{"gene":"UNC45A","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2017,"finding":"UNC-45A functions as a myosin chaperone required for proper folding of non-muscle myosin II (NM-II) heavy chains in vivo. Knockout cells show large fractions of NM-II and myosin-1c failing to fold, and the remaining folded NM-II fails to form functional bipolar filaments. The C-terminal UCS domain is critical for NM-II folding, while the N-terminal TPR domain contributes to stress fiber assembly. UNC-45A knockout causes severe defects in stress fiber assembly, cell morphogenesis, polarity, and migration.","method":"CRISPR/Cas9 knockout, structured-illumination microscopy, gradient centrifugation, proteasome inhibition, deletion mutant analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KO, SIM, fractionation, domain mutants) in single rigorous study with clear phenotypic readouts","pmids":["29055011"],"is_preprint":false},{"year":2015,"finding":"UNC-45A localizes to the NK cell immunological synapse upon activation and is part of the multiprotein complex formed during NK cell activation. UNC-45A is dispensable for immunological synapse formation and lytic granule reorientation but is required for lytic granule exocytosis. Loss of UNC-45A reduces NMIIA binding to actin, indicating UNC-45A promotes actomyosin complex formation required for cytoskeletal dynamics underlying NK cell cytotoxicity.","method":"siRNA knockdown, immunofluorescence microscopy, co-immunoprecipitation, cytotoxicity assays, degranulation assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal functional assays and localization with mechanistic follow-up, single lab","pmids":["26438524"],"is_preprint":false},{"year":2018,"finding":"UNC-45A directly binds to taxol-stabilized microtubules in vitro in the absence of any additional cellular cofactors or other MT-associated proteins, and acts as an ATP-independent microtubule destabilizer. In cells, UNC-45A binds to and destabilizes mitotic spindles; its depletion causes defects in chromosome congression and segregation.","method":"In vitro biophysical reconstitution, total internal reflection fluorescence (TIRF) microscopy, siRNA depletion, chromosome segregation assays","journal":"Molecular cancer research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins plus cellular validation, single lab but multiple orthogonal methods","pmids":["30322860"],"is_preprint":false},{"year":2021,"finding":"UNC-45A binds to the microtubule lattice, causing MT bending, breakage, and depolymerization in vitro and in human and rat cells. This MT-destabilizing activity is independent of its C-terminal NM-II-binding domain and occurs even in the presence of the NM-II inhibitor blebbistatin, establishing UNC-45A as a novel ATP-independent MT-severing protein with activities separable from its myosin chaperone function.","method":"In vitro reconstitution, TIRF microscopy, domain deletion mutants, blebbistatin inhibition, cell-based MT dynamics assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus mutagenesis plus pharmacological separation of functions, replicated across cell types","pmids":["33262310"],"is_preprint":false},{"year":2023,"finding":"UNC-45A preferentially binds to curved regions of microtubules rather than straight regions. UNC-45A overexpression increases MT curvature in cells, and its depletion decreases MT curvature, independently of actomyosin contractility. UNC-45A counteracts the MT-straightening effects of paclitaxel in cells.","method":"In vitro biophysical reconstitution, TIRF microscopy, overexpression and depletion in cells, paclitaxel treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with quantitative enrichment analysis plus cellular validation, single lab with multiple orthogonal approaches","pmids":["37858676"],"is_preprint":false},{"year":2019,"finding":"UNC-45A localizes to the cancer cell nucleus where it up-regulates transcriptional activity of the glucocorticoid receptor (GR), thereby promoting expression of the mitotic kinase NEK7. UNC-45A-deficient cancer cells show pericentrosomal material disorganization, defects in centrosomal separation and mitotic chromosome alignment, metaphase arrest, cytokinesis failure, and mitotic catastrophe; these phenotypes are rescued by heterologous NEK7 expression.","method":"siRNA knockdown, immunofluorescence microscopy, gene microarray, RT-qPCR, transcriptional reporter assays, heterologous rescue experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (microscopy, arrays, reporter assays, rescue) in single lab establishing pathway position","pmids":["30737284"],"is_preprint":false},{"year":2014,"finding":"UNC-45A localizes to centrosomes and binds to and regulates CHK1 nuclear-cytoplasmic localization in an HSP90-independent manner. UNC-45A and CHK1 co-localize at the centrosome by immunocytochemistry and biochemical fractionation. Loss of UNC-45A reduces CHK1 activation and its tethering to the centrosome, causing accumulation of multinucleated cells consistent with centrosome function defects.","method":"Immunocytochemistry, biochemical fractionation, co-localization, siRNA knockdown, cell cycle analysis","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-localization plus fractionation plus functional KD phenotype, single lab","pmids":["25444911"],"is_preprint":false},{"year":2009,"finding":"UNC-45A inhibits signaling through retinoic acid receptor alpha (RAR-alpha). Expression of UNC-45A inhibits retinoic acid-induced proliferation arrest and differentiation of human neuroblastoma cells and suppresses induction of endogenous RAR target genes. UNC-45A also confers resistance to histone deacetylase inhibitors.","method":"Cell-based proliferation and differentiation assays, transcriptional reporter gene assays, gain-of-function genetic screen","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple cell-based assays and reporter assays, single lab","pmids":["19843631"],"is_preprint":false},{"year":2011,"finding":"UNC-45A is alternatively expressed as two isoforms differing by a 15-amino-acid proline-rich N-terminal sequence. The 944-amino-acid isoform is degraded at ~5-fold greater rate than the 929-amino-acid isoform via the ubiquitin-proteasome system. shRNA knockdown of UNC-45A in metastatic breast cancer cells decreases cell proliferation and invasion, with concomitant reduction in myosin II interaction with actin filaments.","method":"shRNA knockdown, cellular metabolic labeling, proteasome inhibition, invasion assays, co-immunoprecipitation","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — labeling experiments establishing differential turnover plus proteasome pathway identification, single lab","pmids":["21802425"],"is_preprint":false},{"year":2008,"finding":"Loss of Unc45a in zebrafish (kurzschluss mutant, identified by positional cloning) causes failure of aortic arches 5 and 6 to form lumenized connections to the lateral dorsal aorta, leading to arteriovenous malformation. Angioblast formation and initial sprouting are normal, implicating UNC-45A specifically in lumenization/connection of aortic arch vessels rather than early angioblast specification, establishing the first in vivo vertebrate developmental role for Unc45a.","method":"Positional cloning, zebrafish genetic mutant analysis, live imaging, vascular morphology assays","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — positional cloning establishing causation plus detailed phenotypic dissection, single study","pmids":["18462713"],"is_preprint":false},{"year":2022,"finding":"UNC-45A acts as a cochaperone for myosin VB (MYO5B). By mass spectrometry, myosin VB was identified as a client of the UNC-45A chaperone and was found misfolded in UNC45A-KO Caco-2 cells. Loss of UNC-45A causes abnormal epithelial morphogenesis, RAB11-positive recycling endosome mislocalization, apical transporter mislocalization, sparse/disorganized microvilli, and microvillus inclusions resembling microvillus inclusion disease. These defects were restored by full-length UNC-45A but not by patient mutant alleles.","method":"Mass spectrometry, CRISPR/Cas9 KO, 3D organoids, confocal microscopy, electron microscopy, rescue with wild-type and mutant UNC45A, zebrafish unc45a morpholino","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — MS client identification, KO with multiple phenotypic assays, organoid model, in vivo zebrafish validation, and mutant rescue in single study","pmids":["35575086"],"is_preprint":false},{"year":2022,"finding":"UNC-45A depletion reduces myosin Vb protein expression in intestinal and hepatic cells, and disrupts two myosin Vb-dependent processes: RAB11A-positive recycling endosome positioning and microvilli development. Reintroduction of UNC-45A or myosin Vb restores these defects. The O2HE patient variant UNC45A-p.V423D impairs UNC45A protein stability but not its ability to promote myosin Vb expression when stable protein is present.","method":"CRISPR-Cas9 KO, site-directed mutagenesis, Western blotting, confocal fluorescence microscopy, scanning electron microscopy, patient variant functional analysis","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal cell biological methods, single lab, functional variant analysis","pmids":["35421597"],"is_preprint":false},{"year":2022,"finding":"UNC-45A is required for intestinal epithelial barrier integrity. CRISPR/Cas9 KO of UNC-45A in intestinal epithelial cells disrupts barrier integrity, impairs assembly of adherens and tight junctions, and attenuates cell migration. Loss of UNC-45A disorganizes actomyosin bundles at epithelial junctions, decreases contractile forces at apical junctions and matrix adhesions. The myosin-binding domain of UNC-45A is required for its role in junctions and motility. Decreased UNC-45 expression also increases gut permeability in Drosophila in vivo.","method":"CRISPR/Cas9 KO, transepithelial resistance measurements, immunofluorescence, traction force microscopy, deletion mutant analysis, Drosophila in vivo model","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KO, force microscopy, domain mutants, in vivo model), clear mechanistic link to myosin-binding domain","pmids":["35344227"],"is_preprint":false},{"year":2024,"finding":"Myosin 1b (MYO1B) is part of the UNC-45A interactome. In the absence of UNC-45A, myosin 1b is degraded and forms aggregates when proteasome activity is inhibited, indicating UNC-45A acts as a chaperone for MYO1B. Loss of MYO1B in 3D Caco-2 cells impairs lumen formation with spindle orientation defects, Golgi fragmentation, and trafficking impairment, placing MYO1B downstream of UNC-45A in intestinal epithelial morphogenesis.","method":"Interactome mass spectrometry, CRISPR/Cas9 KO, proteasome inhibition, 3D organoids, zebrafish myo1b morpholino, confocal microscopy","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS interactome identification plus KO phenotype with mechanistic epistasis, single lab","pmids":["39636728"],"is_preprint":false},{"year":2025,"finding":"The UNC45A p.Leu237Pro O2HE syndrome missense variant retains chaperone activity (prevents myosin aggregation, supports NM-II filament formation) but forms atypically stable oligomers that prevent chaperone-myosin complex dissociation, thereby inhibiting NM-II functions. This causes impaired intracellular trafficking, defective recycling, and abnormal retention of transferrin at endocytic sites. Co-expression of wild-type UNC45A attenuates pathogenic effects of the mutant by inhibiting excessive oligomer formation.","method":"Missense variant functional analysis in patient fibroblasts and U2OS cells, immunofluorescence, transferrin trafficking assays, oligomerization analysis, co-expression rescue","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient cell functional analysis with multiple assays and rescue experiment, single lab","pmids":["40125554"],"is_preprint":false},{"year":2019,"finding":"UNC-45A co-localizes and co-fractionates with microtubules in interphase cells independently of actin or myosin, and localizes to mitotic spindles in clinical tumor specimens. UNC-45A co-fractionates with gamma-tubulin and influences centrosomal positioning.","method":"Immunofluorescence, biochemical co-fractionation, immunohistochemistry of clinical specimens","journal":"Cancer biology & therapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-fractionation and co-localization only, no direct functional manipulation, single lab","pmids":["31328624"],"is_preprint":false},{"year":2025,"finding":"NM2-A motor domain mutation N93K increases interaction of NM2-A with UNC-45A in stress fiber-forming cells (compared to wild type), and in megakaryocytes the N93K mutant forms large aggregates that co-contain wild-type NM2-A and UNC-45A, whereas the tail mutation E1841K forms aggregates that exclude UNC-45A. This suggests UNC-45A recognizes misfolded or aggregation-prone NM-II and co-aggregates with it.","method":"Immunofluorescence, co-immunoprecipitation, expression of NM2-A mutants in multiple cell types including patient-derived megakaryocytes","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single preprint, co-IP and co-localization only, no direct biochemical reconstitution","pmids":["bio_10.1101_2025.05.20.654665"],"is_preprint":true},{"year":2023,"finding":"A variant (c.-98G>T) in the 5'-untranslated region of UNC45A causes reduced UNC45A mRNA and protein expression (reproduced in a CRISPR/Cas9 cell model), and is the causative variant in Aagenaes syndrome. Liver biopsies show mislocalization of hepatobiliary transport proteins BSEP and MRP2, linking UNC45A loss to impaired hepatobiliary transport protein localization.","method":"Whole-genome sequencing, Western blot, PCR, CRISPR/Cas9 cell model, immunohistochemistry of liver biopsies","journal":"Journal of hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR cell model confirms functional impact of UTR variant on expression, IHC links to transport protein mislocalization, replicated across 26 patients","pmids":["37328071"],"is_preprint":false}],"current_model":"UNC-45A is a dual-function cytoskeletal cochaperone that (1) folds non-muscle myosin II heavy chains via its C-terminal UCS domain and promotes actomyosin filament assembly and stress fiber formation via its N-terminal TPR domain, with clients including NM-II, myosin-1c, myosin VB, and myosin 1b; and (2) acts as the only known ATP-independent microtubule-severing protein, preferentially binding curved MT lattice regions to drive bending, breakage, and depolymerization independently of its myosin-binding domain—together, these activities regulate cell division, epithelial morphogenesis and barrier integrity, intracellular trafficking, and NK cell cytotoxicity, while nuclear UNC-45A additionally promotes cancer cell proliferation by activating glucocorticoid receptor-driven NEK7 transcription and CHK1 centrosomal tethering."},"narrative":{"mechanistic_narrative":"UNC45A is a dual-function cytoskeletal cochaperone that couples myosin folding to actomyosin assembly while independently destabilizing microtubules, thereby governing cell division, epithelial morphogenesis, intracellular trafficking, and immune cytotoxicity [PMID:29055011, PMID:33262310, PMID:35575086]. As a myosin chaperone, its C-terminal UCS domain is required for proper folding of non-muscle myosin II heavy chains, while its N-terminal TPR domain drives stress fiber assembly; loss of UNC45A leaves NM-II and myosin-1c misfolded and unable to form functional bipolar filaments, causing defects in cell morphogenesis, polarity, and migration [PMID:29055011]. Its client repertoire extends to myosin VB and myosin 1b, which are degraded or aggregate in its absence, accounting for abnormal epithelial morphogenesis, RAB11-positive recycling endosome mislocalization, apical transporter mislocalization, and microvillus inclusion-like defects [PMID:35575086, PMID:39636728]. Through its myosin-binding activity UNC45A organizes actomyosin bundles at epithelial junctions to maintain barrier integrity and contractile force at adherens and tight junctions [PMID:35344227], and promotes NM-IIA association with actin required for lytic granule exocytosis at the NK cell immunological synapse [PMID:26438524]. Independently of its myosin-binding domain, UNC45A directly binds the microtubule lattice—preferentially at curved regions—and acts as an ATP-independent microtubule-severing protein that drives bending, breakage, and depolymerization, including destabilization of mitotic spindles and effects on chromosome congression and segregation [PMID:30322860, PMID:33262310, PMID:37858676]. Nuclear and centrosomal pools additionally promote cancer cell proliferation by up-regulating glucocorticoid receptor-driven NEK7 transcription and by tethering activated CHK1 to the centrosome [PMID:30737284, PMID:25444911]. Human disease-causing variants establish the physiological importance of these activities: loss-of-expression and missense alleles cause Aagenaes syndrome with hepatobiliary transport protein mislocalization, and O2HE syndrome variants impair myosin-dependent trafficking [PMID:35575086, PMID:40125554, PMID:37328071].","teleology":[{"year":2008,"claim":"Established the first in vivo vertebrate developmental requirement for Unc45a, defining a specific role in vessel lumenization rather than early cell specification.","evidence":"positional cloning of the zebrafish kurzschluss mutant with vascular morphology and live imaging","pmids":["18462713"],"confidence":"Medium","gaps":["Did not define the molecular activity (chaperone vs. cytoskeletal) underlying the vascular defect","No link to specific myosin or microtubule clients"]},{"year":2009,"claim":"Identified a nuclear/signaling role by showing UNC-45A antagonizes nuclear hormone receptor signaling, distinct from any cytoskeletal function.","evidence":"gain-of-function genetic screen with proliferation, differentiation, and reporter assays in neuroblastoma cells","pmids":["19843631"],"confidence":"Medium","gaps":["Mechanism of RAR-alpha inhibition not defined (direct binding vs. indirect)","No structural or biochemical basis"]},{"year":2011,"claim":"Defined isoform-specific turnover and connected UNC-45A levels to cancer cell behavior via myosin-actin interaction.","evidence":"metabolic labeling, proteasome inhibition, shRNA knockdown, and Co-IP in breast cancer cells","pmids":["21802425"],"confidence":"Medium","gaps":["Degron mediating differential degradation not mapped","Direct vs. indirect role in myosin II-actin interaction unresolved"]},{"year":2014,"claim":"Placed UNC-45A at the centrosome regulating checkpoint signaling, showing it controls CHK1 localization and activation independently of HSP90.","evidence":"immunocytochemistry, biochemical fractionation, siRNA knockdown, and cell cycle analysis","pmids":["25444911"],"confidence":"Medium","gaps":["Whether UNC-45A binds CHK1 directly not established","Structural basis of centrosomal tethering unknown"]},{"year":2015,"claim":"Extended UNC-45A function to immune effector cells, distinguishing dispensable (synapse formation) from required (granule exocytosis) steps in cytotoxicity.","evidence":"siRNA knockdown, Co-IP, immunofluorescence, and degranulation/cytotoxicity assays in NK cells","pmids":["26438524"],"confidence":"Medium","gaps":["Direct myosin client at the synapse not biochemically defined","Single-lab functional data"]},{"year":2017,"claim":"Established the core molecular function by demonstrating UNC-45A is a myosin chaperone whose UCS domain folds NM-II and whose TPR domain drives stress fiber assembly.","evidence":"CRISPR/Cas9 knockout, structured-illumination microscopy, gradient fractionation, and domain deletion mutants","pmids":["29055011"],"confidence":"High","gaps":["No in vitro reconstitution of the folding reaction with purified components","ATP/HSP90 dependence of the chaperone cycle not fully resolved"]},{"year":2018,"claim":"Revealed an unexpected second activity by showing UNC-45A binds microtubules directly and destabilizes them in vitro and at the mitotic spindle.","evidence":"in vitro reconstitution with purified protein, TIRF microscopy, siRNA depletion, and chromosome segregation assays","pmids":["30322860"],"confidence":"High","gaps":["Lattice-binding interface not mapped","Relationship between MT activity and myosin chaperone activity unresolved at this stage"]},{"year":2019,"claim":"Connected nuclear UNC-45A to mitotic gene control, defining a transcriptional axis through glucocorticoid receptor and NEK7 that supports cancer proliferation.","evidence":"siRNA knockdown, microarray, RT-qPCR, reporter assays, and heterologous NEK7 rescue","pmids":["30737284"],"confidence":"Medium","gaps":["Whether UNC-45A binds GR or chromatin directly not shown","How a cytoskeletal cochaperone enters the nucleus is undefined"]},{"year":2021,"claim":"Cleanly separated the two functions by showing MT severing is independent of the NM-II-binding domain and persists under myosin inhibition, defining UNC-45A as an ATP-independent MT-severing protein.","evidence":"in vitro reconstitution, TIRF, domain deletion, and blebbistatin treatment across human and rat cells","pmids":["33262310"],"confidence":"High","gaps":["Severing mechanism (lattice deformation vs. tubulin extraction) not resolved","Regulation distinguishing severing from chaperone roles in vivo unknown"]},{"year":2022,"claim":"Broadened the chaperone client list to myosin VB and linked UNC-45A loss to epithelial morphogenesis defects and a human enteropathy via mutant rescue.","evidence":"mass spectrometry, CRISPR/Cas9 KO, 3D organoids, EM, zebrafish morpholino, and wild-type vs. patient-mutant rescue","pmids":["35575086","35421597"],"confidence":"High","gaps":["Direct chaperone-MYO5B folding reaction not reconstituted in vitro","Quantitative client hierarchy among myosins unresolved"]},{"year":2022,"claim":"Demonstrated a tissue-level barrier function, showing the myosin-binding domain is required for junction assembly and contractile force at epithelial junctions in vitro and in vivo.","evidence":"CRISPR/Cas9 KO, transepithelial resistance, traction force microscopy, domain mutants, and Drosophila gut permeability model","pmids":["35344227"],"confidence":"High","gaps":["Which junctional myosin client mediates the effect not pinpointed","Contribution of MT-severing activity to junctions untested"]},{"year":2023,"claim":"Refined the MT-binding model by showing UNC-45A is enriched on curved lattice regions and modulates MT curvature, and identified the causative variant of a human cholestatic syndrome.","evidence":"in vitro reconstitution with curvature analysis, TIRF, overexpression/depletion; and whole-genome sequencing, CRISPR cell model, and liver biopsy IHC for Aagenaes syndrome","pmids":["37858676","37328071"],"confidence":"High","gaps":["Structural basis of curvature preference not defined","Causal chain from UNC45A loss to BSEP/MRP2 mislocalization (chaperone vs. trafficking) not dissected"]},{"year":2024,"claim":"Added myosin 1b as a chaperone client and positioned it downstream of UNC-45A in epithelial lumen formation, extending the client network.","evidence":"interactome mass spectrometry, CRISPR/Cas9 KO, proteasome inhibition, 3D organoids, and zebrafish morpholino","pmids":["39636728"],"confidence":"Medium","gaps":["Direct binding vs. indirect stabilization of MYO1B not separated","In vitro folding assay absent"]},{"year":2025,"claim":"Provided a molecular mechanism for a dominant-acting disease variant: O2HE missense alleles retain folding activity but lock the chaperone-myosin complex via abnormal oligomerization, impairing trafficking.","evidence":"patient fibroblast and U2OS functional assays, transferrin trafficking, oligomerization analysis, and wild-type co-expression rescue","pmids":["40125554"],"confidence":"Medium","gaps":["Structural basis of pathogenic oligomerization not determined","Generalizability across other UNC45A variants untested"]},{"year":null,"claim":"How UNC-45A's chaperone and microtubule-severing activities are spatially and temporally coordinated within a single cell, and how its nuclear localization and transcriptional functions are regulated, remain unresolved.","evidence":"no single study reconciles the cytoskeletal, nuclear, and severing roles mechanistically","pmids":[],"confidence":"Low","gaps":["No structure of UNC-45A bound to a myosin client or microtubule","Mechanism of nuclear import and GR activation undefined","Regulatory switch between myosin folding and MT severing unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,10,13]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,10,13]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2,3,4]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,2,15]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[6,15]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,5,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,10,13]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[10,14,17]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[9,10,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1]}],"complexes":[],"partners":["MYH9","MYO5B","MYO1B","MYO1C","CHEK1","NEK7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H3U1","full_name":"Protein unc-45 homolog A","aliases":["GCUNC-45","Smooth muscle cell-associated protein 1","SMAP-1"],"length_aa":944,"mass_kda":103.1,"function":"Acts as a co-chaperone for HSP90. Prevents the stimulation of HSP90AB1 ATPase activity by AHSA1. Positive factor in promoting PGR function in the cell. May be necessary for proper folding of myosin (Potential). Necessary for normal cell proliferation. Necessary for normal myotube formation and myosin accumulation during muscle cell development. May play a role in erythropoiesis in stroma cells in the spleen (By similarity)","subcellular_location":"Cytoplasm; Cytoplasm, perinuclear region; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9H3U1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/UNC45A","classification":"Not Classified","n_dependent_lines":300,"n_total_lines":1208,"dependency_fraction":0.24834437086092714},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CSNK1A1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/UNC45A","total_profiled":1310},"omim":[{"mim_id":"619377","title":"OSTEOOTOHEPATOENTERIC SYNDROME; OOHE","url":"https://www.omim.org/entry/619377"},{"mim_id":"611220","title":"UNC45 MYOSIN CHAPERONE B; UNC45B","url":"https://www.omim.org/entry/611220"},{"mim_id":"611219","title":"UNC45 MYOSIN CHAPERONE A; UNC45A","url":"https://www.omim.org/entry/611219"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear speckles","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/UNC45A"},"hgnc":{"alias_symbol":["SMAP-1","GC-UNC45"],"prev_symbol":[]},"alphafold":{"accession":"Q9H3U1","domains":[{"cath_id":"1.25.40.10","chopping":"22-148","consensus_level":"high","plddt":89.4648,"start":22,"end":148}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H3U1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H3U1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H3U1-F1-predicted_aligned_error_v6.png","plddt_mean":86.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=UNC45A","jax_strain_url":"https://www.jax.org/strain/search?query=UNC45A"},"sequence":{"accession":"Q9H3U1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H3U1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H3U1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H3U1"}},"corpus_meta":[{"pmid":"18462713","id":"PMC_18462713","title":"Loss of unc45a precipitates arteriovenous shunting in the aortic arches.","date":"2008","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/18462713","citation_count":53,"is_preprint":false},{"pmid":"29429573","id":"PMC_29429573","title":"Loss-of-Function Mutations in UNC45A Cause a Syndrome Associating Cholestasis, Diarrhea, Impaired Hearing, and Bone Fragility.","date":"2018","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29429573","citation_count":46,"is_preprint":false},{"pmid":"29055011","id":"PMC_29055011","title":"UNC-45a promotes myosin folding and stress fiber assembly.","date":"2017","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/29055011","citation_count":40,"is_preprint":false},{"pmid":"30737284","id":"PMC_30737284","title":"The co-chaperone UNC45A is essential for the expression of mitotic kinase NEK7 and tumorigenesis.","date":"2019","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30737284","citation_count":30,"is_preprint":false},{"pmid":"26438524","id":"PMC_26438524","title":"UNC-45A Is a Nonmuscle Myosin IIA Chaperone Required for NK Cell Cytotoxicity via Control of Lytic Granule Secretion.","date":"2015","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/26438524","citation_count":26,"is_preprint":false},{"pmid":"35575086","id":"PMC_35575086","title":"UNC45A deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking.","date":"2022","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/35575086","citation_count":25,"is_preprint":false},{"pmid":"19843631","id":"PMC_19843631","title":"UNC45A confers resistance to histone deacetylase inhibitors and retinoic acid.","date":"2009","source":"Molecular cancer research : 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Biology","url":"https://pubmed.ncbi.nlm.nih.gov/35344227","citation_count":19,"is_preprint":false},{"pmid":"35421597","id":"PMC_35421597","title":"A Functional Relationship Between UNC45A and MYO5B Connects Two Rare Diseases With Shared Enteropathy.","date":"2022","source":"Cellular and molecular gastroenterology and hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/35421597","citation_count":16,"is_preprint":false},{"pmid":"31328624","id":"PMC_31328624","title":"UNC-45A is preferentially expressed in epithelial cells and binds to and co-localizes with interphase MTs.","date":"2019","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/31328624","citation_count":15,"is_preprint":false},{"pmid":"25444911","id":"PMC_25444911","title":"UNC45A localizes to centrosomes and regulates cancer cell proliferation through ChK1 activation.","date":"2014","source":"Cancer 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Part A","url":"https://pubmed.ncbi.nlm.nih.gov/39887522","citation_count":0,"is_preprint":false},{"pmid":"38803956","id":"PMC_38803956","title":"UNC-45A: A potential therapeutic target for malignant tumors.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/38803956","citation_count":0,"is_preprint":false},{"pmid":"40014453","id":"PMC_40014453","title":"Protocol for purifying biologically active microtubule-severing protein UNC-45A from E.coli using GST-affinity and spin columns.","date":"2025","source":"STAR protocols","url":"https://pubmed.ncbi.nlm.nih.gov/40014453","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.20.654665","title":"Molecular Control of Non-Muscle Myosin II-A Aggregation and Intracellular Dynamics by motor- or tail-specific  <i>MYH9</i>  Mutations","date":"2025-05-21","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.20.654665","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15533,"output_tokens":4928,"usd":0.06026,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13104,"output_tokens":4660,"usd":0.09101,"stage2_stop_reason":"end_turn"},"total_usd":0.15127,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"UNC-45A functions as a myosin chaperone required for proper folding of non-muscle myosin II (NM-II) heavy chains in vivo. Knockout cells show large fractions of NM-II and myosin-1c failing to fold, and the remaining folded NM-II fails to form functional bipolar filaments. The C-terminal UCS domain is critical for NM-II folding, while the N-terminal TPR domain contributes to stress fiber assembly. UNC-45A knockout causes severe defects in stress fiber assembly, cell morphogenesis, polarity, and migration.\",\n      \"method\": \"CRISPR/Cas9 knockout, structured-illumination microscopy, gradient centrifugation, proteasome inhibition, deletion mutant analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KO, SIM, fractionation, domain mutants) in single rigorous study with clear phenotypic readouts\",\n      \"pmids\": [\"29055011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"UNC-45A localizes to the NK cell immunological synapse upon activation and is part of the multiprotein complex formed during NK cell activation. UNC-45A is dispensable for immunological synapse formation and lytic granule reorientation but is required for lytic granule exocytosis. Loss of UNC-45A reduces NMIIA binding to actin, indicating UNC-45A promotes actomyosin complex formation required for cytoskeletal dynamics underlying NK cell cytotoxicity.\",\n      \"method\": \"siRNA knockdown, immunofluorescence microscopy, co-immunoprecipitation, cytotoxicity assays, degranulation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal functional assays and localization with mechanistic follow-up, single lab\",\n      \"pmids\": [\"26438524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"UNC-45A directly binds to taxol-stabilized microtubules in vitro in the absence of any additional cellular cofactors or other MT-associated proteins, and acts as an ATP-independent microtubule destabilizer. In cells, UNC-45A binds to and destabilizes mitotic spindles; its depletion causes defects in chromosome congression and segregation.\",\n      \"method\": \"In vitro biophysical reconstitution, total internal reflection fluorescence (TIRF) microscopy, siRNA depletion, chromosome segregation assays\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins plus cellular validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"30322860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UNC-45A binds to the microtubule lattice, causing MT bending, breakage, and depolymerization in vitro and in human and rat cells. This MT-destabilizing activity is independent of its C-terminal NM-II-binding domain and occurs even in the presence of the NM-II inhibitor blebbistatin, establishing UNC-45A as a novel ATP-independent MT-severing protein with activities separable from its myosin chaperone function.\",\n      \"method\": \"In vitro reconstitution, TIRF microscopy, domain deletion mutants, blebbistatin inhibition, cell-based MT dynamics assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus mutagenesis plus pharmacological separation of functions, replicated across cell types\",\n      \"pmids\": [\"33262310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"UNC-45A preferentially binds to curved regions of microtubules rather than straight regions. UNC-45A overexpression increases MT curvature in cells, and its depletion decreases MT curvature, independently of actomyosin contractility. UNC-45A counteracts the MT-straightening effects of paclitaxel in cells.\",\n      \"method\": \"In vitro biophysical reconstitution, TIRF microscopy, overexpression and depletion in cells, paclitaxel treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with quantitative enrichment analysis plus cellular validation, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"37858676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"UNC-45A localizes to the cancer cell nucleus where it up-regulates transcriptional activity of the glucocorticoid receptor (GR), thereby promoting expression of the mitotic kinase NEK7. UNC-45A-deficient cancer cells show pericentrosomal material disorganization, defects in centrosomal separation and mitotic chromosome alignment, metaphase arrest, cytokinesis failure, and mitotic catastrophe; these phenotypes are rescued by heterologous NEK7 expression.\",\n      \"method\": \"siRNA knockdown, immunofluorescence microscopy, gene microarray, RT-qPCR, transcriptional reporter assays, heterologous rescue experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (microscopy, arrays, reporter assays, rescue) in single lab establishing pathway position\",\n      \"pmids\": [\"30737284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"UNC-45A localizes to centrosomes and binds to and regulates CHK1 nuclear-cytoplasmic localization in an HSP90-independent manner. UNC-45A and CHK1 co-localize at the centrosome by immunocytochemistry and biochemical fractionation. Loss of UNC-45A reduces CHK1 activation and its tethering to the centrosome, causing accumulation of multinucleated cells consistent with centrosome function defects.\",\n      \"method\": \"Immunocytochemistry, biochemical fractionation, co-localization, siRNA knockdown, cell cycle analysis\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-localization plus fractionation plus functional KD phenotype, single lab\",\n      \"pmids\": [\"25444911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"UNC-45A inhibits signaling through retinoic acid receptor alpha (RAR-alpha). Expression of UNC-45A inhibits retinoic acid-induced proliferation arrest and differentiation of human neuroblastoma cells and suppresses induction of endogenous RAR target genes. UNC-45A also confers resistance to histone deacetylase inhibitors.\",\n      \"method\": \"Cell-based proliferation and differentiation assays, transcriptional reporter gene assays, gain-of-function genetic screen\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple cell-based assays and reporter assays, single lab\",\n      \"pmids\": [\"19843631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"UNC-45A is alternatively expressed as two isoforms differing by a 15-amino-acid proline-rich N-terminal sequence. The 944-amino-acid isoform is degraded at ~5-fold greater rate than the 929-amino-acid isoform via the ubiquitin-proteasome system. shRNA knockdown of UNC-45A in metastatic breast cancer cells decreases cell proliferation and invasion, with concomitant reduction in myosin II interaction with actin filaments.\",\n      \"method\": \"shRNA knockdown, cellular metabolic labeling, proteasome inhibition, invasion assays, co-immunoprecipitation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — labeling experiments establishing differential turnover plus proteasome pathway identification, single lab\",\n      \"pmids\": [\"21802425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Loss of Unc45a in zebrafish (kurzschluss mutant, identified by positional cloning) causes failure of aortic arches 5 and 6 to form lumenized connections to the lateral dorsal aorta, leading to arteriovenous malformation. Angioblast formation and initial sprouting are normal, implicating UNC-45A specifically in lumenization/connection of aortic arch vessels rather than early angioblast specification, establishing the first in vivo vertebrate developmental role for Unc45a.\",\n      \"method\": \"Positional cloning, zebrafish genetic mutant analysis, live imaging, vascular morphology assays\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — positional cloning establishing causation plus detailed phenotypic dissection, single study\",\n      \"pmids\": [\"18462713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UNC-45A acts as a cochaperone for myosin VB (MYO5B). By mass spectrometry, myosin VB was identified as a client of the UNC-45A chaperone and was found misfolded in UNC45A-KO Caco-2 cells. Loss of UNC-45A causes abnormal epithelial morphogenesis, RAB11-positive recycling endosome mislocalization, apical transporter mislocalization, sparse/disorganized microvilli, and microvillus inclusions resembling microvillus inclusion disease. These defects were restored by full-length UNC-45A but not by patient mutant alleles.\",\n      \"method\": \"Mass spectrometry, CRISPR/Cas9 KO, 3D organoids, confocal microscopy, electron microscopy, rescue with wild-type and mutant UNC45A, zebrafish unc45a morpholino\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — MS client identification, KO with multiple phenotypic assays, organoid model, in vivo zebrafish validation, and mutant rescue in single study\",\n      \"pmids\": [\"35575086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UNC-45A depletion reduces myosin Vb protein expression in intestinal and hepatic cells, and disrupts two myosin Vb-dependent processes: RAB11A-positive recycling endosome positioning and microvilli development. Reintroduction of UNC-45A or myosin Vb restores these defects. The O2HE patient variant UNC45A-p.V423D impairs UNC45A protein stability but not its ability to promote myosin Vb expression when stable protein is present.\",\n      \"method\": \"CRISPR-Cas9 KO, site-directed mutagenesis, Western blotting, confocal fluorescence microscopy, scanning electron microscopy, patient variant functional analysis\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal cell biological methods, single lab, functional variant analysis\",\n      \"pmids\": [\"35421597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UNC-45A is required for intestinal epithelial barrier integrity. CRISPR/Cas9 KO of UNC-45A in intestinal epithelial cells disrupts barrier integrity, impairs assembly of adherens and tight junctions, and attenuates cell migration. Loss of UNC-45A disorganizes actomyosin bundles at epithelial junctions, decreases contractile forces at apical junctions and matrix adhesions. The myosin-binding domain of UNC-45A is required for its role in junctions and motility. Decreased UNC-45 expression also increases gut permeability in Drosophila in vivo.\",\n      \"method\": \"CRISPR/Cas9 KO, transepithelial resistance measurements, immunofluorescence, traction force microscopy, deletion mutant analysis, Drosophila in vivo model\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KO, force microscopy, domain mutants, in vivo model), clear mechanistic link to myosin-binding domain\",\n      \"pmids\": [\"35344227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Myosin 1b (MYO1B) is part of the UNC-45A interactome. In the absence of UNC-45A, myosin 1b is degraded and forms aggregates when proteasome activity is inhibited, indicating UNC-45A acts as a chaperone for MYO1B. Loss of MYO1B in 3D Caco-2 cells impairs lumen formation with spindle orientation defects, Golgi fragmentation, and trafficking impairment, placing MYO1B downstream of UNC-45A in intestinal epithelial morphogenesis.\",\n      \"method\": \"Interactome mass spectrometry, CRISPR/Cas9 KO, proteasome inhibition, 3D organoids, zebrafish myo1b morpholino, confocal microscopy\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS interactome identification plus KO phenotype with mechanistic epistasis, single lab\",\n      \"pmids\": [\"39636728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The UNC45A p.Leu237Pro O2HE syndrome missense variant retains chaperone activity (prevents myosin aggregation, supports NM-II filament formation) but forms atypically stable oligomers that prevent chaperone-myosin complex dissociation, thereby inhibiting NM-II functions. This causes impaired intracellular trafficking, defective recycling, and abnormal retention of transferrin at endocytic sites. Co-expression of wild-type UNC45A attenuates pathogenic effects of the mutant by inhibiting excessive oligomer formation.\",\n      \"method\": \"Missense variant functional analysis in patient fibroblasts and U2OS cells, immunofluorescence, transferrin trafficking assays, oligomerization analysis, co-expression rescue\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient cell functional analysis with multiple assays and rescue experiment, single lab\",\n      \"pmids\": [\"40125554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"UNC-45A co-localizes and co-fractionates with microtubules in interphase cells independently of actin or myosin, and localizes to mitotic spindles in clinical tumor specimens. UNC-45A co-fractionates with gamma-tubulin and influences centrosomal positioning.\",\n      \"method\": \"Immunofluorescence, biochemical co-fractionation, immunohistochemistry of clinical specimens\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-fractionation and co-localization only, no direct functional manipulation, single lab\",\n      \"pmids\": [\"31328624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NM2-A motor domain mutation N93K increases interaction of NM2-A with UNC-45A in stress fiber-forming cells (compared to wild type), and in megakaryocytes the N93K mutant forms large aggregates that co-contain wild-type NM2-A and UNC-45A, whereas the tail mutation E1841K forms aggregates that exclude UNC-45A. This suggests UNC-45A recognizes misfolded or aggregation-prone NM-II and co-aggregates with it.\",\n      \"method\": \"Immunofluorescence, co-immunoprecipitation, expression of NM2-A mutants in multiple cell types including patient-derived megakaryocytes\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single preprint, co-IP and co-localization only, no direct biochemical reconstitution\",\n      \"pmids\": [\"bio_10.1101_2025.05.20.654665\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A variant (c.-98G>T) in the 5'-untranslated region of UNC45A causes reduced UNC45A mRNA and protein expression (reproduced in a CRISPR/Cas9 cell model), and is the causative variant in Aagenaes syndrome. Liver biopsies show mislocalization of hepatobiliary transport proteins BSEP and MRP2, linking UNC45A loss to impaired hepatobiliary transport protein localization.\",\n      \"method\": \"Whole-genome sequencing, Western blot, PCR, CRISPR/Cas9 cell model, immunohistochemistry of liver biopsies\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR cell model confirms functional impact of UTR variant on expression, IHC links to transport protein mislocalization, replicated across 26 patients\",\n      \"pmids\": [\"37328071\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"UNC-45A is a dual-function cytoskeletal cochaperone that (1) folds non-muscle myosin II heavy chains via its C-terminal UCS domain and promotes actomyosin filament assembly and stress fiber formation via its N-terminal TPR domain, with clients including NM-II, myosin-1c, myosin VB, and myosin 1b; and (2) acts as the only known ATP-independent microtubule-severing protein, preferentially binding curved MT lattice regions to drive bending, breakage, and depolymerization independently of its myosin-binding domain—together, these activities regulate cell division, epithelial morphogenesis and barrier integrity, intracellular trafficking, and NK cell cytotoxicity, while nuclear UNC-45A additionally promotes cancer cell proliferation by activating glucocorticoid receptor-driven NEK7 transcription and CHK1 centrosomal tethering.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"UNC45A is a dual-function cytoskeletal cochaperone that couples myosin folding to actomyosin assembly while independently destabilizing microtubules, thereby governing cell division, epithelial morphogenesis, intracellular trafficking, and immune cytotoxicity [#0, #3, #10]. As a myosin chaperone, its C-terminal UCS domain is required for proper folding of non-muscle myosin II heavy chains, while its N-terminal TPR domain drives stress fiber assembly; loss of UNC45A leaves NM-II and myosin-1c misfolded and unable to form functional bipolar filaments, causing defects in cell morphogenesis, polarity, and migration [#0]. Its client repertoire extends to myosin VB and myosin 1b, which are degraded or aggregate in its absence, accounting for abnormal epithelial morphogenesis, RAB11-positive recycling endosome mislocalization, apical transporter mislocalization, and microvillus inclusion-like defects [#10, #13]. Through its myosin-binding activity UNC45A organizes actomyosin bundles at epithelial junctions to maintain barrier integrity and contractile force at adherens and tight junctions [#12], and promotes NM-IIA association with actin required for lytic granule exocytosis at the NK cell immunological synapse [#1]. Independently of its myosin-binding domain, UNC45A directly binds the microtubule lattice—preferentially at curved regions—and acts as an ATP-independent microtubule-severing protein that drives bending, breakage, and depolymerization, including destabilization of mitotic spindles and effects on chromosome congression and segregation [#2, #3, #4]. Nuclear and centrosomal pools additionally promote cancer cell proliferation by up-regulating glucocorticoid receptor-driven NEK7 transcription and by tethering activated CHK1 to the centrosome [#5, #6]. Human disease-causing variants establish the physiological importance of these activities: loss-of-expression and missense alleles cause Aagenaes syndrome with hepatobiliary transport protein mislocalization, and O2HE syndrome variants impair myosin-dependent trafficking [#10, #14, #17].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established the first in vivo vertebrate developmental requirement for Unc45a, defining a specific role in vessel lumenization rather than early cell specification.\",\n      \"evidence\": \"positional cloning of the zebrafish kurzschluss mutant with vascular morphology and live imaging\",\n      \"pmids\": [\"18462713\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the molecular activity (chaperone vs. cytoskeletal) underlying the vascular defect\", \"No link to specific myosin or microtubule clients\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified a nuclear/signaling role by showing UNC-45A antagonizes nuclear hormone receptor signaling, distinct from any cytoskeletal function.\",\n      \"evidence\": \"gain-of-function genetic screen with proliferation, differentiation, and reporter assays in neuroblastoma cells\",\n      \"pmids\": [\"19843631\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of RAR-alpha inhibition not defined (direct binding vs. indirect)\", \"No structural or biochemical basis\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined isoform-specific turnover and connected UNC-45A levels to cancer cell behavior via myosin-actin interaction.\",\n      \"evidence\": \"metabolic labeling, proteasome inhibition, shRNA knockdown, and Co-IP in breast cancer cells\",\n      \"pmids\": [\"21802425\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degron mediating differential degradation not mapped\", \"Direct vs. indirect role in myosin II-actin interaction unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed UNC-45A at the centrosome regulating checkpoint signaling, showing it controls CHK1 localization and activation independently of HSP90.\",\n      \"evidence\": \"immunocytochemistry, biochemical fractionation, siRNA knockdown, and cell cycle analysis\",\n      \"pmids\": [\"25444911\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether UNC-45A binds CHK1 directly not established\", \"Structural basis of centrosomal tethering unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended UNC-45A function to immune effector cells, distinguishing dispensable (synapse formation) from required (granule exocytosis) steps in cytotoxicity.\",\n      \"evidence\": \"siRNA knockdown, Co-IP, immunofluorescence, and degranulation/cytotoxicity assays in NK cells\",\n      \"pmids\": [\"26438524\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct myosin client at the synapse not biochemically defined\", \"Single-lab functional data\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established the core molecular function by demonstrating UNC-45A is a myosin chaperone whose UCS domain folds NM-II and whose TPR domain drives stress fiber assembly.\",\n      \"evidence\": \"CRISPR/Cas9 knockout, structured-illumination microscopy, gradient fractionation, and domain deletion mutants\",\n      \"pmids\": [\"29055011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No in vitro reconstitution of the folding reaction with purified components\", \"ATP/HSP90 dependence of the chaperone cycle not fully resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed an unexpected second activity by showing UNC-45A binds microtubules directly and destabilizes them in vitro and at the mitotic spindle.\",\n      \"evidence\": \"in vitro reconstitution with purified protein, TIRF microscopy, siRNA depletion, and chromosome segregation assays\",\n      \"pmids\": [\"30322860\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lattice-binding interface not mapped\", \"Relationship between MT activity and myosin chaperone activity unresolved at this stage\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected nuclear UNC-45A to mitotic gene control, defining a transcriptional axis through glucocorticoid receptor and NEK7 that supports cancer proliferation.\",\n      \"evidence\": \"siRNA knockdown, microarray, RT-qPCR, reporter assays, and heterologous NEK7 rescue\",\n      \"pmids\": [\"30737284\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether UNC-45A binds GR or chromatin directly not shown\", \"How a cytoskeletal cochaperone enters the nucleus is undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Cleanly separated the two functions by showing MT severing is independent of the NM-II-binding domain and persists under myosin inhibition, defining UNC-45A as an ATP-independent MT-severing protein.\",\n      \"evidence\": \"in vitro reconstitution, TIRF, domain deletion, and blebbistatin treatment across human and rat cells\",\n      \"pmids\": [\"33262310\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Severing mechanism (lattice deformation vs. tubulin extraction) not resolved\", \"Regulation distinguishing severing from chaperone roles in vivo unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Broadened the chaperone client list to myosin VB and linked UNC-45A loss to epithelial morphogenesis defects and a human enteropathy via mutant rescue.\",\n      \"evidence\": \"mass spectrometry, CRISPR/Cas9 KO, 3D organoids, EM, zebrafish morpholino, and wild-type vs. patient-mutant rescue\",\n      \"pmids\": [\"35575086\", \"35421597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct chaperone-MYO5B folding reaction not reconstituted in vitro\", \"Quantitative client hierarchy among myosins unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated a tissue-level barrier function, showing the myosin-binding domain is required for junction assembly and contractile force at epithelial junctions in vitro and in vivo.\",\n      \"evidence\": \"CRISPR/Cas9 KO, transepithelial resistance, traction force microscopy, domain mutants, and Drosophila gut permeability model\",\n      \"pmids\": [\"35344227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which junctional myosin client mediates the effect not pinpointed\", \"Contribution of MT-severing activity to junctions untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Refined the MT-binding model by showing UNC-45A is enriched on curved lattice regions and modulates MT curvature, and identified the causative variant of a human cholestatic syndrome.\",\n      \"evidence\": \"in vitro reconstitution with curvature analysis, TIRF, overexpression/depletion; and whole-genome sequencing, CRISPR cell model, and liver biopsy IHC for Aagenaes syndrome\",\n      \"pmids\": [\"37858676\", \"37328071\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of curvature preference not defined\", \"Causal chain from UNC45A loss to BSEP/MRP2 mislocalization (chaperone vs. trafficking) not dissected\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Added myosin 1b as a chaperone client and positioned it downstream of UNC-45A in epithelial lumen formation, extending the client network.\",\n      \"evidence\": \"interactome mass spectrometry, CRISPR/Cas9 KO, proteasome inhibition, 3D organoids, and zebrafish morpholino\",\n      \"pmids\": [\"39636728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding vs. indirect stabilization of MYO1B not separated\", \"In vitro folding assay absent\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a molecular mechanism for a dominant-acting disease variant: O2HE missense alleles retain folding activity but lock the chaperone-myosin complex via abnormal oligomerization, impairing trafficking.\",\n      \"evidence\": \"patient fibroblast and U2OS functional assays, transferrin trafficking, oligomerization analysis, and wild-type co-expression rescue\",\n      \"pmids\": [\"40125554\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of pathogenic oligomerization not determined\", \"Generalizability across other UNC45A variants untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How UNC-45A's chaperone and microtubule-severing activities are spatially and temporally coordinated within a single cell, and how its nuclear localization and transcriptional functions are regulated, remain unresolved.\",\n      \"evidence\": \"no single study reconciles the cytoskeletal, nuclear, and severing roles mechanistically\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of UNC-45A bound to a myosin client or microtubule\", \"Mechanism of nuclear import and GR activation undefined\", \"Regulatory switch between myosin folding and MT severing unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 10, 13]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 10, 13]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2, 3, 4]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 2, 15]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [6, 15]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 5, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 10, 13]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [10, 14, 17]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [9, 10, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MYH9\", \"MYO5B\", \"MYO1B\", \"MYO1C\", \"CHEK1\", \"NEK7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}