{"gene":"DNAJB6","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1999,"finding":"Mrj (DNAJB6) is essential for chorioallantoic fusion in mouse placental development; null homozygous mutants die at mid-gestation due to failure of this process, with reduced expression of trophoblast-specific transcription factors Err2 and Gcm1 in the chorion.","method":"Gene-trap null allele in mouse; embryological analysis of Mrj−/− conceptuses","journal":"Development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function (null allele), specific developmental phenotype with molecular markers, replicated in follow-up papers from same lab","pmids":["10021343"],"is_preprint":false},{"year":2000,"finding":"DNAJB6 (Mrj) directly binds keratin 18 (K18) through its C-terminus and interacts with Hsp/c70 via its J domain; microinjection of anti-Mrj antibody disorganizes K8/18 intermediate filaments without affecting actin or microtubules, indicating Mrj is required for K8/18 filament organization as a K18-specific co-chaperone.","method":"Yeast two-hybrid, co-immunoprecipitation, immunostaining, anti-Mrj antibody microinjection in HeLa cells","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, yeast two-hybrid, and functional antibody microinjection with specific cytoskeletal phenotype","pmids":["10954706"],"is_preprint":false},{"year":2002,"finding":"DNAJB6 (MRJ) suppresses polyglutamine-dependent protein aggregation, caspase activation, and cellular toxicity in a cell model of Huntington's disease; MRJ is highly enriched in the CNS.","method":"Cell-based aggregation assay (in vitro HD model), overexpression of MRJ in cells, caspase activity assay","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean cellular assay with specific readouts, single lab, single set of methods","pmids":["11896048"],"is_preprint":false},{"year":2005,"finding":"DNAJB6 (Mrj) directly associates with NFATc3 and recruits class II histone deacetylases (HDACs) to repress NFAT transcriptional activity in the nucleus; this requires heat shock stimulation, reduces NFATc3 occupancy at the TNF-α promoter, and blocks calcineurin-induced cardiomyocyte hypertrophic growth. siRNA-mediated knockdown of Mrj augments NFAT activity and induces cardiomyocyte hypertrophy.","method":"Yeast two-hybrid screen, Co-IP, in vitro binding assay, siRNA knockdown, chromatin immunoprecipitation (ChIP), reporter assays","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid, reciprocal Co-IP, in vitro binding, ChIP, and functional siRNA knockdown with cardiomyocyte hypertrophy phenotype","pmids":["16260608"],"is_preprint":false},{"year":2006,"finding":"Drosophila MRJ (dmrj) ortholog of human DNAJB6 suppresses polyglutamine toxicity in neurons and co-localizes with polyQ inclusions; it increases the level of detergent-soluble monomeric polyQ-expanded proteins, a different mechanism from dHDJ1 which promotes cytoplasmic aggregation.","method":"Drosophila transgenic overexpression, eye pigmentation/structural integrity assay, quantitative PCR, colocalization imaging","journal":"Neurobiology of Disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo Drosophila model, multiple orthogonal readouts, single lab","pmids":["16934481"],"is_preprint":false},{"year":2007,"finding":"In Mrj (DNAJB6)-null mouse placenta, absence of Mrj prevents proteasome-dependent degradation of keratin 18 (K18), causing keratin inclusion body formation in chorionic trophoblasts that disrupts chorioallantoic attachment. Genetic reduction of keratin expression in Mrj−/− conceptuses rescues chorioallantoic attachment, demonstrating keratin aggregate cytotoxicity—not loss of normal keratin—is the pathological mechanism.","method":"Genetic rescue experiments (Mrj−/− crossed to K18-null), proteasome inhibitor treatment, mouse knockout analysis","journal":"Development","confidence":"High","confidence_rationale":"Tier 1 / Strong — genetic epistasis by double-mutant rescue with specific molecular mechanism, clean loss-of-function","pmids":["17409114"],"is_preprint":false},{"year":2008,"finding":"DNAJB6 enhances nuclear import of Schlafen1 (Slfn1) and stabilizes it in complex with Hsp70; in DnaJB6 knockdown cells, Slfn1 is sequestered in the cytoplasm and no cell-cycle arrest occurs. Transgenic DnaJB6 expression in T-lineage cells promotes Slfn1 nuclear import, inhibits Slfn1 degradation, downregulates cyclin D1, and suppresses T-cell proliferation.","method":"Co-IP, knockdown/overexpression in T-cell lines, subcellular fractionation, FACS cell cycle analysis, DnaJB6 transgenic mice","journal":"The Biochemical Journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, knockdown and transgenic overexpression, in vivo T-cell model, multiple orthogonal methods","pmids":["18373498"],"is_preprint":false},{"year":2008,"finding":"DNAJB6 large isoform (MRJ-L) contains a functional nuclear localization sequence and when expressed in breast cancer cells reduces migration, invasion, and orthotopic tumor growth; the secreted proteome of MRJ(L)-expressing cells shows reduced osteopontin (SPP1), SPARC, and NPM1 and increased KiSS1.","method":"Ectopic expression in breast cancer cell lines, in vivo nude mouse orthotopic tumor model, mass spectrometry of secreted proteome, NLS identification","journal":"Breast Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo functional assays, proteomics, localization study, single lab","pmids":["18328103"],"is_preprint":false},{"year":2009,"finding":"DNAJB6 is present in the core of Lewy bodies in Parkinson's disease substantia nigra and cortex, suggesting early involvement of this chaperone in the neuronal disease process; DNAJB6 is strongly upregulated in parkinsonian astrocytes.","method":"Immunohistochemistry of post-mortem PD brain tissue with confocal imaging; in silico transcriptome analysis","journal":"Journal of Neuroscience Research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment in human tissue confirmed by multiple staining approaches; no functional follow-up in same paper","pmids":["18711724"],"is_preprint":false},{"year":2009,"finding":"Mrj is required for neural stem cell self-renewal; Mrj−/− neurospheres are significantly smaller and form fewer secondary neurospheres. Loss of Mrj causes neural tube defects and reduces proliferating neuroepithelial cells and expression of progenitor markers (Pax6, Olig2, Hes5) independently of the placental phenotype.","method":"Conditional rescue experiments separating neural from placental phenotype, neurosphere assay, BrdU proliferation analysis, molecular marker expression","journal":"Developmental Dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — null mouse with specific neural phenotype, neurosphere functional assay, marker analysis; single lab","pmids":["19777589"],"is_preprint":false},{"year":2010,"finding":"DNAJB6 large isoform (MRJ-L) induces upregulation of DKK1 (a Wnt/β-catenin signaling inhibitor), causing degradation of β-catenin and downregulation of mesenchymal markers (vimentin, N-cadherin, Twist, Slug) with upregulation of keratin 18, leading to partial reversal of mesenchymal phenotype in cancer cells.","method":"Ectopic expression of MRJ(L) in cancer cell lines, Western blot, immunostaining, morphological analysis","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple molecular readouts with overexpression and specific pathway analysis, single lab","pmids":["20522561"],"is_preprint":false},{"year":2010,"finding":"DNAJB6 (MRJ) interacts with uPAR (urokinase receptor) via its C-terminal region; this complex enhances uPAR-mediated cell adhesion to vitronectin. Full-length MRJ is required for interaction and adhesion enhancement; truncated N- or C-terminal constructs alone are insufficient.","method":"Yeast two-hybrid screen, GST pulldown, co-immunoprecipitation, cell adhesion assay, deletion mapping","journal":"International Journal of Oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid, GST pulldown, Co-IP, functional adhesion assay, deletion mapping; single lab","pmids":["20372789"],"is_preprint":false},{"year":2011,"finding":"Mrj-null trophoblast cells accumulate keratin aggregates, causing collapse of actin cytoskeleton, E-cadherin and β-catenin misexpression, ECM disorganization, and failure to adhere and differentiate into syncytiotrophoblast. Plating Mrj-deficient cells on exogenous laminin-511 normalizes their behavior.","method":"Mrj−/− mouse trophoblast cell analysis, immunofluorescence, in vitro differentiation assay, laminin rescue experiment","journal":"Developmental Dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockout model with molecular and cellular phenotyping and partial rescue, single lab","pmids":["21972064"],"is_preprint":false},{"year":2012,"finding":"Dominant missense mutations in the G/F domain of DNAJB6 (Phe89Ile, Phe93Leu, Phe93Ile) cause LGMD1D. Functional testing in vivo showed mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform (DNAJB6b). In vitro studies showed mutations increase DNAJB6 half-life, extend this effect to the wild-type protein, and reduce its anti-aggregation activity. DNAJB6 interacts with members of the CASA complex including BAG3.","method":"Patient exome sequencing, in vivo Drosophila and cell functional tests, in vitro aggregation assay, co-immunoprecipitation with CASA complex members","journal":"Nature Genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (in vivo and in vitro functional assays, Co-IP), independently validated in companion paper (PMID:22334415), multiple patient families","pmids":["22366786"],"is_preprint":false},{"year":2012,"finding":"DNAJB6 mutations in the G/F domain (Phe93Leu, Pro96Arg) cause dominant myopathy with abnormal aggregation of TDP-43 and DNAJB6 itself in affected muscle, consistent with impaired anti-aggregation chaperone function.","method":"Exome sequencing, linkage analysis, muscle histochemistry and immunohistochemistry for TDP-43 and DNAJB6","journal":"Annals of Neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic identification plus tissue pathological analysis; replicates and extends PMID:22366786","pmids":["22334415"],"is_preprint":false},{"year":2012,"finding":"DNAJB6 binds HSPA8 (HSC70) via its J domain and recruits protein phosphatase PP2A to dephosphorylate GSK3β at Ser9, thereby activating GSK3β, promoting β-catenin degradation, reducing TCF/LEF activity, and suppressing osteopontin (OPN) expression. Deletion of the J domain abolishes assembly of this multiprotein complex.","method":"Co-immunoprecipitation of DNAJB6–HSPA8–PP2A complex, Western blot for GSK3β phosphorylation, deletion mutagenesis of J domain, in vitro and in vivo tumor assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP of multiprotein complex, domain deletion mutagenesis, phosphorylation readout, in vitro and in vivo functional validation","pmids":["22266849"],"is_preprint":false},{"year":2012,"finding":"DNAJB6 suppresses tumor metastasis and EMT by upregulating DKK1 transcription. Mechanistically, β-catenin stabilization (from DNAJB6 silencing) drives MSX1 transcription, which then suppresses DKK1 at its promoter. This defines a β-catenin/MSX1/DKK1 negative feedback loop regulated by DNAJB6.","method":"siRNA knockdown, ChIP assay on MSX1 and DKK1 promoters, reporter assays, overexpression studies","journal":"The Biochemical Journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, reporter, and knockdown with multiple molecular readouts; single lab","pmids":["22455953"],"is_preprint":false},{"year":2012,"finding":"miR-632 targets the coding region of DNAJB6, downregulates DNAJB6 protein levels, and increases invasive ability of breast cancer cells. Silencing endogenous miR-632 abrogates invasive ability and promotes epithelial characteristics.","method":"miRNA target prediction, luciferase reporter assay, miRNA overexpression and silencing, invasion assay","journal":"Laboratory Investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assay validating miR-632 targeting, functional invasion phenotype, single lab","pmids":["22710984"],"is_preprint":false},{"year":2012,"finding":"MRJ(S) (cytoplasmic DNAJB6b) translocates to the nucleus in response to heat shock and hypoxia via a NLS-independent mechanism mediated by a 20-amino-acid C-terminal stress-sensing region; constitutive nuclear localization of MRJ(S) promotes proliferation and invasiveness.","method":"Deletion analysis, stress induction experiments, subcellular fractionation and imaging","journal":"Experimental Cell Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — deletion mapping with functional localization data and cellular phenotype; single lab","pmids":["22504047"],"is_preprint":false},{"year":2013,"finding":"Purified DNAJB6 forms large heterogeneous oligomers (unlike dimeric DNAJB1) and, at substoichiometric molar ratios, directly suppresses fibrillation of polyQ45 peptides in vitro independently of HSPA1 and ATP. The suppression is a direct protein-protein interaction between DNAJB6 and polyQ peptides.","method":"Protein purification, thioflavin T fibrillation assay in vitro, comparison with DNAJB1, ATP/HSPA1 independence tests","journal":"Cell Stress & Chaperones","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro with purified proteins, mechanistic delineation from ATP/Hsp70 independence, replicated in companion cellular study","pmids":["23904097"],"is_preprint":false},{"year":2013,"finding":"DNAJB6 and DNAJB8, but not HSPA/Hsp70 or DNAJB1, prevent intracellular aggregation of polyglutamine peptides expressed in cells; DNAJB6 and DNAJB8 affect soluble polyQ peptide levels, indicating direct inhibition of polyQ peptide aggregation (not via indirect cellular effects).","method":"Cellular polyQ peptide aggregation assay, fluorescence microscopy, filter trap assay","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean cell-based functional comparison across multiple DNAJ family members, replicated with in vitro data in companion paper","pmids":["23612975"],"is_preprint":false},{"year":2014,"finding":"DNAJB6 inhibits amyloid-β42 (Aβ42) fibrillation at highly sub-stoichiometric molar ratios by interacting with aggregated (not monomeric) forms of Aβ42, preventing growth of aggregated species and inhibiting both primary and secondary nucleation. DNAJB6 is gradually incorporated into growing fibrils and depleted from solution.","method":"Thioflavin T fluorescence, far-UV CD spectroscopy, quantitative kinetic analysis, immunochemistry","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins, kinetic analysis, multiple orthogonal biophysical methods","pmids":["25217638"],"is_preprint":false},{"year":2014,"finding":"DNAJB6 (MRJ) forms a triple complex with HSP70 and uPAR; MRJ overexpression enhances HSP70–uPAR interaction while MRJ knockdown reduces soluble uPAR and triple complex formation. Knockdown of HSP70 and/or MRJ inhibits uPAR-mediated cell adhesion, invasion, migration, and MMP2/MMP9 expression via the MAPK/ERK and FAK pathways.","method":"Co-immunoprecipitation, overexpression, siRNA knockdown, transwell invasion assay, wound-healing assay, Western blot","journal":"BMC Cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, functional knockdown assays, molecular pathway analysis; single lab","pmids":["25175595"],"is_preprint":false},{"year":2015,"finding":"The J domain HPD motif of DNAJB6a is required for its tumor-suppressive effects; DNAJB6a forms a complex with AKT1 in living cells (detected by bimolecular fluorescence complementation) and reduces AKT signaling. Loss of DNAJB6a results in upregulation of AKT signaling; nuclear DNAJB6 localization is required for its anti-proliferative effects in cancer cells.","method":"Bimolecular fluorescence complementation, xenograft tumor assay, immunoblot of AKT signaling, shRNA knockdown, NLS mutant overexpression","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 / Strong — bimolecular fluorescence complementation for live-cell interaction, multiple cancer cell lines, in vivo xenograft, domain mutagenesis (HPD motif)","pmids":["26302489"],"is_preprint":false},{"year":2016,"finding":"DNAJB6b (long isoform, dnajb6b(L)) acts as a cardioprotective gene in zebrafish and mouse cardiomyopathy models; loss-of-function exacerbates cardiomyopathy, and the deleterious effects are ameliorated by inhibition of ER stress. Overexpression of dnajb6(L) exerts cardioprotective effects.","method":"Zebrafish insertional mutagenesis screen, doxorubicin stress assay, ER stress inhibitor treatment, mouse cardiomyopathy model","journal":"JCI Insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — forward genetic screen in zebrafish, ER stress pathway placement, mouse model validation; single lab","pmids":["27642634"],"is_preprint":false},{"year":2016,"finding":"Overexpression of DNAJB6 in cytoplasmic inclusions of LGMD1D muscle is associated with markers of defective chaperone-assisted selective autophagy (CASA) including BAG3, ubiquitin, TDP-43, p62, and SMI-31, indicating DNAJB6 mutations impair CASA-dependent protein quality control at the sarcomere.","method":"Immunohistochemistry, electron microscopy, extensive protein marker panel on LGMD1D patient muscle biopsies","journal":"Acta Neuropathologica Communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — detailed tissue pathology with co-localization of multiple CASA pathway markers; no direct biochemical reconstitution","pmids":["26847086"],"is_preprint":false},{"year":2017,"finding":"DNAJB6 suppression of α-synuclein aggregation requires its J domain (H31Q catalytically inactive mutant cannot suppress aggregation) and is dependent on Hsp70 (the co-chaperone partnership). CRISPR/Cas9 knockout of DNAJB6 in HEK293T-α-syn cells causes massive α-syn aggregation, reversed by DNAJB6 re-introduction.","method":"CRISPR/Cas9 KO, re-introduction with J-domain mutant (H31Q), fluorescence microscopy for aggregation","journal":"Scientific Reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean CRISPR KO rescue experiment, active-site mutagenesis (H31Q), Hsp70-dependence established, specific phenotypic readout","pmids":["28831037"],"is_preprint":false},{"year":2018,"finding":"Conserved S/T residues in the unique S/T-rich region of DNAJB6 are required for efficient inhibition of Aβ42 amyloid fibril formation; progressive S/T-to-A substitutions progressively reduce suppression, particularly of primary nucleation kinetics. S/T residues mediate binding to Aβ42 (measured by SPR and microscale thermophoresis), and DNAJB6 keeps monomeric Aβ42 soluble over extended time.","method":"Thioflavin T fibrillation assay, surface plasmon resonance, microscale thermophoresis, NMR spectroscopy, systematic S/T substitution mutagenesis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biophysical methods, systematic mutagenesis, in vitro reconstitution with purified proteins","pmids":["30024736"],"is_preprint":false},{"year":2018,"finding":"The DNAJB6 oligomeric structure includes a peptide-binding cleft lined with conserved S/T residues at the dimer interface; oligomers are dynamic and exchange subunits. Elongated particles (160×120 Å) were detected by EM, and crosslinking MS provided distance constraints supporting a dimer model.","method":"Lysine-specific crosslinking MS, homology modeling, docking, mixed isotope crosslinking, SAXS, negative-stain EM with single particle reconstruction","journal":"Scientific Reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple structural methods (SAXS, EM, crosslinking MS) with functional validation, single lab but orthogonal approaches","pmids":["29581438"],"is_preprint":false},{"year":2018,"finding":"Morpholino-mediated isoform switching of DNAJB6/MRJ (reducing MRJ-L relative to MRJ-S) suppresses HIV-1 and RSV replication; CstF64 reduction correlates with increase of MRJ-L in macrophages. MRJ-L depletion reduced viral mRNA/genome production.","method":"Morpholino oligonucleotide treatment, viral replication assay (RT-qPCR for viral genome), CstF64 manipulation","journal":"Molecular Therapy. Nucleic Acids","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — morpholino isoform-specific knockdown with viral replication readout; single lab, single method type","pmids":["30641477"],"is_preprint":false},{"year":2019,"finding":"DNAJB6 mutations in the J domain (p.A50V, p.E54A) cause dominant distal/proximo-distal myopathy with histology similar to G/F domain mutations; both J-domain mutations show reduced anti-aggregation capacity by filter trap assay and TDP-43 disaggregation assays. Structural modeling shows mutated J-domain residues are in close proximity to G/F domain residues.","method":"Targeted gene sequencing, filter trap assay, TDP-43 disaggregation assay, structural protein modeling","journal":"Neuromuscular Disorders","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional in vitro aggregation assays validate mutation effect, genetic co-segregation; single lab","pmids":["31955980"],"is_preprint":false},{"year":2019,"finding":"DNAJB6 CRISPR knockout in HEK293T-α-syn cells increases seeded α-syn aggregation induced by recombinant preformed fibrils (PFFs); this increase is strongly reduced by proteasomal inhibitor MG132, suggesting DNAJB6 targets aggregated/misfolded α-syn for proteasomal degradation. DNAJB6b but not DNAJB1 suppresses seeded α-syn aggregation.","method":"CRISPR/Cas9 KO, α-syn PFF seeding assay, fluorescence microscopy and FRET analysis, proteasome inhibitor (MG132) treatment","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO with pharmacological pathway dissection, FRET quantification; single lab","pmids":["31514384"],"is_preprint":false},{"year":2019,"finding":"Hsp40 DNAJB6 interacts with JEV NS3 protein (identified by yeast two-hybrid and confirmed by co-localization and Co-IP); loss of DNAJB6 function increases JEV replication without affecting viral binding or internalization, indicating DNAJB6 negatively regulates post-entry viral replication.","method":"Yeast two-hybrid screen, co-immunoprecipitation, co-localization imaging, DNAJB6 knockout/knockdown with viral replication assay","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP and functional KO assay; single lab","pmids":["31739611"],"is_preprint":false},{"year":2019,"finding":"DNAJB6 knockout in myoblasts leads to accumulation of sarcomeric proteins and hypertrophic myotubes with enhanced fusion index, correlating with diminished GSK3β activity. In contrast, LGMD1D DNAJB6 mutations (e.g., F93L) enhance GSK3β activation and suppress β-catenin and NFAT3c signaling. GSK3β inhibition with lithium chloride improves muscle size and strength in a DNAJB6b-F93L LGMD1D mouse model.","method":"CRISPR/Cas9 KO myoblasts, stable isotope labeling/quantitative mass spectrometry, histochemistry, immunohistochemistry, grip strength and inverted wire hang tests in LGMD1D mouse model, lithium chloride treatment","journal":"Neurology. Genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO and knockin mouse model, quantitative proteomics, in vivo therapeutic rescue, multiple orthogonal methods","pmids":["31123706"],"is_preprint":false},{"year":2019,"finding":"DnaJB6 is a RanGTP-regulated protein that interacts with dynactin subunit p150Glued (DCTN1) in a RanGTP-dependent manner specifically during M-phase; it promotes spindle pole focusing and dynein force generation during mitosis.","method":"Co-immunoprecipitation (M-phase specific), RanGTP dependency assay, spindle pole focusing analysis, dynein force generation assay","journal":"Journal of Cell Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with RanGTP specificity, functional spindle assay; single lab","pmids":["31064815"],"is_preprint":false},{"year":2020,"finding":"Loss of DNAJB6 expression during neuronal differentiation (confirmed in iPSC-derived neurons and in vivo) explains neuronal hypersensitivity to polyQ aggregation in SCA3 and HD. DNAJB6 upregulation in neurons antagonizes glutamate-induced polyQ aggregation; DNAJB6 knockdown in neural progenitors causes spontaneous polyQ aggregation.","method":"iPSC generation and neuronal differentiation, glutamate treatment aggregation assay, DNAJB6 overexpression and siRNA knockdown in neurons/progenitors, in vivo expression confirmation","journal":"Molecular Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — iPSC patient-derived neurons, knockdown and overexpression rescue, in vivo confirmation, multiple patient lines","pmids":["32268123"],"is_preprint":false},{"year":2020,"finding":"DNAJB6 directly captures oligomeric forms of Aβ (Aβ1-40) via its S/T residues, as detected by native mass spectrometry; WT DNAJB6 reduces signals from Aβ oligomers and appears to form dimers/trimers bound to Aβ, while S/T-to-A mutant does not. This confirms oligomeric Aβ (not monomers) as the primary DNAJB6 substrate for inhibiting primary nucleation.","method":"Native mass spectrometry-based detection of Aβ oligomers with WT vs S/T mutant DNAJB6","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biophysical detection by native MS, mutagenesis validation, mechanistically consistent with prior kinetic studies","pmids":["32350108"],"is_preprint":false},{"year":2020,"finding":"DNAJB6 knockout in HEK293 cells causes a 5-fold increase in polyQ74-huntingtin aggregation and increased cell death; DNAJA1 KO causes a 4-fold decrease in aggregation, demonstrating that DNAJA1 and DNAJB6 modulate polyQ aggregation in opposite directions. DNAJB1 KO had no effect.","method":"CRISPR/Cas9 KO of DNAJB6, DNAJA1, DNAJB1, fluorescence microscopy, filter trap assay, trypan blue and PI cell death assays","journal":"Scientific Reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean CRISPR KO with multiple orthogonal assays, comparison across three DNAJ family members in parallel","pmids":["32424160"],"is_preprint":false},{"year":2021,"finding":"Overexpression of DNAJB6b in rat substantia nigra via AAV suppresses α-syn aggregation induced by α-syn overexpression, strongly reduces dopaminergic cell death, and rescues motor behavior deficits (stepping test) in an in vivo Parkinson's disease model.","method":"AAV6-mediated overexpression in rat SNpc, α-syn PFF seeding cell assay, immunohistochemistry for dopaminergic neurons, behavioral assessment","journal":"Neurobiology of Disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo AAV overexpression in animal model with both molecular (cell death) and behavioral readouts, replicates cellular findings","pmids":["34390836"],"is_preprint":false},{"year":2022,"finding":"DNAJB6 forms foci near nuclear pore complexes (NPCs) and localizes inside herniations at NPC biogenesis intermediates (confirmed by immunoelectron tomography). DNAJB6 binds FG-Nups and prevents aggregation of FG regions in cells and in vitro. Loss of DNAJB6 causes accumulation of cytosolic annulate lamellae, demonstrating a role in interphase NPC biogenesis quality control.","method":"Immunoelectron tomography, live imaging, in vitro FG-Nup aggregation assay, DNAJB6 knockout with NPC biogenesis readout, Co-IP of FG-Nups","journal":"Nature Cell Biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — immunoelectron tomography for localization, in vitro reconstitution, KO phenotype, Co-IP, multiple orthogonal methods in one study","pmids":["36302971"],"is_preprint":false},{"year":2022,"finding":"Disease-associated LGMDD1 mutations in DNAJB6 G/F domain alter function in a substrate/conformer-specific manner: they change the structure of client aggregates, interfere with the Hsp70 ATPase cycle, reduce dimerization, and impair substrate processing in a dominant-negative manner (poisoning WT Sis1 function in yeast prion model).","method":"Yeast prion model (Sis1 as functional homolog), in vitro chaperone activity assays, Hsp70 ATPase cycle assay, dimerization analysis","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro chaperone reconstitution assays, ATPase cycle measurement, dominant-negative mechanistic evidence in yeast model","pmids":["35931773"],"is_preprint":false},{"year":2022,"finding":"The T193A mutation in the C-terminal domain (CTD) of DNAJB6 reduces self-oligomerization and anti-aggregation activity. NMR shows the mutation has minimal effects on β-stranded CTD structure but increases population and rate of a partially folded state via β-strand peptide plane flips at ≈100 μs timescale, altering ability to oligomerize.","method":"NMR spectroscopy including relaxation-based methods, oligomerization assays","journal":"Angewandte Chemie","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR with relaxation-based dynamics analysis, mutagenesis linking structure to oligomerization to function","pmids":["35247211","38505697"],"is_preprint":false},{"year":2022,"finding":"Dnajb6 is required for cardiac pacemaker function; GBT411 zebrafish insertional mutant (trapping Dnajb6) shows sick sinus syndrome (SSS)-like cardiac arrhythmia. Dnajb6 is expressed in a subpopulation of sinus node pacemaker cells overlapping with HCN4 expression.","method":"Zebrafish insertional mutagenesis (gene-break transposon), electrocardiographic measurement, in situ expression analysis, mouse model validation","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — forward genetic screen with electrophysiological phenotyping and expression analysis; single lab","pmids":["36255053"],"is_preprint":false},{"year":2023,"finding":"LGMDD1 disease-mutant DNAJB6 proteins show no reduction in aggregation-prevention activity in vitro; instead, solution NMR reveals structural changes in the G/F domain that disrupt regulation of Hsp70 binding. While WT DNAJB6 contains a helical element that regulates its ability to activate Hsp70, LGMDD1 mutants lack this regulation and hyperactivate Hsp70 in an unregulated manner, depleting Hsp70 in myocytes. Interfering with DNAJB6–Hsp70 binding reverses the disease phenotype.","method":"Solution NMR (structural characterization), in vitro aggregation assay, biochemical Hsp70 interaction assays, myocyte Hsp70 level measurement, disease phenotype reversal experiment","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structural characterization, in vitro biochemistry, disease model phenotype rescue; identifies novel toxic-gain-of-function mechanism distinct from prior loss-of-function model","pmids":["37923706"],"is_preprint":false},{"year":2023,"finding":"The C-terminal domain (CTD) of DNAJB6 (specifically the first two β-strands) is sufficient to inhibit secondary nucleation of Aβ42 fibril formation by binding Aβ42 fibrils, but inhibition of primary nucleation requires the full-length protein including regions outside the CTD. The S/T residues in the CTD are required for CTD dimerization/stability but not for secondary nucleation inhibition per se.","method":"CTD constructs grafted onto scaffold protein, thioflavin T kinetics, native MS, chemical crosslinking, surface plasmon resonance","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — domain dissection with multiple biophysical methods, mechanistic delineation of primary vs secondary nucleation inhibition","pmids":["37797698"],"is_preprint":false},{"year":2023,"finding":"FBXL21 ubiquitinates DNAJB6 and its client protein Desmin for proteasomal degradation; LGMDD1 DNAJB6 mutations render resistance to FBXL21-directed degradation. Fbxl21 KO causes aberrant Desmin accumulation and aggravated cytoplasmic TDP-43 accumulation during heat shock.","method":"Ubiquitination assay, proteasomal degradation assay, Fbxl21 KO cells, LGMDD1 mutation functional test, TDP-43 localization under stress","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical ubiquitination assay, KO phenotype, mutation resistance experiment; preprint, single lab","pmids":["42239455"],"is_preprint":true},{"year":2025,"finding":"DNAJB6 co-phase separates with FUS-containing condensates and locks them into a loose gel-like state that prevents fibrilization, thereby suppressing FUS-ALS toxicity. Domain mapping and deep mutational scan identify key residues required for this activity. DNAJB6 overexpression prevents motor neuron loss and microglial activation in a mouse model of FUS-ALS.","method":"Yeast multiplex genetic screen, biophysical examination of co-phase separation, domain mapping, deep mutational scan, mouse FUS-ALS model with motor neuron counting","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — biophysical reconstitution of condensate behavior, deep mutational scan, in vivo mouse model validation","pmids":["41271702"],"is_preprint":false},{"year":2025,"finding":"The conserved intrinsically disordered region (IDR) of DNAJB6 promotes stable gel-like assemblies that prevent aberrant phase transitions of FG-Nups; DNAJB6, DNAJB2, and DNAJB8 all prevent FG-Nup aberrant phase transitions. Mutant analysis shows the sequence space for the IDR is narrow and optimized to avoid self-aggregation while providing anti-amyloidogenic capacity.","method":"Phase separation assay for FG-Nups, DNAJB6 IDR mutant analysis, gel-like assembly characterization","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional mutant analysis with phase separation assays; preprint, extends prior published NPC findings","pmids":[],"is_preprint":true},{"year":2025,"finding":"The GF-linker of DNAJB6 (as with DNAJB1) creates a hydrophobic partially collapsed cluster with the J-domain that provides autoinhibition of Hsp70 binding; disruption of this cluster destabilizes autoinhibition. The GF-linker is also recognized by the substrate-binding domain of Hsc70 and dictates the lifetime of the JDP–Hsc70 complex; both functions are DNAJB-class member-specific.","method":"NMR spectroscopy of autoinhibited DNAJB6 constructs, allosteric communication analysis, Hsc70 binding assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR structural analysis, Hsc70 binding assay; preprint, single lab","pmids":[],"is_preprint":true}],"current_model":"DNAJB6 is an oligomeric HSP40/DNAJ co-chaperone that directly binds aggregation-prone substrates (polyglutamine peptides, Aβ42 oligomers, α-synuclein, FG-nucleoporins, FUS condensates) via conserved S/T residues in its C-terminal domain and a dynamic GF linker that autoinhibits Hsp70 activation; disease mutations (LGMDD1) structurally disrupt the GF-domain helix that regulates Hsp70 binding, causing hyperactivation and Hsp70 depletion rather than a simple loss of anti-aggregation activity, while also impairing the CASA/BAG3 protein quality-control complex in muscle; additionally, the DNAJB6b cytoplasmic isoform supports NPC biogenesis by surveilling FG-Nup phase transitions, regulates sarcomeric proteostasis through GSK3β-dependent myogenic signaling, and the nuclear DNAJB6a isoform suppresses Wnt/β-catenin and AKT signaling to restrain tumor progression."},"narrative":{"mechanistic_narrative":"DNAJB6 is an oligomeric HSP40/J-domain co-chaperone that functions as a generalist suppressor of pathological protein aggregation and phase transitions across development, muscle, and neurons [PMID:11896048, PMID:23904097, PMID:36302971]. It directly binds aggregation-prone substrates at substoichiometric ratios — polyglutamine peptides, amyloid-β42, and α-synuclein — through conserved S/T residues in a C-terminal peptide-binding cleft formed at its oligomeric dimer interface, blocking primary and secondary nucleation of amyloid fibrils [PMID:23904097, PMID:25217638, PMID:30024736, PMID:29581438, PMID:32350108]. Anti-aggregation activity for some clients (e.g. α-synuclein) requires the J domain and Hsp70 partnership and routes misfolded species toward proteasomal degradation, whereas direct fibrillation suppression of polyQ and Aβ proceeds independently of ATP and Hsp70 [PMID:23904097, PMID:28831037, PMID:31514384]. The protein exists as cytoplasmic (DNAJB6b) and nuclear (DNAJB6a) isoforms with distinct roles: the cytoplasmic isoform surveils FG-nucleoporin phase transitions to support interphase nuclear pore complex biogenesis and locks FUS condensates into a non-fibrillar gel-like state [PMID:36302971, PMID:41271702], while the nuclear isoform restrains tumor progression by assembling Hsp70/PP2A complexes that activate GSK3β and by suppressing Wnt/β-catenin and AKT signaling [PMID:22266849, PMID:22455953, PMID:26302489]. In development, DNAJB6 prevents cytotoxic keratin-18 aggregation required for chorioallantoic fusion and supports neural stem cell self-renewal [PMID:17409114, PMID:19777589]. Dominant missense mutations in the G/F and J domains cause limb-girdle muscular dystrophy (LGMDD1); these mutations disrupt a G/F-domain helical element that normally regulates Hsp70 binding, producing unregulated Hsp70 hyperactivation and depletion and impairing CASA/BAG3-dependent sarcomeric protein quality control, rather than a simple loss of anti-aggregation activity [PMID:22366786, PMID:26847086, PMID:37923706].","teleology":[{"year":1999,"claim":"Established DNAJB6 as an essential developmental gene before any molecular activity was known, anchoring its in vivo importance.","evidence":"Gene-trap null allele in mouse with embryological analysis of placental development","pmids":["10021343"],"confidence":"High","gaps":["Did not define the molecular activity underlying the chorioallantoic fusion defect","Did not distinguish isoform-specific contributions"]},{"year":2000,"claim":"Defined DNAJB6 as a substrate-specific co-chaperone, linking its J domain to Hsp70 and its C-terminus to a client (keratin 18) for cytoskeletal organization.","evidence":"Yeast two-hybrid, reciprocal Co-IP, and anti-Mrj antibody microinjection in HeLa cells","pmids":["10954706"],"confidence":"High","gaps":["Did not resolve whether keratin binding reflects folding or degradation triage","No structural basis for C-terminal client binding"]},{"year":2007,"claim":"Resolved the developmental mechanism: DNAJB6 prevents cytotoxic keratin aggregation rather than maintaining normal keratin, established by genetic epistasis.","evidence":"Mrj−/− × K18-null double-mutant rescue and proteasome inhibition in mouse placenta","pmids":["17409114"],"confidence":"High","gaps":["Did not show direct biochemical disaggregation of keratin in vitro","Aggregate-clearance route only inferred from proteasome dependence"]},{"year":2006,"claim":"Extended anti-aggregation function to neurodegeneration-relevant polyQ proteins and showed it acts by raising soluble monomeric client, distinct from aggregation-promoting J proteins.","evidence":"Cell-based HD aggregation assay and Drosophila polyQ neurodegeneration model with colocalization","pmids":["11896048","16934481"],"confidence":"Medium","gaps":["Mechanism of solubilization not biochemically defined at this stage","Overexpression-based; endogenous requirement not tested"]},{"year":2008,"claim":"Revealed DNAJB6 as a regulator of client nuclear import and stability, and uncovered an isoform/localization dimension with opposing cancer outcomes.","evidence":"Co-IP, knockdown/transgenic T-cell models for Slfn1 import; ectopic large-isoform expression in breast cancer with secretome MS","pmids":["18373498","18328103"],"confidence":"High","gaps":["Did not unify how chaperone activity drives transcriptional/secretome changes","Isoform-specific structural determinants of localization unresolved"]},{"year":2012,"claim":"Identified DNAJB6 G/F-domain mutations as the cause of LGMD1D and tied disease specifically to the cytoplasmic isoform with reduced anti-aggregation activity and CASA/BAG3 association.","evidence":"Patient exome sequencing, Drosophila and cell functional tests, in vitro aggregation assay, Co-IP with CASA members; replicated with TDP-43 muscle pathology","pmids":["22366786","22334415"],"confidence":"High","gaps":["Initial framing as loss of anti-aggregation activity (later revised)","Half-life extension mechanism not structurally explained at this point"]},{"year":2012,"claim":"Defined a nuclear/cancer signaling axis in which DNAJB6 assembles an Hsp70-PP2A complex to activate GSK3β and drives a β-catenin/MSX1/DKK1 feedback loop to suppress EMT and metastasis.","evidence":"Co-IP of multiprotein complex, J-domain deletion, GSK3β phospho readouts, ChIP/reporter assays, tumor assays","pmids":["22266849","22455953"],"confidence":"High","gaps":["Whether co-chaperone activity per se versus scaffolding drives signaling not separated","Connection between aggregation surveillance and transcriptional control unclear"]},{"year":2013,"claim":"Demonstrated that DNAJB6 is an oligomer that directly and substoichiometrically suppresses amyloid fibrillation independently of ATP and Hsp70, defining an intrinsic anti-aggregation mechanism.","evidence":"Purified-protein thioflavin T fibrillation assays with polyQ45, ATP/HSPA1 independence tests, comparison to dimeric DNAJB1; parallel cellular polyQ assays","pmids":["23904097","23612975"],"confidence":"High","gaps":["Did not localize the substrate-binding surface on the protein","Oligomer architecture not yet resolved"]},{"year":2014,"claim":"Established that DNAJB6 targets aggregated/oligomeric Aβ species and inhibits both primary and secondary nucleation, becoming incorporated into fibrils.","evidence":"Thioflavin T kinetics, CD, quantitative kinetic analysis, immunochemistry with purified Aβ42; triple complex Co-IP with Hsp70/uPAR in cancer cells","pmids":["25217638","25175595"],"confidence":"High","gaps":["Residues mediating Aβ binding not identified","Stoichiometry of fibril incorporation not quantified"]},{"year":2018,"claim":"Mapped the molecular basis of anti-aggregation to conserved S/T residues lining a peptide-binding cleft at the oligomer dimer interface.","evidence":"Systematic S/T-to-A mutagenesis with SPR, microscale thermophoresis, NMR; crosslinking MS, SAXS, and negative-stain EM of oligomers","pmids":["30024736","29581438"],"confidence":"High","gaps":["High-resolution structure of the full oligomer not obtained","How dynamic subunit exchange couples to client capture unresolved"]},{"year":2017,"claim":"Showed J-domain/Hsp70-dependent handling of α-synuclein and that endogenous DNAJB6 is required to prevent aggregation, defining a degradation-coupled route distinct from ATP-independent fibrillation blockade.","evidence":"CRISPR KO with rescue, catalytically dead H31Q J-domain mutant, and MG132 proteasome inhibition in α-syn cell models","pmids":["28831037","31514384"],"confidence":"High","gaps":["Why some clients require Hsp70 while others do not is not mechanistically unified","In vivo relevance shown only later"]},{"year":2019,"claim":"Connected DNAJB6 to muscle and cytoskeletal physiology through GSK3β-dependent sarcomeric proteostasis, mitotic spindle function, and a J-domain disease class with reduced disaggregation activity.","evidence":"CRISPR KO myoblasts with quantitative proteomics and LGMD1D mouse lithium rescue; RanGTP-dependent p150Glued Co-IP and spindle assays; J-domain mutation filter-trap/TDP-43 disaggregation assays","pmids":["31123706","31064815","31955980"],"confidence":"High","gaps":["Direct substrate at the sarcomere not biochemically identified","Mechanistic link between chaperone activity and GSK3β regulation in muscle incomplete"]},{"year":2020,"claim":"Identified oligomeric Aβ as the captured species by native MS and explained neuronal vulnerability via developmental loss of DNAJB6 expression.","evidence":"Native mass spectrometry with WT vs S/T mutant DNAJB6; iPSC-derived neuron differentiation with knockdown/overexpression rescue and parallel CRISPR comparison to DNAJA1/DNAJB1","pmids":["32350108","32268123","32424160"],"confidence":"High","gaps":["Why DNAJA1 and DNAJB6 act oppositely on polyQ not fully explained","Transcriptional control of neuronal DNAJB6 downregulation unresolved"]},{"year":2022,"claim":"Defined a cell-biological surveillance role at nuclear pore complexes and refined the disease mechanism toward dominant-negative substrate/Hsp70-cycle perturbation.","evidence":"Immunoelectron tomography, in vitro FG-Nup aggregation assays and Co-IP for NPC biogenesis; yeast Sis1 prion model with ATPase-cycle and dimerization assays for LGMDD1 mutants","pmids":["36302971","35931773"],"confidence":"High","gaps":["How FG-Nup surveillance is spatially restricted to NPC intermediates not fully defined","Reconciliation of substrate-specific versus Hsp70-cycle disease effects pending"]},{"year":2023,"claim":"Resolved the LGMDD1 mechanism as a toxic gain of function: mutations disrupt a G/F-domain helix regulating Hsp70 binding, causing unregulated Hsp70 hyperactivation and depletion that is reversible by interfering with the DNAJB6–Hsp70 interaction.","evidence":"Solution NMR structural characterization, in vitro aggregation and Hsp70 interaction assays, myocyte Hsp70 measurement, disease phenotype reversal; FBXL21 ubiquitination of DNAJB6/Desmin (preprint)","pmids":["37923706","37797698","42239455"],"confidence":"High","gaps":["FBXL21 regulation reported only in preprint without independent confirmation","Therapeutic window for Hsp70-interaction disruption in vivo not established"]},{"year":2025,"claim":"Generalized DNAJB6 function to control of biomolecular condensates, showing it co-phase-separates with FUS and FG-Nups to maintain non-fibrillar gel-like states via its intrinsically disordered region.","evidence":"Biophysical co-phase-separation and deep mutational scanning with FUS-ALS mouse model; FG-Nup phase transition assays with IDR mutants (preprint) and NMR of GF-linker autoinhibition (preprint)","pmids":["41271702"],"confidence":"High","gaps":["IDR phase-control mechanism and GF-linker autoinhibition reported partly in preprints awaiting peer review","How condensate control integrates with classical Hsp70 co-chaperone cycling unresolved"]},{"year":null,"claim":"It remains unresolved how DNAJB6's intrinsic, Hsp70-independent anti-aggregation activity is mechanistically coordinated with its Hsp70-dependent, degradation-coupled and signaling roles, and how isoform localization and oligomer dynamics partition these functions across tissues.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of a client-bound oligomer","No unified model linking condensate control, Hsp70 cycling, and transcriptional/signaling outputs","Isoform-specific structural switch governing nuclear vs cytoplasmic function undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[1,19,26,27,39]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,6,26]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[19,21,36,46]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,15,43,48]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[13,18,39]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,6,18,23]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[39]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[34]}],"pathway":[{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[18,19,21,27]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[5,26,31]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[15,16,23,33]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,5,9]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[39]}],"complexes":["CASA/BAG3 complex","DNAJB6–HSPA8–PP2A complex","DNAJB6–HSP70–uPAR complex"],"partners":["HSPA8","BAG3","KRT18","NFATC3","AKT1","DCTN1","PLAUR","FBXL21"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75190","full_name":"DnaJ homolog subfamily B member 6","aliases":["HHDJ1","Heat shock protein J2","HSJ-2","MRJ","MSJ-1"],"length_aa":326,"mass_kda":36.1,"function":"Has a stimulatory effect on the ATPase activity of HSP70 in a dose-dependent and time-dependent manner and hence acts as a co-chaperone of HSP70 (PubMed:10954706, PubMed:28233300). Plays an indispensable role in the organization of KRT8/KRT18 filaments (PubMed:10954706). Acts as an endogenous molecular chaperone for neuronal proteins including huntingtin (PubMed:11896048, PubMed:22366786). Suppresses aggregation and toxicity of polyglutamine-containing, aggregation-prone proteins (PubMed:20159555, PubMed:22366786). Also reduces cellular toxicity and caspase-3 activity (PubMed:11896048) Isoform B but not isoform A inhibits huntingtin aggregation","subcellular_location":"Cytoplasm, perinuclear region; Nucleus; Cytoplasm, myofibril, sarcomere, Z line","url":"https://www.uniprot.org/uniprotkb/O75190/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DNAJB6","classification":"Not Classified","n_dependent_lines":421,"n_total_lines":1208,"dependency_fraction":0.3485099337748344},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000105993","cell_line_id":"CID000020","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nuclear_punctae","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"RNF219","stoichiometry":0.2},{"gene":"HSPH1","stoichiometry":0.2},{"gene":"MLK4","stoichiometry":0.2},{"gene":"HSP90AB2P","stoichiometry":0.2},{"gene":"HSP90AB4P","stoichiometry":0.2},{"gene":"TUBA1A;TUBA3E","stoichiometry":0.2},{"gene":"TUBB4B","stoichiometry":0.2},{"gene":"HSPA2","stoichiometry":0.2},{"gene":"ALDH18A1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000020","total_profiled":1310},"omim":[{"mim_id":"621078","title":"MYOPATHY, MYOFIBRILLAR, 13, WITH RIMMED VACUOLES; MFM13","url":"https://www.omim.org/entry/621078"},{"mim_id":"611332","title":"DNAJ/HSP40 HOMOLOG, SUBFAMILY B, MEMBER 6; DNAJB6","url":"https://www.omim.org/entry/611332"},{"mim_id":"603511","title":"MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL DOMINANT 1; LGMDD1","url":"https://www.omim.org/entry/603511"},{"mim_id":"158590","title":"NEURONOPATHY, DISTAL HEREDITARY MOTOR, AUTOSOMAL DOMINANT 2; HMND2","url":"https://www.omim.org/entry/158590"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DNAJB6"},"hgnc":{"alias_symbol":["MRJ"],"prev_symbol":["LGMD1D"]},"alphafold":{"accession":"O75190","domains":[{"cath_id":"1.10.287.110","chopping":"5-76_92-115","consensus_level":"high","plddt":80.7608,"start":5,"end":115},{"cath_id":"-","chopping":"190-246","consensus_level":"high","plddt":83.6196,"start":190,"end":246}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75190","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75190-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75190-F1-predicted_aligned_error_v6.png","plddt_mean":62.97},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DNAJB6","jax_strain_url":"https://www.jax.org/strain/search?query=DNAJB6"},"sequence":{"accession":"O75190","fasta_url":"https://rest.uniprot.org/uniprotkb/O75190.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75190/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75190"}},"corpus_meta":[{"pmid":"22366786","id":"PMC_22366786","title":"Mutations 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\"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, yeast two-hybrid, and functional antibody microinjection with specific cytoskeletal phenotype\",\n      \"pmids\": [\"10954706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DNAJB6 (MRJ) suppresses polyglutamine-dependent protein aggregation, caspase activation, and cellular toxicity in a cell model of Huntington's disease; MRJ is highly enriched in the CNS.\",\n      \"method\": \"Cell-based aggregation assay (in vitro HD model), overexpression of MRJ in cells, caspase activity assay\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean cellular assay with specific readouts, single lab, single set of methods\",\n      \"pmids\": [\"11896048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"DNAJB6 (Mrj) directly associates with NFATc3 and recruits class II histone deacetylases (HDACs) to repress NFAT transcriptional activity in the nucleus; this requires heat shock stimulation, reduces NFATc3 occupancy at the TNF-α promoter, and blocks calcineurin-induced cardiomyocyte hypertrophic growth. siRNA-mediated knockdown of Mrj augments NFAT activity and induces cardiomyocyte hypertrophy.\",\n      \"method\": \"Yeast two-hybrid screen, Co-IP, in vitro binding assay, siRNA knockdown, chromatin immunoprecipitation (ChIP), reporter assays\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid, reciprocal Co-IP, in vitro binding, ChIP, and functional siRNA knockdown with cardiomyocyte hypertrophy phenotype\",\n      \"pmids\": [\"16260608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Drosophila MRJ (dmrj) ortholog of human DNAJB6 suppresses polyglutamine toxicity in neurons and co-localizes with polyQ inclusions; it increases the level of detergent-soluble monomeric polyQ-expanded proteins, a different mechanism from dHDJ1 which promotes cytoplasmic aggregation.\",\n      \"method\": \"Drosophila transgenic overexpression, eye pigmentation/structural integrity assay, quantitative PCR, colocalization imaging\",\n      \"journal\": \"Neurobiology of Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo Drosophila model, multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"16934481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In Mrj (DNAJB6)-null mouse placenta, absence of Mrj prevents proteasome-dependent degradation of keratin 18 (K18), causing keratin inclusion body formation in chorionic trophoblasts that disrupts chorioallantoic attachment. Genetic reduction of keratin expression in Mrj−/− conceptuses rescues chorioallantoic attachment, demonstrating keratin aggregate cytotoxicity—not loss of normal keratin—is the pathological mechanism.\",\n      \"method\": \"Genetic rescue experiments (Mrj−/− crossed to K18-null), proteasome inhibitor treatment, mouse knockout analysis\",\n      \"journal\": \"Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genetic epistasis by double-mutant rescue with specific molecular mechanism, clean loss-of-function\",\n      \"pmids\": [\"17409114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DNAJB6 enhances nuclear import of Schlafen1 (Slfn1) and stabilizes it in complex with Hsp70; in DnaJB6 knockdown cells, Slfn1 is sequestered in the cytoplasm and no cell-cycle arrest occurs. Transgenic DnaJB6 expression in T-lineage cells promotes Slfn1 nuclear import, inhibits Slfn1 degradation, downregulates cyclin D1, and suppresses T-cell proliferation.\",\n      \"method\": \"Co-IP, knockdown/overexpression in T-cell lines, subcellular fractionation, FACS cell cycle analysis, DnaJB6 transgenic mice\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, knockdown and transgenic overexpression, in vivo T-cell model, multiple orthogonal methods\",\n      \"pmids\": [\"18373498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DNAJB6 large isoform (MRJ-L) contains a functional nuclear localization sequence and when expressed in breast cancer cells reduces migration, invasion, and orthotopic tumor growth; the secreted proteome of MRJ(L)-expressing cells shows reduced osteopontin (SPP1), SPARC, and NPM1 and increased KiSS1.\",\n      \"method\": \"Ectopic expression in breast cancer cell lines, in vivo nude mouse orthotopic tumor model, mass spectrometry of secreted proteome, NLS identification\",\n      \"journal\": \"Breast Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo functional assays, proteomics, localization study, single lab\",\n      \"pmids\": [\"18328103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DNAJB6 is present in the core of Lewy bodies in Parkinson's disease substantia nigra and cortex, suggesting early involvement of this chaperone in the neuronal disease process; DNAJB6 is strongly upregulated in parkinsonian astrocytes.\",\n      \"method\": \"Immunohistochemistry of post-mortem PD brain tissue with confocal imaging; in silico transcriptome analysis\",\n      \"journal\": \"Journal of Neuroscience Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment in human tissue confirmed by multiple staining approaches; no functional follow-up in same paper\",\n      \"pmids\": [\"18711724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mrj is required for neural stem cell self-renewal; Mrj−/− neurospheres are significantly smaller and form fewer secondary neurospheres. Loss of Mrj causes neural tube defects and reduces proliferating neuroepithelial cells and expression of progenitor markers (Pax6, Olig2, Hes5) independently of the placental phenotype.\",\n      \"method\": \"Conditional rescue experiments separating neural from placental phenotype, neurosphere assay, BrdU proliferation analysis, molecular marker expression\",\n      \"journal\": \"Developmental Dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — null mouse with specific neural phenotype, neurosphere functional assay, marker analysis; single lab\",\n      \"pmids\": [\"19777589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"DNAJB6 large isoform (MRJ-L) induces upregulation of DKK1 (a Wnt/β-catenin signaling inhibitor), causing degradation of β-catenin and downregulation of mesenchymal markers (vimentin, N-cadherin, Twist, Slug) with upregulation of keratin 18, leading to partial reversal of mesenchymal phenotype in cancer cells.\",\n      \"method\": \"Ectopic expression of MRJ(L) in cancer cell lines, Western blot, immunostaining, morphological analysis\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple molecular readouts with overexpression and specific pathway analysis, single lab\",\n      \"pmids\": [\"20522561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"DNAJB6 (MRJ) interacts with uPAR (urokinase receptor) via its C-terminal region; this complex enhances uPAR-mediated cell adhesion to vitronectin. Full-length MRJ is required for interaction and adhesion enhancement; truncated N- or C-terminal constructs alone are insufficient.\",\n      \"method\": \"Yeast two-hybrid screen, GST pulldown, co-immunoprecipitation, cell adhesion assay, deletion mapping\",\n      \"journal\": \"International Journal of Oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid, GST pulldown, Co-IP, functional adhesion assay, deletion mapping; single lab\",\n      \"pmids\": [\"20372789\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Mrj-null trophoblast cells accumulate keratin aggregates, causing collapse of actin cytoskeleton, E-cadherin and β-catenin misexpression, ECM disorganization, and failure to adhere and differentiate into syncytiotrophoblast. Plating Mrj-deficient cells on exogenous laminin-511 normalizes their behavior.\",\n      \"method\": \"Mrj−/− mouse trophoblast cell analysis, immunofluorescence, in vitro differentiation assay, laminin rescue experiment\",\n      \"journal\": \"Developmental Dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout model with molecular and cellular phenotyping and partial rescue, single lab\",\n      \"pmids\": [\"21972064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Dominant missense mutations in the G/F domain of DNAJB6 (Phe89Ile, Phe93Leu, Phe93Ile) cause LGMD1D. Functional testing in vivo showed mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform (DNAJB6b). In vitro studies showed mutations increase DNAJB6 half-life, extend this effect to the wild-type protein, and reduce its anti-aggregation activity. DNAJB6 interacts with members of the CASA complex including BAG3.\",\n      \"method\": \"Patient exome sequencing, in vivo Drosophila and cell functional tests, in vitro aggregation assay, co-immunoprecipitation with CASA complex members\",\n      \"journal\": \"Nature Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (in vivo and in vitro functional assays, Co-IP), independently validated in companion paper (PMID:22334415), multiple patient families\",\n      \"pmids\": [\"22366786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DNAJB6 mutations in the G/F domain (Phe93Leu, Pro96Arg) cause dominant myopathy with abnormal aggregation of TDP-43 and DNAJB6 itself in affected muscle, consistent with impaired anti-aggregation chaperone function.\",\n      \"method\": \"Exome sequencing, linkage analysis, muscle histochemistry and immunohistochemistry for TDP-43 and DNAJB6\",\n      \"journal\": \"Annals of Neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic identification plus tissue pathological analysis; replicates and extends PMID:22366786\",\n      \"pmids\": [\"22334415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DNAJB6 binds HSPA8 (HSC70) via its J domain and recruits protein phosphatase PP2A to dephosphorylate GSK3β at Ser9, thereby activating GSK3β, promoting β-catenin degradation, reducing TCF/LEF activity, and suppressing osteopontin (OPN) expression. Deletion of the J domain abolishes assembly of this multiprotein complex.\",\n      \"method\": \"Co-immunoprecipitation of DNAJB6–HSPA8–PP2A complex, Western blot for GSK3β phosphorylation, deletion mutagenesis of J domain, in vitro and in vivo tumor assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP of multiprotein complex, domain deletion mutagenesis, phosphorylation readout, in vitro and in vivo functional validation\",\n      \"pmids\": [\"22266849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DNAJB6 suppresses tumor metastasis and EMT by upregulating DKK1 transcription. Mechanistically, β-catenin stabilization (from DNAJB6 silencing) drives MSX1 transcription, which then suppresses DKK1 at its promoter. This defines a β-catenin/MSX1/DKK1 negative feedback loop regulated by DNAJB6.\",\n      \"method\": \"siRNA knockdown, ChIP assay on MSX1 and DKK1 promoters, reporter assays, overexpression studies\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, reporter, and knockdown with multiple molecular readouts; single lab\",\n      \"pmids\": [\"22455953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"miR-632 targets the coding region of DNAJB6, downregulates DNAJB6 protein levels, and increases invasive ability of breast cancer cells. Silencing endogenous miR-632 abrogates invasive ability and promotes epithelial characteristics.\",\n      \"method\": \"miRNA target prediction, luciferase reporter assay, miRNA overexpression and silencing, invasion assay\",\n      \"journal\": \"Laboratory Investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assay validating miR-632 targeting, functional invasion phenotype, single lab\",\n      \"pmids\": [\"22710984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MRJ(S) (cytoplasmic DNAJB6b) translocates to the nucleus in response to heat shock and hypoxia via a NLS-independent mechanism mediated by a 20-amino-acid C-terminal stress-sensing region; constitutive nuclear localization of MRJ(S) promotes proliferation and invasiveness.\",\n      \"method\": \"Deletion analysis, stress induction experiments, subcellular fractionation and imaging\",\n      \"journal\": \"Experimental Cell Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — deletion mapping with functional localization data and cellular phenotype; single lab\",\n      \"pmids\": [\"22504047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Purified DNAJB6 forms large heterogeneous oligomers (unlike dimeric DNAJB1) and, at substoichiometric molar ratios, directly suppresses fibrillation of polyQ45 peptides in vitro independently of HSPA1 and ATP. The suppression is a direct protein-protein interaction between DNAJB6 and polyQ peptides.\",\n      \"method\": \"Protein purification, thioflavin T fibrillation assay in vitro, comparison with DNAJB1, ATP/HSPA1 independence tests\",\n      \"journal\": \"Cell Stress & Chaperones\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro with purified proteins, mechanistic delineation from ATP/Hsp70 independence, replicated in companion cellular study\",\n      \"pmids\": [\"23904097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DNAJB6 and DNAJB8, but not HSPA/Hsp70 or DNAJB1, prevent intracellular aggregation of polyglutamine peptides expressed in cells; DNAJB6 and DNAJB8 affect soluble polyQ peptide levels, indicating direct inhibition of polyQ peptide aggregation (not via indirect cellular effects).\",\n      \"method\": \"Cellular polyQ peptide aggregation assay, fluorescence microscopy, filter trap assay\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean cell-based functional comparison across multiple DNAJ family members, replicated with in vitro data in companion paper\",\n      \"pmids\": [\"23612975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DNAJB6 inhibits amyloid-β42 (Aβ42) fibrillation at highly sub-stoichiometric molar ratios by interacting with aggregated (not monomeric) forms of Aβ42, preventing growth of aggregated species and inhibiting both primary and secondary nucleation. DNAJB6 is gradually incorporated into growing fibrils and depleted from solution.\",\n      \"method\": \"Thioflavin T fluorescence, far-UV CD spectroscopy, quantitative kinetic analysis, immunochemistry\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins, kinetic analysis, multiple orthogonal biophysical methods\",\n      \"pmids\": [\"25217638\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DNAJB6 (MRJ) forms a triple complex with HSP70 and uPAR; MRJ overexpression enhances HSP70–uPAR interaction while MRJ knockdown reduces soluble uPAR and triple complex formation. Knockdown of HSP70 and/or MRJ inhibits uPAR-mediated cell adhesion, invasion, migration, and MMP2/MMP9 expression via the MAPK/ERK and FAK pathways.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, siRNA knockdown, transwell invasion assay, wound-healing assay, Western blot\",\n      \"journal\": \"BMC Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, functional knockdown assays, molecular pathway analysis; single lab\",\n      \"pmids\": [\"25175595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The J domain HPD motif of DNAJB6a is required for its tumor-suppressive effects; DNAJB6a forms a complex with AKT1 in living cells (detected by bimolecular fluorescence complementation) and reduces AKT signaling. Loss of DNAJB6a results in upregulation of AKT signaling; nuclear DNAJB6 localization is required for its anti-proliferative effects in cancer cells.\",\n      \"method\": \"Bimolecular fluorescence complementation, xenograft tumor assay, immunoblot of AKT signaling, shRNA knockdown, NLS mutant overexpression\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — bimolecular fluorescence complementation for live-cell interaction, multiple cancer cell lines, in vivo xenograft, domain mutagenesis (HPD motif)\",\n      \"pmids\": [\"26302489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DNAJB6b (long isoform, dnajb6b(L)) acts as a cardioprotective gene in zebrafish and mouse cardiomyopathy models; loss-of-function exacerbates cardiomyopathy, and the deleterious effects are ameliorated by inhibition of ER stress. Overexpression of dnajb6(L) exerts cardioprotective effects.\",\n      \"method\": \"Zebrafish insertional mutagenesis screen, doxorubicin stress assay, ER stress inhibitor treatment, mouse cardiomyopathy model\",\n      \"journal\": \"JCI Insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — forward genetic screen in zebrafish, ER stress pathway placement, mouse model validation; single lab\",\n      \"pmids\": [\"27642634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Overexpression of DNAJB6 in cytoplasmic inclusions of LGMD1D muscle is associated with markers of defective chaperone-assisted selective autophagy (CASA) including BAG3, ubiquitin, TDP-43, p62, and SMI-31, indicating DNAJB6 mutations impair CASA-dependent protein quality control at the sarcomere.\",\n      \"method\": \"Immunohistochemistry, electron microscopy, extensive protein marker panel on LGMD1D patient muscle biopsies\",\n      \"journal\": \"Acta Neuropathologica Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — detailed tissue pathology with co-localization of multiple CASA pathway markers; no direct biochemical reconstitution\",\n      \"pmids\": [\"26847086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DNAJB6 suppression of α-synuclein aggregation requires its J domain (H31Q catalytically inactive mutant cannot suppress aggregation) and is dependent on Hsp70 (the co-chaperone partnership). CRISPR/Cas9 knockout of DNAJB6 in HEK293T-α-syn cells causes massive α-syn aggregation, reversed by DNAJB6 re-introduction.\",\n      \"method\": \"CRISPR/Cas9 KO, re-introduction with J-domain mutant (H31Q), fluorescence microscopy for aggregation\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean CRISPR KO rescue experiment, active-site mutagenesis (H31Q), Hsp70-dependence established, specific phenotypic readout\",\n      \"pmids\": [\"28831037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Conserved S/T residues in the unique S/T-rich region of DNAJB6 are required for efficient inhibition of Aβ42 amyloid fibril formation; progressive S/T-to-A substitutions progressively reduce suppression, particularly of primary nucleation kinetics. S/T residues mediate binding to Aβ42 (measured by SPR and microscale thermophoresis), and DNAJB6 keeps monomeric Aβ42 soluble over extended time.\",\n      \"method\": \"Thioflavin T fibrillation assay, surface plasmon resonance, microscale thermophoresis, NMR spectroscopy, systematic S/T substitution mutagenesis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biophysical methods, systematic mutagenesis, in vitro reconstitution with purified proteins\",\n      \"pmids\": [\"30024736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The DNAJB6 oligomeric structure includes a peptide-binding cleft lined with conserved S/T residues at the dimer interface; oligomers are dynamic and exchange subunits. Elongated particles (160×120 Å) were detected by EM, and crosslinking MS provided distance constraints supporting a dimer model.\",\n      \"method\": \"Lysine-specific crosslinking MS, homology modeling, docking, mixed isotope crosslinking, SAXS, negative-stain EM with single particle reconstruction\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple structural methods (SAXS, EM, crosslinking MS) with functional validation, single lab but orthogonal approaches\",\n      \"pmids\": [\"29581438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Morpholino-mediated isoform switching of DNAJB6/MRJ (reducing MRJ-L relative to MRJ-S) suppresses HIV-1 and RSV replication; CstF64 reduction correlates with increase of MRJ-L in macrophages. MRJ-L depletion reduced viral mRNA/genome production.\",\n      \"method\": \"Morpholino oligonucleotide treatment, viral replication assay (RT-qPCR for viral genome), CstF64 manipulation\",\n      \"journal\": \"Molecular Therapy. Nucleic Acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — morpholino isoform-specific knockdown with viral replication readout; single lab, single method type\",\n      \"pmids\": [\"30641477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DNAJB6 mutations in the J domain (p.A50V, p.E54A) cause dominant distal/proximo-distal myopathy with histology similar to G/F domain mutations; both J-domain mutations show reduced anti-aggregation capacity by filter trap assay and TDP-43 disaggregation assays. Structural modeling shows mutated J-domain residues are in close proximity to G/F domain residues.\",\n      \"method\": \"Targeted gene sequencing, filter trap assay, TDP-43 disaggregation assay, structural protein modeling\",\n      \"journal\": \"Neuromuscular Disorders\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional in vitro aggregation assays validate mutation effect, genetic co-segregation; single lab\",\n      \"pmids\": [\"31955980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DNAJB6 CRISPR knockout in HEK293T-α-syn cells increases seeded α-syn aggregation induced by recombinant preformed fibrils (PFFs); this increase is strongly reduced by proteasomal inhibitor MG132, suggesting DNAJB6 targets aggregated/misfolded α-syn for proteasomal degradation. DNAJB6b but not DNAJB1 suppresses seeded α-syn aggregation.\",\n      \"method\": \"CRISPR/Cas9 KO, α-syn PFF seeding assay, fluorescence microscopy and FRET analysis, proteasome inhibitor (MG132) treatment\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO with pharmacological pathway dissection, FRET quantification; single lab\",\n      \"pmids\": [\"31514384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Hsp40 DNAJB6 interacts with JEV NS3 protein (identified by yeast two-hybrid and confirmed by co-localization and Co-IP); loss of DNAJB6 function increases JEV replication without affecting viral binding or internalization, indicating DNAJB6 negatively regulates post-entry viral replication.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, co-localization imaging, DNAJB6 knockout/knockdown with viral replication assay\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP and functional KO assay; single lab\",\n      \"pmids\": [\"31739611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DNAJB6 knockout in myoblasts leads to accumulation of sarcomeric proteins and hypertrophic myotubes with enhanced fusion index, correlating with diminished GSK3β activity. In contrast, LGMD1D DNAJB6 mutations (e.g., F93L) enhance GSK3β activation and suppress β-catenin and NFAT3c signaling. GSK3β inhibition with lithium chloride improves muscle size and strength in a DNAJB6b-F93L LGMD1D mouse model.\",\n      \"method\": \"CRISPR/Cas9 KO myoblasts, stable isotope labeling/quantitative mass spectrometry, histochemistry, immunohistochemistry, grip strength and inverted wire hang tests in LGMD1D mouse model, lithium chloride treatment\",\n      \"journal\": \"Neurology. Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO and knockin mouse model, quantitative proteomics, in vivo therapeutic rescue, multiple orthogonal methods\",\n      \"pmids\": [\"31123706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DnaJB6 is a RanGTP-regulated protein that interacts with dynactin subunit p150Glued (DCTN1) in a RanGTP-dependent manner specifically during M-phase; it promotes spindle pole focusing and dynein force generation during mitosis.\",\n      \"method\": \"Co-immunoprecipitation (M-phase specific), RanGTP dependency assay, spindle pole focusing analysis, dynein force generation assay\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with RanGTP specificity, functional spindle assay; single lab\",\n      \"pmids\": [\"31064815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss of DNAJB6 expression during neuronal differentiation (confirmed in iPSC-derived neurons and in vivo) explains neuronal hypersensitivity to polyQ aggregation in SCA3 and HD. DNAJB6 upregulation in neurons antagonizes glutamate-induced polyQ aggregation; DNAJB6 knockdown in neural progenitors causes spontaneous polyQ aggregation.\",\n      \"method\": \"iPSC generation and neuronal differentiation, glutamate treatment aggregation assay, DNAJB6 overexpression and siRNA knockdown in neurons/progenitors, in vivo expression confirmation\",\n      \"journal\": \"Molecular Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — iPSC patient-derived neurons, knockdown and overexpression rescue, in vivo confirmation, multiple patient lines\",\n      \"pmids\": [\"32268123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DNAJB6 directly captures oligomeric forms of Aβ (Aβ1-40) via its S/T residues, as detected by native mass spectrometry; WT DNAJB6 reduces signals from Aβ oligomers and appears to form dimers/trimers bound to Aβ, while S/T-to-A mutant does not. This confirms oligomeric Aβ (not monomers) as the primary DNAJB6 substrate for inhibiting primary nucleation.\",\n      \"method\": \"Native mass spectrometry-based detection of Aβ oligomers with WT vs S/T mutant DNAJB6\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biophysical detection by native MS, mutagenesis validation, mechanistically consistent with prior kinetic studies\",\n      \"pmids\": [\"32350108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DNAJB6 knockout in HEK293 cells causes a 5-fold increase in polyQ74-huntingtin aggregation and increased cell death; DNAJA1 KO causes a 4-fold decrease in aggregation, demonstrating that DNAJA1 and DNAJB6 modulate polyQ aggregation in opposite directions. DNAJB1 KO had no effect.\",\n      \"method\": \"CRISPR/Cas9 KO of DNAJB6, DNAJA1, DNAJB1, fluorescence microscopy, filter trap assay, trypan blue and PI cell death assays\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean CRISPR KO with multiple orthogonal assays, comparison across three DNAJ family members in parallel\",\n      \"pmids\": [\"32424160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Overexpression of DNAJB6b in rat substantia nigra via AAV suppresses α-syn aggregation induced by α-syn overexpression, strongly reduces dopaminergic cell death, and rescues motor behavior deficits (stepping test) in an in vivo Parkinson's disease model.\",\n      \"method\": \"AAV6-mediated overexpression in rat SNpc, α-syn PFF seeding cell assay, immunohistochemistry for dopaminergic neurons, behavioral assessment\",\n      \"journal\": \"Neurobiology of Disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo AAV overexpression in animal model with both molecular (cell death) and behavioral readouts, replicates cellular findings\",\n      \"pmids\": [\"34390836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DNAJB6 forms foci near nuclear pore complexes (NPCs) and localizes inside herniations at NPC biogenesis intermediates (confirmed by immunoelectron tomography). DNAJB6 binds FG-Nups and prevents aggregation of FG regions in cells and in vitro. Loss of DNAJB6 causes accumulation of cytosolic annulate lamellae, demonstrating a role in interphase NPC biogenesis quality control.\",\n      \"method\": \"Immunoelectron tomography, live imaging, in vitro FG-Nup aggregation assay, DNAJB6 knockout with NPC biogenesis readout, Co-IP of FG-Nups\",\n      \"journal\": \"Nature Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — immunoelectron tomography for localization, in vitro reconstitution, KO phenotype, Co-IP, multiple orthogonal methods in one study\",\n      \"pmids\": [\"36302971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Disease-associated LGMDD1 mutations in DNAJB6 G/F domain alter function in a substrate/conformer-specific manner: they change the structure of client aggregates, interfere with the Hsp70 ATPase cycle, reduce dimerization, and impair substrate processing in a dominant-negative manner (poisoning WT Sis1 function in yeast prion model).\",\n      \"method\": \"Yeast prion model (Sis1 as functional homolog), in vitro chaperone activity assays, Hsp70 ATPase cycle assay, dimerization analysis\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro chaperone reconstitution assays, ATPase cycle measurement, dominant-negative mechanistic evidence in yeast model\",\n      \"pmids\": [\"35931773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The T193A mutation in the C-terminal domain (CTD) of DNAJB6 reduces self-oligomerization and anti-aggregation activity. NMR shows the mutation has minimal effects on β-stranded CTD structure but increases population and rate of a partially folded state via β-strand peptide plane flips at ≈100 μs timescale, altering ability to oligomerize.\",\n      \"method\": \"NMR spectroscopy including relaxation-based methods, oligomerization assays\",\n      \"journal\": \"Angewandte Chemie\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR with relaxation-based dynamics analysis, mutagenesis linking structure to oligomerization to function\",\n      \"pmids\": [\"35247211\", \"38505697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Dnajb6 is required for cardiac pacemaker function; GBT411 zebrafish insertional mutant (trapping Dnajb6) shows sick sinus syndrome (SSS)-like cardiac arrhythmia. Dnajb6 is expressed in a subpopulation of sinus node pacemaker cells overlapping with HCN4 expression.\",\n      \"method\": \"Zebrafish insertional mutagenesis (gene-break transposon), electrocardiographic measurement, in situ expression analysis, mouse model validation\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — forward genetic screen with electrophysiological phenotyping and expression analysis; single lab\",\n      \"pmids\": [\"36255053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LGMDD1 disease-mutant DNAJB6 proteins show no reduction in aggregation-prevention activity in vitro; instead, solution NMR reveals structural changes in the G/F domain that disrupt regulation of Hsp70 binding. While WT DNAJB6 contains a helical element that regulates its ability to activate Hsp70, LGMDD1 mutants lack this regulation and hyperactivate Hsp70 in an unregulated manner, depleting Hsp70 in myocytes. Interfering with DNAJB6–Hsp70 binding reverses the disease phenotype.\",\n      \"method\": \"Solution NMR (structural characterization), in vitro aggregation assay, biochemical Hsp70 interaction assays, myocyte Hsp70 level measurement, disease phenotype reversal experiment\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structural characterization, in vitro biochemistry, disease model phenotype rescue; identifies novel toxic-gain-of-function mechanism distinct from prior loss-of-function model\",\n      \"pmids\": [\"37923706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The C-terminal domain (CTD) of DNAJB6 (specifically the first two β-strands) is sufficient to inhibit secondary nucleation of Aβ42 fibril formation by binding Aβ42 fibrils, but inhibition of primary nucleation requires the full-length protein including regions outside the CTD. The S/T residues in the CTD are required for CTD dimerization/stability but not for secondary nucleation inhibition per se.\",\n      \"method\": \"CTD constructs grafted onto scaffold protein, thioflavin T kinetics, native MS, chemical crosslinking, surface plasmon resonance\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — domain dissection with multiple biophysical methods, mechanistic delineation of primary vs secondary nucleation inhibition\",\n      \"pmids\": [\"37797698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FBXL21 ubiquitinates DNAJB6 and its client protein Desmin for proteasomal degradation; LGMDD1 DNAJB6 mutations render resistance to FBXL21-directed degradation. Fbxl21 KO causes aberrant Desmin accumulation and aggravated cytoplasmic TDP-43 accumulation during heat shock.\",\n      \"method\": \"Ubiquitination assay, proteasomal degradation assay, Fbxl21 KO cells, LGMDD1 mutation functional test, TDP-43 localization under stress\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical ubiquitination assay, KO phenotype, mutation resistance experiment; preprint, single lab\",\n      \"pmids\": [\"42239455\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DNAJB6 co-phase separates with FUS-containing condensates and locks them into a loose gel-like state that prevents fibrilization, thereby suppressing FUS-ALS toxicity. Domain mapping and deep mutational scan identify key residues required for this activity. DNAJB6 overexpression prevents motor neuron loss and microglial activation in a mouse model of FUS-ALS.\",\n      \"method\": \"Yeast multiplex genetic screen, biophysical examination of co-phase separation, domain mapping, deep mutational scan, mouse FUS-ALS model with motor neuron counting\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biophysical reconstitution of condensate behavior, deep mutational scan, in vivo mouse model validation\",\n      \"pmids\": [\"41271702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The conserved intrinsically disordered region (IDR) of DNAJB6 promotes stable gel-like assemblies that prevent aberrant phase transitions of FG-Nups; DNAJB6, DNAJB2, and DNAJB8 all prevent FG-Nup aberrant phase transitions. Mutant analysis shows the sequence space for the IDR is narrow and optimized to avoid self-aggregation while providing anti-amyloidogenic capacity.\",\n      \"method\": \"Phase separation assay for FG-Nups, DNAJB6 IDR mutant analysis, gel-like assembly characterization\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional mutant analysis with phase separation assays; preprint, extends prior published NPC findings\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The GF-linker of DNAJB6 (as with DNAJB1) creates a hydrophobic partially collapsed cluster with the J-domain that provides autoinhibition of Hsp70 binding; disruption of this cluster destabilizes autoinhibition. The GF-linker is also recognized by the substrate-binding domain of Hsc70 and dictates the lifetime of the JDP–Hsc70 complex; both functions are DNAJB-class member-specific.\",\n      \"method\": \"NMR spectroscopy of autoinhibited DNAJB6 constructs, allosteric communication analysis, Hsc70 binding assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural analysis, Hsc70 binding assay; preprint, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"DNAJB6 is an oligomeric HSP40/DNAJ co-chaperone that directly binds aggregation-prone substrates (polyglutamine peptides, Aβ42 oligomers, α-synuclein, FG-nucleoporins, FUS condensates) via conserved S/T residues in its C-terminal domain and a dynamic GF linker that autoinhibits Hsp70 activation; disease mutations (LGMDD1) structurally disrupt the GF-domain helix that regulates Hsp70 binding, causing hyperactivation and Hsp70 depletion rather than a simple loss of anti-aggregation activity, while also impairing the CASA/BAG3 protein quality-control complex in muscle; additionally, the DNAJB6b cytoplasmic isoform supports NPC biogenesis by surveilling FG-Nup phase transitions, regulates sarcomeric proteostasis through GSK3β-dependent myogenic signaling, and the nuclear DNAJB6a isoform suppresses Wnt/β-catenin and AKT signaling to restrain tumor progression.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DNAJB6 is an oligomeric HSP40/J-domain co-chaperone that functions as a generalist suppressor of pathological protein aggregation and phase transitions across development, muscle, and neurons [#2, #19, #39]. It directly binds aggregation-prone substrates at substoichiometric ratios — polyglutamine peptides, amyloid-\\u03b242, and \\u03b1-synuclein — through conserved S/T residues in a C-terminal peptide-binding cleft formed at its oligomeric dimer interface, blocking primary and secondary nucleation of amyloid fibrils [#19, #21, #27, #28, #36]. Anti-aggregation activity for some clients (e.g. \\u03b1-synuclein) requires the J domain and Hsp70 partnership and routes misfolded species toward proteasomal degradation, whereas direct fibrillation suppression of polyQ and A\\u03b2 proceeds independently of ATP and Hsp70 [#19, #26, #31]. The protein exists as cytoplasmic (DNAJB6b) and nuclear (DNAJB6a) isoforms with distinct roles: the cytoplasmic isoform surveils FG-nucleoporin phase transitions to support interphase nuclear pore complex biogenesis and locks FUS condensates into a non-fibrillar gel-like state [#39, #46], while the nuclear isoform restrains tumor progression by assembling Hsp70/PP2A complexes that activate GSK3\\u03b2 and by suppressing Wnt/\\u03b2-catenin and AKT signaling [#15, #16, #23]. In development, DNAJB6 prevents cytotoxic keratin-18 aggregation required for chorioallantoic fusion and supports neural stem cell self-renewal [#5, #9]. Dominant missense mutations in the G/F and J domains cause limb-girdle muscular dystrophy (LGMDD1); these mutations disrupt a G/F-domain helical element that normally regulates Hsp70 binding, producing unregulated Hsp70 hyperactivation and depletion and impairing CASA/BAG3-dependent sarcomeric protein quality control, rather than a simple loss of anti-aggregation activity [#13, #25, #43].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established DNAJB6 as an essential developmental gene before any molecular activity was known, anchoring its in vivo importance.\",\n      \"evidence\": \"Gene-trap null allele in mouse with embryological analysis of placental development\",\n      \"pmids\": [\"10021343\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the molecular activity underlying the chorioallantoic fusion defect\", \"Did not distinguish isoform-specific contributions\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined DNAJB6 as a substrate-specific co-chaperone, linking its J domain to Hsp70 and its C-terminus to a client (keratin 18) for cytoskeletal organization.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, and anti-Mrj antibody microinjection in HeLa cells\",\n      \"pmids\": [\"10954706\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether keratin binding reflects folding or degradation triage\", \"No structural basis for C-terminal client binding\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolved the developmental mechanism: DNAJB6 prevents cytotoxic keratin aggregation rather than maintaining normal keratin, established by genetic epistasis.\",\n      \"evidence\": \"Mrj\\u2212/\\u2212 \\u00d7 K18-null double-mutant rescue and proteasome inhibition in mouse placenta\",\n      \"pmids\": [\"17409114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show direct biochemical disaggregation of keratin in vitro\", \"Aggregate-clearance route only inferred from proteasome dependence\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended anti-aggregation function to neurodegeneration-relevant polyQ proteins and showed it acts by raising soluble monomeric client, distinct from aggregation-promoting J proteins.\",\n      \"evidence\": \"Cell-based HD aggregation assay and Drosophila polyQ neurodegeneration model with colocalization\",\n      \"pmids\": [\"11896048\", \"16934481\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of solubilization not biochemically defined at this stage\", \"Overexpression-based; endogenous requirement not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed DNAJB6 as a regulator of client nuclear import and stability, and uncovered an isoform/localization dimension with opposing cancer outcomes.\",\n      \"evidence\": \"Co-IP, knockdown/transgenic T-cell models for Slfn1 import; ectopic large-isoform expression in breast cancer with secretome MS\",\n      \"pmids\": [\"18373498\", \"18328103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not unify how chaperone activity drives transcriptional/secretome changes\", \"Isoform-specific structural determinants of localization unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified DNAJB6 G/F-domain mutations as the cause of LGMD1D and tied disease specifically to the cytoplasmic isoform with reduced anti-aggregation activity and CASA/BAG3 association.\",\n      \"evidence\": \"Patient exome sequencing, Drosophila and cell functional tests, in vitro aggregation assay, Co-IP with CASA members; replicated with TDP-43 muscle pathology\",\n      \"pmids\": [\"22366786\", \"22334415\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Initial framing as loss of anti-aggregation activity (later revised)\", \"Half-life extension mechanism not structurally explained at this point\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined a nuclear/cancer signaling axis in which DNAJB6 assembles an Hsp70-PP2A complex to activate GSK3\\u03b2 and drives a \\u03b2-catenin/MSX1/DKK1 feedback loop to suppress EMT and metastasis.\",\n      \"evidence\": \"Co-IP of multiprotein complex, J-domain deletion, GSK3\\u03b2 phospho readouts, ChIP/reporter assays, tumor assays\",\n      \"pmids\": [\"22266849\", \"22455953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether co-chaperone activity per se versus scaffolding drives signaling not separated\", \"Connection between aggregation surveillance and transcriptional control unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated that DNAJB6 is an oligomer that directly and substoichiometrically suppresses amyloid fibrillation independently of ATP and Hsp70, defining an intrinsic anti-aggregation mechanism.\",\n      \"evidence\": \"Purified-protein thioflavin T fibrillation assays with polyQ45, ATP/HSPA1 independence tests, comparison to dimeric DNAJB1; parallel cellular polyQ assays\",\n      \"pmids\": [\"23904097\", \"23612975\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not localize the substrate-binding surface on the protein\", \"Oligomer architecture not yet resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established that DNAJB6 targets aggregated/oligomeric A\\u03b2 species and inhibits both primary and secondary nucleation, becoming incorporated into fibrils.\",\n      \"evidence\": \"Thioflavin T kinetics, CD, quantitative kinetic analysis, immunochemistry with purified A\\u03b242; triple complex Co-IP with Hsp70/uPAR in cancer cells\",\n      \"pmids\": [\"25217638\", \"25175595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Residues mediating A\\u03b2 binding not identified\", \"Stoichiometry of fibril incorporation not quantified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Mapped the molecular basis of anti-aggregation to conserved S/T residues lining a peptide-binding cleft at the oligomer dimer interface.\",\n      \"evidence\": \"Systematic S/T-to-A mutagenesis with SPR, microscale thermophoresis, NMR; crosslinking MS, SAXS, and negative-stain EM of oligomers\",\n      \"pmids\": [\"30024736\", \"29581438\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the full oligomer not obtained\", \"How dynamic subunit exchange couples to client capture unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed J-domain/Hsp70-dependent handling of \\u03b1-synuclein and that endogenous DNAJB6 is required to prevent aggregation, defining a degradation-coupled route distinct from ATP-independent fibrillation blockade.\",\n      \"evidence\": \"CRISPR KO with rescue, catalytically dead H31Q J-domain mutant, and MG132 proteasome inhibition in \\u03b1-syn cell models\",\n      \"pmids\": [\"28831037\", \"31514384\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why some clients require Hsp70 while others do not is not mechanistically unified\", \"In vivo relevance shown only later\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected DNAJB6 to muscle and cytoskeletal physiology through GSK3\\u03b2-dependent sarcomeric proteostasis, mitotic spindle function, and a J-domain disease class with reduced disaggregation activity.\",\n      \"evidence\": \"CRISPR KO myoblasts with quantitative proteomics and LGMD1D mouse lithium rescue; RanGTP-dependent p150Glued Co-IP and spindle assays; J-domain mutation filter-trap/TDP-43 disaggregation assays\",\n      \"pmids\": [\"31123706\", \"31064815\", \"31955980\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrate at the sarcomere not biochemically identified\", \"Mechanistic link between chaperone activity and GSK3\\u03b2 regulation in muscle incomplete\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified oligomeric A\\u03b2 as the captured species by native MS and explained neuronal vulnerability via developmental loss of DNAJB6 expression.\",\n      \"evidence\": \"Native mass spectrometry with WT vs S/T mutant DNAJB6; iPSC-derived neuron differentiation with knockdown/overexpression rescue and parallel CRISPR comparison to DNAJA1/DNAJB1\",\n      \"pmids\": [\"32350108\", \"32268123\", \"32424160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why DNAJA1 and DNAJB6 act oppositely on polyQ not fully explained\", \"Transcriptional control of neuronal DNAJB6 downregulation unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined a cell-biological surveillance role at nuclear pore complexes and refined the disease mechanism toward dominant-negative substrate/Hsp70-cycle perturbation.\",\n      \"evidence\": \"Immunoelectron tomography, in vitro FG-Nup aggregation assays and Co-IP for NPC biogenesis; yeast Sis1 prion model with ATPase-cycle and dimerization assays for LGMDD1 mutants\",\n      \"pmids\": [\"36302971\", \"35931773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How FG-Nup surveillance is spatially restricted to NPC intermediates not fully defined\", \"Reconciliation of substrate-specific versus Hsp70-cycle disease effects pending\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved the LGMDD1 mechanism as a toxic gain of function: mutations disrupt a G/F-domain helix regulating Hsp70 binding, causing unregulated Hsp70 hyperactivation and depletion that is reversible by interfering with the DNAJB6\\u2013Hsp70 interaction.\",\n      \"evidence\": \"Solution NMR structural characterization, in vitro aggregation and Hsp70 interaction assays, myocyte Hsp70 measurement, disease phenotype reversal; FBXL21 ubiquitination of DNAJB6/Desmin (preprint)\",\n      \"pmids\": [\"37923706\", \"37797698\", \"42239455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"FBXL21 regulation reported only in preprint without independent confirmation\", \"Therapeutic window for Hsp70-interaction disruption in vivo not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Generalized DNAJB6 function to control of biomolecular condensates, showing it co-phase-separates with FUS and FG-Nups to maintain non-fibrillar gel-like states via its intrinsically disordered region.\",\n      \"evidence\": \"Biophysical co-phase-separation and deep mutational scanning with FUS-ALS mouse model; FG-Nup phase transition assays with IDR mutants (preprint) and NMR of GF-linker autoinhibition (preprint)\",\n      \"pmids\": [\"41271702\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IDR phase-control mechanism and GF-linker autoinhibition reported partly in preprints awaiting peer review\", \"How condensate control integrates with classical Hsp70 co-chaperone cycling unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how DNAJB6's intrinsic, Hsp70-independent anti-aggregation activity is mechanistically coordinated with its Hsp70-dependent, degradation-coupled and signaling roles, and how isoform localization and oligomer dynamics partition these functions across tissues.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of a client-bound oligomer\", \"No unified model linking condensate control, Hsp70 cycling, and transcriptional/signaling outputs\", \"Isoform-specific structural switch governing nuclear vs cytoplasmic function undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [1, 19, 26, 27, 39]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 6, 26]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [19, 21, 36, 46]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 15, 43, 48]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [13, 18, 39]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 6, 18, 23]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [39]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [34]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [18, 19, 21, 27]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [5, 26, 31]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [15, 16, 23, 33]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 5, 9]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [39]}\n    ],\n    \"complexes\": [\"CASA/BAG3 complex\", \"DNAJB6\\u2013HSPA8\\u2013PP2A complex\", \"DNAJB6\\u2013HSP70\\u2013uPAR complex\"],\n    \"partners\": [\"HSPA8\", \"BAG3\", \"KRT18\", \"NFATC3\", \"AKT1\", \"DCTN1\", \"PLAUR\", \"FBXL21\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}