"pmids\": [\"12163372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Multiple stress inducers (DNA damage, apoptotic stimuli) cause translocation of cytoplasmic CLIC4 to the nucleus. CLIC4 associates with Ran, NTF2, and Importin-α nuclear import complexes. Deletion or mutation of the C-terminal nuclear localization signal abolishes nuclear translocation; N-terminal deletion enhances it. Nuclear-targeted CLIC4 accelerates apoptosis and induces apoptosis even in Apaf-null fibroblasts or Bcl-2-overexpressing keratinocytes.\",\n      \"method\": \"Immunogold EM, confocal microscopy, co-immunoprecipitation, deletion/mutation analysis, adenoviral nuclear targeting, apoptosis assays in Apaf-null and Bcl-2-overexpressing cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (EM, IP, mutagenesis, genetic epistasis) characterizing nuclear import mechanism\",\n      \"pmids\": [\"14610078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CLIC4 colocalizes with AKAP350 at the centrosome and midbody in cultured mammalian cells, and with AKAP350 and the tight junction protein ZO-1 in the apical region of polarized epithelial cells. CLIC4 is enriched in mitochondria, cortical actin-based structures, and the nuclear matrix, and associates with microtubule cytoskeletal proteins biochemically.\",\n      \"method\": \"Immunofluorescence microscopy, subcellular fractionation, biochemical co-sedimentation\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by immunofluorescence and fractionation, but functional consequence not fully established in this study\",\n      \"pmids\": [\"14569596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structure of CLIC4 resolved at 1.8 Å by X-ray crystallography. CLIC4 is monomeric and adopts a GST fold, similar to CLIC1 but with differences in helix 2 of the glutaredoxin-like N-terminal domain. Purified recombinant CLIC4 binds artificial lipid bilayers, induces chloride efflux when associated with liposomes, and forms a 30 pS ion channel in artificial bilayers. Oxidation enhances membrane binding; no channels were observed under reducing conditions.\",\n      \"method\": \"X-ray crystallography (1.8 Å), lipid bilayer reconstitution, chloride efflux assay, tip-dip electrophysiology, oxidation/reduction manipulation\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus reconstituted ion channel activity with redox regulation established in vitro\",\n      \"pmids\": [\"16176272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CLIC4 protein levels are upregulated by c-Myc; Myc binds directly to the CLIC4 gene promoter and activates its transcription (by quantitative proteomics and ChIP). Suppression of CLIC4 by RNAi inhibits Myc-induced apoptosis under stress conditions and abolishes cooperative induction of apoptosis by Myc and Bax.\",\n      \"method\": \"Isotope-coded affinity tag quantitative proteomics, chromatin immunoprecipitation, RNAi knockdown, apoptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP demonstrates direct promoter binding by Myc; functional RNAi epistasis with multiple stress conditions\",\n      \"pmids\": [\"16316993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CLIC4 expression decreases during VEGF-A-induced endothelial cell tubular morphogenesis. siRNA- or antisense-mediated suppression of CLIC4 arrests tubular morphogenesis in vitro, establishing a required role for CLIC4 in endothelial tube/lumen formation.\",\n      \"method\": \"2D proteomics, antisense and siRNA knockdown, in vitro tubulogenesis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA and antisense knockdown with specific tubulogenesis phenotype in a single lab\",\n      \"pmids\": [\"16239224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Reconstituted recombinant CLIC4 in planar lipid bilayers forms redox-regulated, poorly selective ion channels (maximum ~15 pS in KCl). A truncated version comprising only the N-terminal 61 residues (containing the predicted transmembrane domain) also forms non-selective channels with reduced conductance that retain trans-redox sensitivity and can be blocked by trans (not cis) thiol-reactive DTNB, suggesting the predicted TMD forms oligomeric pores and the trans cysteine is at the external pore entrance.\",\n      \"method\": \"Planar lipid bilayer reconstitution, site-specific truncation mutant analysis, redox manipulation, thiol-reactive DTNB block from trans/cis sides\",\n      \"journal\": \"Molecular membrane biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis/truncation approach probing pore topology, single lab\",\n      \"pmids\": [\"17453412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CLIC1 and CLIC5 channel activity in planar bilayers is strongly and reversibly inhibited by F-actin; CLIC4 channels are NOT inhibited by F-actin under the same conditions, demonstrating differential actin regulation among CLIC family members.\",\n      \"method\": \"Planar lipid bilayer reconstitution with addition of F-actin; cytochalasin reversal experiment\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro reconstitution assay with pharmacological reversal; negative result for CLIC4 is itself mechanistically informative\",\n      \"pmids\": [\"18028448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CLIC4 expression is reduced in multiple human epithelial cancers and excluded from the nucleus in cancer cells. In xenografts, adenoviral introduction of CLIC4 or nuclear-targeted CLIC4 into breast cancer cells inhibits tumor growth, whereas overexpression of CLIC4 in stromal cells enhances tumor growth.\",\n      \"method\": \"Tissue microarray, adenoviral transduction into xenografts, CLIC4 overexpression in stromal cells, in vivo tumor growth assay\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo functional data with nuclear targeting; single lab\",\n      \"pmids\": [\"17200346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CLIC4 physically interacts with the C-terminus of the histamine H3 receptor, confirmed by in vitro pull-down, co-immunoprecipitation from rat brain lysate, and immunofluorescence co-localization in rat cerebellar neurons. CLIC4 enhances cell surface expression of H3R, but not a mutant H3R that cannot interact with CLIC4, as measured by flow cytometry, radioligand binding, and cell-based ELISA.\",\n      \"method\": \"In vitro pull-down, co-immunoprecipitation from rat brain, immunofluorescence, flow cytometry, radioligand binding assay, cell-based ELISA\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods confirming interaction and functional consequence (receptor surface expression) in the same study\",\n      \"pmids\": [\"18302930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TGF-β promotes CLIC4 and Schnurri-2 expression, their cytoplasmic association, and their co-translocation to the nucleus. In the nucleus, CLIC4 associates with phospho-Smad2 and phospho-Smad3, protecting them from dephosphorylation by nuclear phosphatases, thereby sustaining TGF-β signaling. In the absence of CLIC4 or Schnurri-2, TGF-β signaling is abrogated; direct nuclear targeting of CLIC4 removes the requirement for Schnurri-2.\",\n      \"method\": \"Co-immunoprecipitation, nuclear fractionation, siRNA knockdown, adenoviral nuclear targeting, TGF-β signaling reporter assays, phospho-Smad stabilization assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, knockdown, nuclear targeting epistasis, signaling assays) in a high-impact journal\",\n      \"pmids\": [\"19448624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of Gα13-coupled, RhoA-activating receptors (LPA, thrombin, S1P). This translocation is strictly dependent on Gα13-mediated RhoA activation and F-actin integrity but not Rho kinase activity. Mutational analysis reveals dependence on at least six conserved residues including reactive Cys35. Membrane-targeted CLIC4 does not modulate transmembrane chloride currents.\",\n      \"method\": \"Live-cell imaging, pharmacological inhibitors (Y-27632, cytochalasin), dominant-negative/constitutively active RhoA constructs, site-directed mutagenesis, electrophysiology\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging with multiple genetic and pharmacological perturbations and mutagenesis in the same study\",\n      \"pmids\": [\"19776349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CLIC4 is directly S-nitrosylated on a cysteine residue (detected by biotin switch assay), and this modification induces conformational unfolding (CD spectra, trypsinolysis) and enhanced association with importin-α and Ran, promoting nuclear translocation. TNF-α-induced nuclear translocation of CLIC4 depends on nitric oxide synthase activity, and NOS inhibition blocks TNF-α-induced CLIC4 nitrosylation and nuclear import. Cysteine mutants show altered nitrosylation, nuclear residence, and stability.\",\n      \"method\": \"Biotin switch assay (S-nitrosylation detection), CD spectroscopy, limited trypsinolysis, co-immunoprecipitation with importin-α/Ran, NOS inhibition, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical detection of PTM with multiple orthogonal methods (biotin switch, CD, IP) and mutagenesis\",\n      \"pmids\": [\"20504765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CLIC4-null macrophages show reduced accumulation of phosphorylated IRF3 upon LPS stimulation, while CLIC4 overexpression enhances LPS-mediated IRF3 phosphorylation. CLIC4-null mice are protected from LPS-induced death with reduced serum inflammatory cytokines, and are impaired in Listeria monocytogenes clearance. Deletion of CLIC4 had little effect on MAPK and NF-κB activation, placing CLIC4 specifically in the IRF3 arm of LPS signaling.\",\n      \"method\": \"CLIC4-null mouse generation, LPS challenge in vivo, Western blot for phospho-IRF3/MAPK/NF-κB, stable CLIC4-overexpressing macrophage lines, Listeria infection assay\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with in vivo phenotype and mechanistic epistasis (IRF3 vs MAPK/NF-κB), replicated by overexpression\",\n      \"pmids\": [\"21469130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CLIC4-null mice exhibit delayed wound reepithelialization and corneal wound healing, reduced β4 integrin and p21 expression in wounded skin, reduced TGF-β-induced phospho-Smad2 in CLIC4-null keratinocytes, slower keratinocyte migration, and failure to increase migration in response to TGF-β, placing CLIC4 upstream of TGF-β signaling in epidermal wound healing.\",\n      \"method\": \"CLIC4-null mouse (C57Bl/6 background), full-thickness skin wound and corneal wound assays, Western blot, keratinocyte migration assay, TGF-β stimulation of cultured keratinocytes\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple in vivo and in vitro phenotypic readouts establishing TGF-β pathway dependency\",\n      \"pmids\": [\"22613027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In metabolically stressed keratinocytes, CLIC4 is S-nitrosylated and translocates to the nucleus, where it enhances TGF-β signaling by protecting phospho-Smad2/3 from dephosphorylation. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear CLIC4 translocation. Adenoviral nuclear targeting of CLIC4 in squamous cancer cells enhances TGF-β transcriptional activity and inhibits growth in vitro and in orthograft tumors.\",\n      \"method\": \"Adenoviral nuclear targeting, TGF-β reporter assay, S-nitrosylation assay, tumor orthograft model, transgenic epidermis overexpression model\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo and in vitro experiments with mechanistic pathway dissection (S-nitrosylation → nuclear import → TGF-β/Smad signaling)\",\n      \"pmids\": [\"22387366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CLIC4 is required for TGF-β-induced activation of p38 MAPK in stromal fibroblasts, and this requirement involves interaction of CLIC4 with PPM1a, the selective phosphatase of activated p38. Genetic ablation of CLIC4 in primary fibroblasts prevents TGF-β-induced expression of α-SMA and extracellular matrix components. Conditioned media from CLIC4-overexpressing fibroblasts increases tumor cell migration/invasion and promotes EMT in a TGF-β-dependent manner.\",\n      \"method\": \"CLIC4 knockout primary fibroblasts, co-immunoprecipitation (CLIC4–PPM1a), p38 MAPK phosphorylation assay, conditioned medium experiments, migration/invasion assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic ablation plus Co-IP identifying PPM1a as a CLIC4-binding phosphatase, multiple functional readouts\",\n      \"pmids\": [\"23416981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CLIC4 knockdown in HeLa and MDA-MB-231 cells decreases cell-matrix adhesion, cell spreading, and integrin signaling, while increasing cell motility. LPA stimulates recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both internalization and serum/LPA-induced recycling of β1 integrin (but not EGFR). CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking.\",\n      \"method\": \"siRNA knockdown, live-cell imaging, co-immunoprecipitation, integrin internalization/recycling assays, Rab35 activity assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal assays (trafficking, signaling, GTPase activity) with receptor-specific controls in a single study\",\n      \"pmids\": [\"25344254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Clic4 silencing or β-cell-specific knockout reduces cytokine-induced apoptosis, associated with increased expression and stability of Bcl-2, Bad, and phosphorylated Bad. Mass spectrometry of co-immunoprecipitated proteins found no direct association of CLIC4 with Bcl-2 family proteins, but CLIC4 co-purified with proteasome components, suggesting CLIC4 regulates Bcl-2/Bad stability via proteasomal degradation.\",\n      \"method\": \"siRNA knockdown, β-cell-specific knockout mice, co-immunoprecipitation with mass spectrometry, Bcl-2/Bad half-life measurement, cytokine-induced apoptosis assay\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and Co-IP/MS; mechanism (proteasome association) is suggestive rather than fully demonstrated, single lab\",\n      \"pmids\": [\"25830089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CLIC4 is required for apical exocytosis and lumen formation in renal tubulogenesis. CLIC4-null embryos have impaired renal tubulogenesis; in MDCK 3D cultures, CLIC4 localizes to early endosomes, recycling endosomes, and apical transport carriers before reaching apical membrane at steady state. CLIC4 suppression impairs apical vesicle coalescence and lumen formation, rescued by Rab8 and Cdc42. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating branched actin formation on early endosomes.\",\n      \"method\": \"CLIC4-null mouse embryos, MDCK 3D culture lumenogenesis assay, live confocal imaging, Rab8/Cdc42 rescue experiments, retromer knockdown, actin branching analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with in vivo phenotype, mechanistic dissection of retromer/actin pathway, rescue experiments in a high-impact journal\",\n      \"pmids\": [\"26786190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Elevated CLIC4 induces expression of Smad7Δ, a novel truncated/alternatively spliced form of Smad7 missing 94 bp in exon 4 (predicted to lack the TGF-β inhibitory MH2 domain). Smad7Δ acts as a dominant-negative inhibitor of full-length Smad7, thereby further enhancing TGF-β signaling. TGF-β treatment also enhances Smad7Δ expression, amplified by CLIC4.\",\n      \"method\": \"CLIC4 overexpression in multiple cell types, RT-PCR splice variant identification, TGF-β reporter assay, proliferation assay, Smad phosphorylation analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — identification of splice variant with functional dominant-negative assay; single lab\",\n      \"pmids\": [\"27536941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CLIC4 activates ERM proteins (ezrin, radixin, moesin) in glomerular endothelial cells; CLIC4 silencing reduces ERM phosphorylation and cytoskeletal association, and exogenous CLIC4 rescues ERM dephosphorylation. Mice lacking both CLIC4 and CLIC5 show profound reduction of glomerular EC ERM phosphorylation and develop glomerular capillary architectural defects, proteinuria, and glomerular cell proliferation.\",\n      \"method\": \"CLIC4/CLIC5 knockout mice (single and double), CLIC4 siRNA in cultured glomerular EC, exogenous CLIC4 expression rescue, ERM phosphorylation assay, histopathology\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — double knockout genetic model with in vitro rescue and specific molecular readout (ERM phosphorylation)\",\n      \"pmids\": [\"27582103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Upon LPS stimulation of macrophages, CLIC4 translocates to the nucleus and cellular membrane (detected by confocal microscopy and cell fractionation). siRNA knockdown of CLIC4 in BMDMs impairs IL-1β transcription, ASC speck formation, and secretion of mature IL-1β in LPS/ATP-stimulated cells, showing CLIC4 participates in both the priming signal (IL-1β transcription) and the NLRP3 inflammasome activation step.\",\n      \"method\": \"Confocal microscopy, cell fractionation, siRNA knockdown, IL-1β transcription assay, ASC speck formation assay, ELISA for mature IL-1β\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal assays (fractionation, imaging, siRNA, inflammasome assembly readouts) in primary macrophages\",\n      \"pmids\": [\"28576828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CLIC4 translocation to the plasma membrane upon LPA or EGF stimulation requires RhoA activation via the RhoA effector mDia2 and depends on F-actin polymerization. CLIC4 binds the G-actin-binding protein profilin-1 via residues required for CLIC4 trafficking. Profilin-1 silencing impairs agonist-induced CLIC4 trafficking and mDia2-dependent filopodium formation. CLIC4 knockdown increases filopodium formation (rescued by wild-type CLIC4 but not trafficking-incompetent CLIC4(C35A)), and CLIC4 accelerates LPA-induced filopodium retraction.\",\n      \"method\": \"Live-cell imaging, siRNA knockdown, co-immunoprecipitation, profilin-1 pull-down, mDia2 overexpression, filopodium quantification, CLIC4 mutant rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct profilin binding shown by pull-down, multiple genetic and imaging approaches, mutant rescue experiments\",\n      \"pmids\": [\"30381396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CLIC4 localizes to the cytokinetic cleavage furrow and midbody in a RhoA-dependent manner. Mutations of GST activity-related residues (C35A, F37D) abolish cell-cycle-dependent CLIC4 localization. CLIC4 interacts with ezrin, anillin, and ALIX at the cleavage furrow and midbody. CLIC4 facilitates ezrin activation at the cleavage furrow; conversely, inhibition of ezrin activation reduces CLIC4 translocation. CLIC4 and CLIC1 double knockout causes abnormal polar cortex blebbing and cleavage furrow regression, resulting in multinucleated cells.\",\n      \"method\": \"Live-cell imaging, immunofluorescence, CLIC1/CLIC4 knockout, site-directed mutagenesis (C35A, F37D), co-immunoprecipitation identifying ezrin/anillin/ALIX, ezrin phosphorylation assay\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with specific cytokinesis phenotype, Co-IP partners, mutagenesis, and reciprocal functional dependency with ezrin\",\n      \"pmids\": [\"31879279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Proteomic analysis of CLIC4-interacting proteins in pulmonary artery endothelial cells identified Arf6 GTPase-activating proteins and clathrin as binding partners. CLIC4 overexpression promotes Arf6-mediated reduction of gyrating clathrin and increased lysosomal targeting of BMPRII, reducing BMPRII expression and signaling. Arf6 siRNA, Arf inhibitor SecinH3, and clathrin-mediated endocytosis inhibitors restore BMPRII expression, but chloride channel inhibitor IAA-94 does not, placing CLIC4 in an Arf6-dependent trafficking pathway independent of its channel activity.\",\n      \"method\": \"Proteomic interactome (Co-IP/MS), Arf6 siRNA, pharmacological inhibitors (SecinH3, IAA-94, clathrin inhibitors), BMPRII expression and signaling assays, sugen/hypoxia and monocrotaline animal models\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proteomic binding partners validated by siRNA epistasis and multiple inhibitors; in vivo disease models replicate findings\",\n      \"pmids\": [\"30582444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLIC4 localizes to the cytokinetic cleavage furrow and is required for successful completion of mitotic cell division. CLIC4 recruits MST4 kinase (STK26) to the cleavage furrow and regulates ezrin phosphorylation, thereby remodeling the sub-plasma-membrane actomyosin network during cytokinesis.\",\n      \"method\": \"Live-cell imaging, CLIC4 knockdown, MST4 kinase recruitment assay, ezrin phosphorylation assay, co-immunoprecipitation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with functional KD and molecular readout (MST4 recruitment, ezrin phosphorylation); single lab, limited orthogonal validation of direct interaction\",\n      \"pmids\": [\"32184265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLIC4 silencing enhances filopodium formation induced by constitutively active mDia2 mutants. CLIC4 binds the actin-regulatory region of mDia2 in vitro (pull-down assay), suggesting CLIC4 modulates the activity of the open conformation of mDia2 to inhibit filopodium formation.\",\n      \"method\": \"siRNA knockdown, in vitro pull-down (CLIC4 with mDia2 actin-regulatory region), filopodium quantification with constitutively active mDia2 mutants\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct binding shown by pull-down plus functional filopodium assay; single lab, limited mechanistic depth in abstract\",\n      \"pmids\": [\"32145706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CLIC1 and CLIC4 in endothelial cells are required for S1P-induced activation of Rac1 downstream of S1PR1. CLIC1 and CLIC4 transiently translocate to the plasma membrane in response to S1P. Only CLIC1 (not CLIC4) was essential for S1P-induced RhoA activation downstream of S1PR2/S1PR3. CLIC1 and CLIC4 are not functionally interchangeable and are critical for S1P-induced endothelial barrier function.\",\n      \"method\": \"siRNA knockdown of CLIC1/CLIC4, live-cell imaging of membrane translocation, Rac1/RhoA GTPase activation assays, endothelial barrier assay, rescue experiments\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — specific GTPase activation assays with receptor-specific knockdown and rescue, establishing GPCR pathway specificity\",\n      \"pmids\": [\"33879602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CLIC4 is present in mitochondrial-associated membranes (MAMs) of cardiomyocytes. CLIC4 loss increases myocardial infarction and reduces cardiac function after ischemia-reperfusion injury. CLIC4-null cardiomyocytes show increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation. CLIC4 modulates ER and mitochondrial calcium homeostasis under physiological and pathological conditions.\",\n      \"method\": \"MAM fractionation, CLIC4-null mouse IR injury model, echocardiography, cardiomyocyte hypoxia-reoxygenation assay, calcium imaging, apoptosis assay\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — subcellular fractionation demonstrating MAM localization combined with in vivo KO phenotype and in vitro calcium measurements\",\n      \"pmids\": [\"36269835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FTO-mediated m6A demethylation stabilizes CLIC4 mRNA; FTO depletion increases m6A methylation on CLIC4 mRNA and reduces its stability, leading to decreased CLIC4 protein.\",\n      \"method\": \"MeRIP-RT-qPCR, mRNA stability assay, RNA-seq, FTO knockdown/overexpression\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct m6A mapping on CLIC4 mRNA with stability assay; single lab\",\n      \"pmids\": [\"35397614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CLIC4 deletion from murine breast tumor cells using CRISPR enhances ROS accumulation, sensitizes cells to H2O2-induced apoptosis, and is associated with mitochondrial hyperactivity (increased membrane potential, organelle enlargement, increased superoxide). In the absence of CLIC4, H2O2-induced apoptosis involves degradation of Bcl2 and UCP2. CLIC4 therefore maintains redox homeostasis and mitochondrial function, consistent with a glutaredoxin-like enzymatic activity.\",\n      \"method\": \"CRISPR knockout, ROS measurement, mitochondrial membrane potential assay, apoptosis assay, transcriptomic profiling, Bcl2/UCP2 protein analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with multiple orthogonal mitochondrial/redox readouts; single lab\",\n      \"pmids\": [\"35863434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CLIC4 is required for thrombin/PAR1-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Thrombin promotes CLIC4 (but not CLIC1) relocalization to HUVEC membranes. Endothelial-specific CLIC4 deletion in mice reduces lung edema and microvascular permeability induced by PAR1 activating peptide. CLIC1 was not required for thrombin-mediated barrier disruption but contributed to barrier recovery.\",\n      \"method\": \"CLIC4/CLIC1 siRNA knockdown, endothelial-specific conditional Clic4 knockout mice (PAR1 peptide-induced lung permeability), RhoA activation assay, ERM phosphorylation assay, endothelial barrier assay\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional in vivo KO with in vitro mechanistic dissection; receptor- and CLIC-specific functional differentiation\",\n      \"pmids\": [\"37317855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CLIC4 is expressed on the plasma membrane of sperm cells and is required for IAA-94-sensitive chloride currents (genetic ablation of CLIC4 eliminates these currents). CLIC4 regulates cell volume during sperm maturation without altering membrane potential, motility, or acrosome reaction. CLIC4 channel activity in sperm is modulated by Protein Kinase C (PKC).\",\n      \"method\": \"Patch-clamp electrophysiology, CLIC4-null mouse sperm, IAA-94 pharmacology, PKC modulation assay, sperm morphology and functional assays\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct channel recording with genetic KO ablation of currents and PKC modulation; single lab\",\n      \"pmids\": [\"41715063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CLIC4 C-terminus (not N-terminus) encodes CLIC4-specific functions required for S1P-induced Rac1 activation in endothelial cells. The CLIC4 N-terminus encodes determinants for S1P-induced plasma membrane relocalization, but is dispensable for Rac1 activation when the C-terminus is targeted to the membrane via a heterologous signal. The postulated ion channel and thiol-transferase (GST) activities of CLIC4 are NOT required for Rac1 activation.\",\n      \"method\": \"Structure-function mutagenesis (N/C-terminal domain swaps, heterologous membrane targeting), Rac1 activation assay, live-cell imaging of plasma membrane translocation, endothelial cell siRNA rescue experiments\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-function analysis with domain-specific rescue; preprint, not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CLIC4 is a multifunctional, metamorphic protein that adopts a soluble GST-omega fold (crystal structure at 1.8 Å) and can auto-insert into membranes to form redox-regulated, poorly selective ion channels; it traffics constitutively between the cytoplasm, plasma membrane (recruited by RhoA/Gα13-coupled receptor activation via mDia2 and profilin-1 binding), mitochondria/MAMs, and nucleus (via S-nitrosylation of Cys35 and importin-α/Ran-mediated import), where nuclear CLIC4 stabilizes phospho-Smad2/3 against dephosphorylation to sustain TGF-β signaling, interacts with Schnurri-2, promotes cell cycle arrest and apoptosis (in part through mitochondrial membrane potential dissipation and cytochrome c release), and regulates cytokinesis by recruiting MST4 kinase and activating ezrin at the cleavage furrow; at the plasma membrane CLIC4 suppresses Rab35 activity to control β1 integrin trafficking, counteracts mDia2-driven filopodium formation, mediates GPCR (S1PR1, PAR1) coupling to Rac1 and RhoA via its C-terminus independently of ion channel or thiol-transferase activity, and promotes NLRP3 inflammasome priming; CLIC4 is also regulated post-translationally by PKC phosphorylation (in sperm) and at the mRNA level by FTO-dependent m6A demethylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CLIC4 is a metamorphic, multifunctional protein that interconverts between a soluble GST-omega/glutaredoxin-like fold (resolved by crystallography at 1.8 Å) and a membrane-inserted form, redistributing among the cytoplasm, plasma membrane, mitochondria, and nucleus to couple cellular redox state and small-GTPase signaling to cytoskeletal dynamics, membrane trafficking, apoptosis, and TGF-β signaling [#7, #15, #16]. Reconstituted recombinant CLIC4 auto-inserts into lipid bilayers under oxidizing conditions to form poorly selective, redox-regulated ion channels whose pore is built by an N-terminal transmembrane segment with the C-terminus facing the cytoplasm [#3, #7, #10]; CLIC4 supplies IAA-94-sensitive chloride currents in native sperm where it controls cell volume and is modulated by PKC [#37]. A defining feature is stress- and agonist-triggered translocation: S-nitrosylation of a reactive cysteine unfolds CLIC4 and promotes importin-α/Ran-dependent nuclear import, where CLIC4 protects phospho-Smad2/3 from dephosphorylation—acting with Schnurri-2 and PPM1a—to sustain TGF-β/p38 signaling, drive myofibroblast conversion and wound reepithelialization, and execute proapoptotic programs converging on mitochondria [#5, #14, #16, #19, #20, #18, #2]. At the plasma membrane, Gα13/RhoA-coupled receptor activation (LPA, thrombin/PAR1, S1P/S1PR1) recruits CLIC4 via mDia2- and profilin-1-dependent actin dynamics, and CLIC4 then transduces these signals to Rac1 and RhoA activation, ERM-protein phosphorylation, and endothelial barrier control through its C-terminus independently of ion-channel or thiol-transferase activity [#15, #27, #32, #36, #38]. CLIC4 also governs endosomal trafficking—suppressing Rab35 to control β1-integrin internalization and recycling and modulating retromer/Arf6-dependent apical transport during lumenogenesis—and is required for cytokinesis, where it localizes to the cleavage furrow, recruits MST4 kinase, and activates ezrin to remodel the cortical actomyosin network [#21, #23, #29, #28, #30]. CLIC4 expression is controlled transcriptionally by p53 and c-Myc and post-transcriptionally by FTO-dependent m6A demethylation [#2, #8, #34].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established CLIC4 as a membrane-associating, channel-forming protein subject to PKC phosphorylation, framing the foundational question of whether it is a bona fide ion channel.\",\n      \"evidence\": \"ER localization in transfected cells plus planar lipid bilayer recordings and in vitro PKC assay\",\n      \"pmids\": [\"9295337\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Channel formation shown for ER-derived vesicles, not purified protein\", \"Physiological ionic substrate and gating unresolved\", \"Functional consequence of PKC phosphorylation unaddressed\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified the first direct cytoskeletal and adaptor partners, placing CLIC4 at the interface of vesicle trafficking and the actin/tubulin cytoskeleton.\",\n      \"evidence\": \"Affinity chromatography, mass spectrometry, reciprocal Co-IP and gel overlay from rat brain identifying dynamin I, 14-3-3, tubulin, actin\",\n      \"pmids\": [\"11563969\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional roles of individual interactions not dissected\", \"Caveolar endocytosis role inferred from colocalization only\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined a mitochondrial, p53/TNF-α-inducible proapoptotic role distinct from Bax, and demonstrated low-conductance plasma membrane channels with cytoplasmic C-terminus topology.\",\n      \"evidence\": \"Mitochondrial fractionation, membrane-potential/cytochrome c/caspase assays with antisense epistasis; patch-clamp with sided antibody block\",\n      \"pmids\": [\"11997498\", \"12237120\", \"12163372\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking membrane insertion to mitochondrial depolarization unresolved\", \"Whether channel activity drives apoptosis untested\", \"TGF-β upregulation of CLIC4 mechanistically uncharacterized at this stage\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Revealed regulated nuclear translocation via importin-α/Ran with a C-terminal NLS, establishing nuclear CLIC4 as a stress-responsive proapoptotic species.\",\n      \"evidence\": \"Immunogold EM, Co-IP with Ran/NTF2/importin-α, NLS deletion/mutation, adenoviral nuclear targeting in Apaf-null and Bcl-2 cells; plus centrosome/midbody/AKAP350 colocalization\",\n      \"pmids\": [\"14610078\", \"14569596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal triggering import not yet defined\", \"Nuclear effectors downstream of CLIC4 unknown at this stage\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Provided the atomic GST-fold structure and reconstituted redox-gated channel activity from purified protein, and placed CLIC4 under c-Myc transcriptional control with required roles in apoptosis and tubulogenesis.\",\n      \"evidence\": \"1.8 Å X-ray structure with bilayer reconstitution/chloride efflux under oxidation; quantitative proteomics and ChIP for Myc; siRNA/antisense tubulogenesis assays\",\n      \"pmids\": [\"16176272\", \"16316993\", \"16239224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure is of the soluble form only; membrane conformation unresolved\", \"How a soluble GST-fold monomer becomes a channel structurally unexplained\", \"Enzymatic substrate of the glutaredoxin-like fold not identified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapped the N-terminal transmembrane segment as the pore-forming, redox-sensitive element and distinguished CLIC4 from CLIC1/CLIC5 by its insensitivity to F-actin inhibition.\",\n      \"evidence\": \"Bilayer reconstitution with N-terminal truncation, trans/cis DTNB block, and comparative F-actin addition experiments\",\n      \"pmids\": [\"17453412\", \"18028448\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Oligomeric stoichiometry of the pore not determined\", \"Physiological relevance of in vitro channel still open\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed CLIC4 acts as a chaperone for GPCR surface delivery, expanding its role beyond channel/cytoskeletal functions.\",\n      \"evidence\": \"Pull-down, Co-IP from rat brain, immunofluorescence, and surface-expression assays with H3 receptor binding-deficient mutant control\",\n      \"pmids\": [\"18302930\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trafficking step at which CLIC4 acts not pinpointed\", \"Generality to other GPCRs untested here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined CLIC4 as a nuclear stabilizer of phospho-Smad2/3 acting with Schnurri-2 to sustain TGF-β signaling, and identified Gα13/RhoA-dependent recruitment to the plasma membrane.\",\n      \"evidence\": \"Co-IP, nuclear fractionation, siRNA, nuclear-targeting epistasis and reporter assays; live imaging with RhoA constructs and Cys35 mutagenesis\",\n      \"pmids\": [\"19448624\", \"19776349\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which CLIC4 blocks Smad dephosphorylation unresolved\", \"Identity of the nuclear phosphatase not defined\", \"Function of membrane-targeted CLIC4 (non-channel) initially unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Connected nitric-oxide signaling to CLIC4 conformational change and nuclear import, defining S-nitrosylation as the molecular switch upstream of TGF-β stabilization.\",\n      \"evidence\": \"Biotin-switch assay, CD spectroscopy, limited trypsinolysis, importin-α/Ran Co-IP, and NOS inhibition with cysteine mutants\",\n      \"pmids\": [\"20504765\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise nitrosylated cysteine residue contribution among multiple cysteines\", \"Reversal/denitrosylation mechanism unaddressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated in vivo immune function by placing CLIC4 selectively in the IRF3 arm of LPS/TLR signaling, separate from MAPK/NF-κB.\",\n      \"evidence\": \"CLIC4-null mice with LPS challenge, phospho-IRF3/MAPK/NF-κB blots, overexpressing macrophages, Listeria clearance\",\n      \"pmids\": [\"21469130\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target of CLIC4 in the IRF3 pathway unidentified\", \"Whether channel or trafficking activity mediates the effect unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed CLIC4 is required in vivo for epidermal/corneal wound healing through TGF-β/Smad signaling, linking S-nitrosylation-driven nuclear CLIC4 to tissue repair and tumor suppression.\",\n      \"evidence\": \"CLIC4-null wound-healing models, keratinocyte migration and phospho-Smad2 assays; S-nitrosylation/nuclear-targeting orthograft tumor experiments\",\n      \"pmids\": [\"22613027\", \"22387366\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream NOS isoform in keratinocytes not specified\", \"Stromal vs epithelial context-dependence of TGF-β output unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified PPM1a as a CLIC4-binding phosphatase and extended TGF-β dependence to p38 MAPK and stromal myofibroblast/EMT-promoting functions.\",\n      \"evidence\": \"CLIC4-knockout primary fibroblasts, CLIC4–PPM1a Co-IP, p38 phosphorylation assays, conditioned-medium migration/invasion experiments\",\n      \"pmids\": [\"23416981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CLIC4 directly inhibits PPM1a catalytic activity not shown\", \"Subcellular site of CLIC4–PPM1a interaction undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established CLIC4 as a Rab35 suppressor controlling β1-integrin internalization and recycling, linking GPCR activation to adhesion and motility.\",\n      \"evidence\": \"siRNA, live imaging, Co-IP, integrin trafficking assays with EGFR control, and Rab35 activity assay\",\n      \"pmids\": [\"25344254\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Rab35 suppression (GAP/GEF vs sequestration) unknown\", \"Direct vs indirect CLIC4–Rab35 relationship unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined CLIC4 roles in retromer/actin-dependent apical lumenogenesis and in amplifying TGF-β via a dominant-negative Smad7 splice form, broadening its trafficking and signaling repertoire.\",\n      \"evidence\": \"CLIC4-null embryos, MDCK 3D lumen assays with Rab8/Cdc42 rescue and retromer knockdown; RT-PCR splice-variant identification with reporter assays\",\n      \"pmids\": [\"26786190\", \"27536941\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CLIC4 negatively regulates branched actin on endosomes mechanistically open\", \"Trans-acting factor producing Smad7Δ splicing unidentified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed CLIC4 activates ERM proteins in glomerular endothelium, with CLIC4/CLIC5 redundancy required for glomerular capillary integrity.\",\n      \"evidence\": \"CLIC4/CLIC5 single and double knockout mice, glomerular EC siRNA with CLIC4 rescue, ERM phosphorylation and histopathology\",\n      \"pmids\": [\"27582103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase/phosphatase through which CLIC4 controls ERM phosphorylation undefined\", \"Direct CLIC4–ERM interaction not established here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Mechanistically resolved agonist-induced membrane recruitment as mDia2/profilin-1/F-actin-dependent and assigned CLIC4 an antagonist role in filopodium dynamics.\",\n      \"evidence\": \"Live imaging, profilin-1 pull-down, mDia2 overexpression, filopodium quantification, and CLIC4(C35A) trafficking-mutant rescue\",\n      \"pmids\": [\"30381396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CLIC4 limits mDia2-driven actin elongation biochemically unclear at this point\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Placed CLIC4 at the cytokinetic furrow with ezrin/anillin/ALIX partners and identified an Arf6/clathrin-dependent BMPRII-degradation pathway relevant to pulmonary hypertension, both independent of channel activity.\",\n      \"evidence\": \"CLIC1/CLIC4 knockout cytokinesis phenotyping with C35A/F37D mutants and Co-IP; interactome with Arf6-GAP/clathrin, Arf6 siRNA, SecinH3/IAA-94/clathrin inhibitors, in vivo PH models\",\n      \"pmids\": [\"31879279\", \"30582444\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs scaffolded nature of ezrin/anillin/ALIX interactions not fully resolved\", \"How CLIC4 modulates Arf6-GAP activity mechanistically open\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified MST4 kinase recruitment as the means by which furrow CLIC4 controls ezrin phosphorylation and cytokinetic actomyosin remodeling, and clarified mDia2 inhibition via direct binding.\",\n      \"evidence\": \"Live imaging with CLIC4 knockdown, MST4 recruitment and ezrin phosphorylation assays, Co-IP; in vitro pull-down with mDia2 actin-regulatory region and constitutively active mDia2 filopodium assays\",\n      \"pmids\": [\"32184265\", \"32145706\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Directness of CLIC4–MST4 interaction not orthogonally validated\", \"Structural basis of CLIC4–mDia2 binding unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated non-interchangeable, receptor-specific roles for CLIC4 and CLIC1 in S1P-driven Rac1 versus RhoA activation and endothelial barrier function.\",\n      \"evidence\": \"CLIC1/CLIC4 siRNA, membrane translocation imaging, Rac1/RhoA activation assays, barrier assays and rescue\",\n      \"pmids\": [\"33879602\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of CLIC4 specificity for Rac1 not defined here\", \"Effector linking CLIC4 to Rac1-GEF unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended CLIC4 to MAM/mitochondrial calcium and redox homeostasis with cardioprotective and ROS-buffering functions consistent with its glutaredoxin-like fold.\",\n      \"evidence\": \"MAM fractionation, CLIC4-null IR injury and hypoxia-reoxygenation models, calcium imaging; CRISPR knockout with ROS/membrane-potential/Bcl2/UCP2 readouts; plus FTO/m6A mRNA-stability mapping\",\n      \"pmids\": [\"36269835\", \"35863434\", \"35397614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CLIC4 directly catalyzes a redox reaction in vivo unproven\", \"Mechanism of CLIC4 control over ER-mitochondrial calcium flux undefined\", \"m6A reader linking FTO to CLIC4 stability not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Confirmed in vivo endothelial CLIC4 requirement for thrombin/PAR1-driven RhoA activation, ERM phosphorylation, and vascular permeability, with CLIC-isoform specificity.\",\n      \"evidence\": \"Endothelial-specific Clic4 knockout with PAR1-peptide lung permeability, CLIC4/CLIC1 siRNA, RhoA activation and barrier assays\",\n      \"pmids\": [\"37317855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct CLIC4 effector coupling PAR1 to RhoA-GEF unknown\", \"How membrane-recruited CLIC4 selectively engages RhoA vs Rac1 unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Confirmed CLIC4 supplies a native IAA-94-sensitive chloride conductance regulating sperm volume under PKC control, and localized GPCR-to-Rac1 coupling to the CLIC4 C-terminus independent of channel and GST activities.\",\n      \"evidence\": \"Patch-clamp on CLIC4-null sperm with PKC modulation; domain-swap/heterologous-targeting structure-function with Rac1 activation assays (one preprint)\",\n      \"pmids\": [\"41715063\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a soluble protein generates native sperm currents structurally unresolved\", \"C-terminal effector(s) for Rac1 activation not identified\", \"Domain-swap GPCR-coupling findings remain in preprint form\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how CLIC4 mechanistically converts between its soluble GST-fold and its membrane/signaling forms to select among its many outputs—channel activity, GTPase coupling, Smad stabilization, and trafficking—and which endogenous enzymatic substrate (if any) the glutaredoxin-like fold acts upon.\",\n      \"evidence\": \"No discovery in the corpus reconciles the membrane-inserted conformation with the soluble structure or identifies an in vivo catalytic substrate\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of the membrane-inserted conformation\", \"No defined endogenous redox substrate\", \"Logic determining which downstream function dominates in a given context unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 3, 7, 10, 37]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [14, 20, 21, 29, 32]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [13, 14, 28, 30]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [27, 28, 31]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5, 15]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 15, 21, 36]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 14, 16, 19]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [2, 33, 35]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 33]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [21, 23]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [6, 28]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [14, 15, 32, 36]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 5, 8, 35]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [17, 26]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [21, 23, 29]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [28, 30]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [36]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SMAD2\", \"SMAD3\", \"PPM1a\", \"EZR\", \"MST4\", \"PFN1\", \"DIAPH3\", \"RHOA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}