Metrics details Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been reported to cause distal myopathy dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3 we studied all three variants and compared them to the BAG3_Glu455Lys mutant We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function which causes these variants to accumulate in the form of insoluble HDAC6- and vimentin-positive aggresomes The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70 promote the so-called chaperone-assisted selective autophagy (CASA) As a consequence of their increased aggregation-proneness mutant BAG3 trapped ubiquitinylated client proteins at the aggresome these data show that all BAG3_Pro209 mutants irrespective of their different clinical phenotypes are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis BAG3_Pro209 mutations cause cytoplasmic aggregation (a) Schematic representation of the structure of BAG3 The known interactors of each motif are shown at the top and the missense mutations that were studied in this manuscript are shown at the bottom in red (b) HEK293T cells stably expressing HSPB8-V5 were transiently transfected with BAG3-GFP constructs Six random fields were selected for analysis The mean number of cells counted per field was 95 and thus over 400 cells per genotype were counted (scale bar = 10 μm) (c) Quantification of BAG3-GFP inclusions using Flow cytometric analysis of inclusions (FloIT) Transiently transfected HEK293T cells were collected and stained with DAPI prior to 0.1% Triton X-100 treatment The intracellular BAG3-GFP inclusions and Hoechst-positive nuclei are subsequently quantified using flow cytometry Bar graph represents the means of BAG3-GFP cytoplasmic inclusions per 100 transfected cells One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis (d,e) Bio-informatic analysis of (d) the solubility of wild type or mutant BAG3 with CamSol and (e) of the aggregation propensity with Tango software (f) Western blot analysis of the NP-40 soluble fraction from HEK293T cells stably expressing HSPB8-V5 and transiently transfected with BAG3-GFP constructs The constructs were abbreviated as followed: wild type (WT) One of three representative western blots is shown (g) Filter retardation assay (FRA) analysis of the NP-40 insoluble fraction Anti-GFP and anti-HSPB8 antibodies were used to detect insoluble levels of BAG3 (wild type or mutants) and HSPB8 Relative optical densities are reported in the graphs as means ± SD of normalized values One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis (n = 3) The constructs were abbreviated as followed: non-transfected (NT) As also other members of the HSPB family are capable of binding to BAG3 it is thought that in case HSPB8 would be unable to fulfil its role (e.g these other sHSPs could partly replace its function by binding to BAG3 Such compensatory mechanisms would ensure that BAG3-sHSP interactions are maintained even under compromising conditions and underscore the importance of this interaction By clustering the different components into a single complex substrates are likely handed over faster to reduce the potentially dangerous dwell time further supporting the interpretation that these mutations may affect BAG3 PQC functions we stably overexpressed HSPB8 in HEK293T cells which are characterized by low expression levels of HSPB8 and abundant Hsp70 subtle differences were detected between the three Pro209 mutants; as the Pro209Leu mutation caused aggregation in a slightly higher number of cells compared to the other mutants Similar to what we observed with fluorescence microscopy the Pro209Leu mutant formed a higher amount of aggregates these data demonstrate that all three mutants affecting the IPV-motif cause protein aggregation a phenotype that seems unique to IPV-mutants as the BAG-domain Glu455Lys mutant and BAG3 wild type protein remained diffusely distributed in the cytoplasm BAG3_Pro209 mutants also aggregate in muscle (C2C12) and motoneuron-like cells (NSC-34) We transiently transfected GFP-tagged BAG3 wild type or mutant constructs in C2C12 and NSC-34 cells We then verified protein aggregation by separating the soluble fraction (western blot) and insoluble fraction (filter retardation assay (FRA)) (a,c) or verified protein aggregation by immunofluorescence (b,d) The FRA analysis is displayed for the NP-40 insoluble fraction Combined these data demonstrate that all BAG3_Pro209 mutants have a decreased protein solubility and this gives rise to large protein aggregates in the cytosol BAG3_Pro209 mutants accumulate at aggresomes Co-localization was assessed between BAG3-GFP and aggresome-markers in HEK293T cells stably expressing HSPB8-V5 and transiently transfected for 24 h with BAG3-GFP constructs As markers for aggresomes we used: (a) FLAG-HDAC6 (c) Live-cell time-lapse imaging of GFP-tagged BAG3_Pro209Leu in HEK293T cells BAG3_Pro209 mutants sequester other members of the CASA-complex in aggresomes HEK293T cells that stably overexpress HSPB8-V5 were transiently transfected with wild type or mutant BAG3-GFP constructs to assess the interaction between BAG3 and components of the CASA-complex (a) Co-immunoprecipitation of BAG3-GFP and the CASA-complex using the GFP-trap system The amount of interacting proteins was quantified and corrected for the amount of immunoprecipitated BAG3 as represented in the graph bar (means ± SD) The wild type (WT) or mutants were abbreviated as followed: Pro209Ser (PS) (b–d) Immunocytochemistry of BAG3-GFP constructs to assess colocalization with (b) endogenous Hsp70 (e,f) Live-cell time-lapse imaging of GFP-tagged BAG3_Pro209Leu and RFP-tagged SQSTM1/p62 or Hsp70 HeLa cells were transiently transfected with mutant BAG3-GFP constructs and (e) mCherry-tagged SQSTM1/p62 or (f) mScarlet-tagged Hsp70 Other members of the CASA-complex thus relocate to the aggresome in cells expressing BAG3_Pro209 mutants This supports the interpretation that Hsp70 and SQSTM1/p62 associate with BAG3 already in the early stages of the aggregation process as tagging the small protein with a fluorescent protein of the same size could potentially interfere with its functioning as a consequence of its increased aggregation propensity potentially decreasing their availability and compromising their functioning BAG3_Pro209 mutants are trapped in long-lasting aggresome structures due to reduced subunit exchange (a) Protein degradation rates were determined with a cycloheximide wash-out experiment HEK293T cells that stably overexpress HSPB8-V5 were transiently transfected with wild type or mutant BAG3-GFP constructs and subjected to cycloheximide treatment (50 µg/ml) for the indicated time Protein turnover of BAG3-GFP was determined by western blot after separation of the soluble from insoluble fraction (b–d) Fluorescence recovery after photobleaching (FRAP) analysis was performed on HeLa cells that were transiently transfected with BAG3-GFP and mScarlet-Hsp70 or SQSTM1/p62-mCherry constructs Bleaching was performed either on (b) BAG3-GFP Quantification of the fluorescence intensity over time was plotted for cells overexpressing WT and mutant BAG3 Graph bar shows the means (±SD) over time (n = 6) indicating that the presence of SQSTM/p62 is not influencing BAG3 mobility these data show that two distinct pools of mutant BAG3 exist: one pool of mutant BAG3 is trapped in aggresome-associated structures with drastically reduced subunit exchange compared to wild type BAG3 while a second pool of mutant BAG3_Pro209Leu is moving freely within the cytosol Due to a reduced exchange with the cytosolic (soluble) fraction initial engagement with pre-aggresome bodies commits mutant BAG3 towards the aggresome where it holds a residence time in the range of hours This process occurs independently of SQSTM1/p62 recruitment at the BAG3 pre-aggresome bodies (2019) showed that BAG3_Pro209 mutants fail to stimulate Hsp70-dependent client processing leading to the sequestration of ubiquitinylated Hsp70-bound clients into aggregates We verified whether the aggresomes formed by all BAG3_Pro209 mutants were enriched for ubiquitinylated proteins which would suggest a failure to degrade Hsp70-bound clients BAG3_Pro209 mutations cause a failure in chaperone-function of the CASA-complex Chaperone-activity was assessed in HEK293T cells stably expressing HSPB8-V5 and transiently transfected with wild type or mutant BAG3-GFP constructs (a) Aggregation of ubiquitinylated clients was verified by separation of the soluble and insoluble fraction Both fractions were analyzed by western blot with anti-ubiquitin as marker for the accumulation of ubiquitinylated-proteins (b) Immunocytochemistry of BAG3-GFP constructs to assess colocalization with ubiquitinylated proteins Scale bar = 10 µm (c) Protein aggregation assay by transient transfection of model client protein SOD1_G93A The same total protein lysates were analyzed by western blot and filter retardation assay (FRA) Relative optical densities are reported in the graph as means ± SD of normalized values (d) Autophagic activity was determined by western blot before and after starvation by serum depletion plus 10 nM bafilomycin A1 for 2 hours Protein lysates were analyzed by SDS-PAGE with LC3B-II as a marker for autophagosomes Following abbreviations were used: non-transfected (NT) the autophagic pathway is not impaired by BAG3_Pro209 mutants suggesting that the accumulation of ubiquitinylated proteins cannot be explained by impairment of autophagy and supporting the idea that the CASA-complexes composed of BAG3_Pro209 mutants fail to release the bound client from Hsp70 for degradation by autophagosomes This interpretation is in line with Meister-Broekema et al who showed that BAG3_Pro209Leu fails to stimulate Hsp70-dependent client processing HDAC6-inhibition with tubastatin A or HDAC6-depletion with shRNA does not rescue BAG3_Pro209-associated phenotypes The protein aggregation and aggresome formation of BAG3_Pro209 mutants was assessed in HEK293T cells stably expressing HSPB8-V5 and transiently transfected with wild type or mutant BAG3-GFP constructs before and after HDAC6 inhibition (a,b) Following abbreviations were used: wild type (WT) neither pharmacological inhibition nor genetic depletion of HDAC6 prevented aggresome formation in BAG3_Pro209 mutant cells Inhibition of HDAC6 may therefore not offer the desired therapeutic potential to rescue the compromised chaperone-function in cells expressing BAG3_Pro209 mutants these data suggest that BAG3_Pro209 mutants induce aggresome formation downstream of HDAC6 or from an independent pathway This effort to group misfolded proteins at one well-determined spot ensures that potentially toxic proteins are removed from the remaining cytosol and protects the cell from adverse effects The aggresome is therefore rich in ubiquitinylated proteins and requires chaperones and autophagosomes to remove and degrade these components in a controlled manner Misfolded proteins are captured by the CASA-complex and transported to the MTOC where autophagosomes are concentrated and efficiently degrade the misfolded cargo BAG3_Pro209 mutations destabilize the protein’s intrinsic stability and lead to BAG3 aggregation Pro209Leu BAG3 impairs the functional chaperone-cycle of Hsp70 (Meister-Broekema et al. the CASA-complexes that contain mutant BAG3 accumulate at the aggresome with their bound clients and co-factors preventing on the one hand the degradation of the Hsp70-bound misfolded cargo and sequestering important proteostasis factors such as HSPB8 This may then provide new insights in the diverse compositions and functions of the CASA-complex and help in understanding why IPV-mutations give rise to such diverse clinical phenotypes A limitation in studying the CASA-complex is that the substrate repertoire has not yet been fully elucidated Assessing the activity of the CASA-complex is therefore limited to model substrates which are often mutant proteins that misfold and aggregate A concern to such approaches is that the overexpression of mutant BAG3 and mutant model substrates may by themselves overwhelm the degradation systems while the PQC systems in patients with BAG3 mutations are typically not challenged by an additional mutant protein (such as SOD1_G93A or poly-GA) It will therefore be an important step in the future to assess whether the decrease in the activity of the CASA-complex can be translated to the affected tissues in vivo the possibility that other modifying or (epi-) genetic factors contribute to clinical differences in both BAG3 and SQSTM1/p62 linked diseases cannot be excluded despite the distinct phenotypes associated with Pro209 mutations in BAG3 they all seem to induce aggresome formation causing the sequestration of PQC factors if a therapy for one of the Pro209-associated diseases can be identified it may also be beneficial to other Pro209-associated phenotypes Mutations were introduced through site-directed mutagenesis using the wild type BAG3-GSGS-GFP construct in the pEGFP-N1 vector (a kind gift of Josée N Point mutations were introduced with following primers: Fw: CGCGGGGGTACATCTCCATTTCGGTGATACACGAGCAGAA Rv: TTCTGCTCGTGTATCACCGAAATGGAGATGTACCCCCGCG Fw: CGCGGGGGTACATCTCCATTCTGGTGATACACGAGCAGAA Rv: TTCTGCTCGTGTATCACCAGAATGGAGATGTACCCCCGCG Fw: CGCGGGGGTACATCTCCATTCAGGTGATACACGAGCAGAA Rv: TTCTGCTCGTGTATCACCTGAATGGAGATGTACCCCCGCG Fw: AAAAAGTACCTGATGATCAAAGAGTATTTGACCAAAGAGC Rv: GCTCTTTGGTCAAATACTCTTTGATCATCAGGTACTTTTT Incorporation of the respective mutations was verified by Sanger sequencing HEK293T cells were transduced with lentivirus containing the HSPB8 ORF (NM_014365) in pLENTI6/V5 (Life Technologies UK) were transiently transfected with packaging (pCMV dR8.91) envelope (pMD2-VSV) and pLenti6/V5 plasmids using linear polyethylenimine (PEI) (23966-1 the virus containing supernatant was collected filtered and transferred to fresh HEK293T cells for infection Positive cells were selected by blasticidine selection Cells were cultured at 37 °C and 5% CO2 in DMEM (Life Technologies USA) supplemented with 10% Fetal Bovine Serum 1% Glutamine and 1% Penicillin-Streptomycin (Life Technologies HEK293T cells that were lentiviral transduced with HSPB8-V5 and were plated in 24-well plates at 75,000 cells/well cells were transiently transfected using Lipofectamine3000/P3000 reagent medium was removed and cells were harvested in PBS with 10% FBS (Gibco USA) and centrifuged for 5 min at 100 g at 4 °C Cells were resuspended in PBS with 10% FBS (Gibco USA) and an aliquot was analyzed by flow cytometry to determine the transfection efficiency in respect to untransfected control cells Flow cytometry was performed using NovoCyte Flow Cytometer 3000 (ACEA Biosciences Inc. USA) and results were analyzed by NovoExpress software 1.2.5 (ACEA Biosciences Inc. excitation wavelengths and emission collection windows were FITC (488 nm a solution of PBS containing 1% (v/v) Triton X-100 a cocktail of Protease inhibitors (Sigma-Aldrich USA) and DAPI (0.02 µg/µl) was added to a final concentration of 0.5% (v/v) Triton X-100 and DAPI 0.01 µg/µl After two minutes incubation at room temperature the cell lysates were analyzed by flow cytometry Three untransfected control samples without DAPI were analyzed to set gates on nuclei population 373/482 (FITC for cell transfection or inclusion analysis respectively) Nuclei were counted based on the Pacific Blue positive population Inclusions were identified for fluorescence and FSC compared to cells transfected with eGFPN1 vector as control Following the equation set by Whiten et al (2016) the number of inclusions was normalized to the number of counted nuclei and reported as inclusions/100 transfected cells Nuclei population was analyzed based on FITC fluorescence and a percentage of nuclei enriched with GFP-positive particles was determined For Tango, we inserted a protein sequence of 70 amino acids spanning the second IPV-motif (SQSPAASDCSSSSSSASLPSSGRSSLGSHQLPRGYISIPVIHEQNVTRPAAQPSFHQAQKTHYPAQQGEY)(Fig. 1e) The parameters were as following: no protection at the N-terminus or C-terminus of the peptide sequence We selected and plotted Beta-aggregation for both the wild type sequence as the three IPV-mutants (Ser/Leu/Gln) HEK293T stable cell lines for HSPB8-V5 were transiently transfected with different wild type or mutant BAG3-GFP constructs using PEI (23966-1 the reverse experiment was performed by transiently transfecting HeLa cells cells were lysed with lysis buffer [20 mM Tris-HCl pH 7.4 Complete Protease inhibitor (Roche Applied Science Samples were centrifuged for 10 min at 20,000 g and equal amounts of supernatant (NP40-soluble fraction only) was loaded on GFP-Trap beads (gta-20 Beads were incubated with the protein lysate for 1 h at 4 °C and washed three times with wash buffer [20 mM Tris-HCl pH 7.4 Proteins were eluted from the beads with Sarkosyl elution buffer (140 mM NaCl 10% glycerol) before being supplemented with NuPAGE LDS sample buffer (Life Technologies USA) and loaded on 4-12% NuPAGE gels (Life Technologies Proteins were transferred to nitrocellulose membranes (Hybond-P; GE Healthcare USA) and decorated with antibodies against GFP (ab290 Samples were detected using enhanced chemiluminescent ECL Plus (Pierce HeLa cells were transfected using Lipofectamine 2000 reagent (Invitrogen USA) with empty vector or BAG3-GFP constructs (wild type or mutants) 24 h post-transfection cells were lysed in lysis buffer (150 mM NaCl The cell lysates were centrifuged and cleared with A/G beads (Santa Cruz Biotechnology Rabbit TrueBlot beads (Tebu-bio) were incubated at 4 °C for 1 h with home-made rabbit HSPB8 antibody (Carra et al Rabbit TrueBlot beads complexed with the specific antibodies were added to the precleared lysates Beads were washed four times with the lysis buffer; both co-immunoprecipitated proteins and input fractions were resolved on SDS-PAGE followed by western blot NSC-34 or C2C12 cells were plated in 24-well plates containing poly-D lysine (P-7280 USA) coated coverslips and then transfected with wild type or mutant BAG3-GFP constructs as described above for WB and FRA experiments For protein aggregation-prone behaviour evaluation Cells were then fixed using a 1:1 solution of 4% paraformaldehyde (PFA) and 4% sucrose in 0.2 N PB (0.06 M KH2PO4 0.31 M Na2HPO4; pH 7.4) for 25 min at 37 °C Nuclei were stained with Hoechst (1:2000 in PBS; 33342 Images were captured by Axiovert 200 microscope (Zeiss Germany) with a photometric CoolSnap CCD camera (Ropper Scientific Images were processed using Metamorph software (Universal Imaging Six different fields were captured for each sample of which each field of view contained an average of 95 cells This summed up to a total of WT = 687 cells HeLa cells were transfected with BAG3-GFP wild type or mutant constructs and with either P62-mCherry and mScarlet-HSP70 constructs and imaged 48 hours after transfection in a μ-slide 8-well (80826 Germany) in FluoroBrite DMEM medium (Life Technologies USA) supplemented with 10% fetal bovine serum and 4mM L-glutamine at 37 °C and 5% CO2 FRAP measurements were performed on a Zeiss LSM700 laser scanning confocal microscope using a PlanApochromat 63×/1.4 NA objective Imaging and photobleaching settings were kept identical for all wild type and mutant BAG3 cells within the three different FRAP experiments For western blot analysis of autophagic flux HEK293T cells stably transduced with HSPB8-V5 were transiently transfected using PEI MAX (24765-1 cells were cultured in serum-deprived medium with bafilomycin A1 (10 nM) for two hours Proteins were extracted with RIPA buffer [1% Nonidet P-40 cOmplete Protease Inhibitor Cocktail (Roche Applied Science Phospho-STOP inhibitor mix (05 892 970 001 Equal amounts of protein were then loaded on 12% NuPAGE gels (Life Technologies Statistical analyses have been performed using the statistical tests as stated in each figure legend This comprised Student T tests or One-Way ANOVA with Bonferroni’s multiple comparisons tests The statistical analysis was performed using PRISM software (GraphPad Software Balchin, D., Hayer-Hartl, M. & Hartl, F. U. In vivo aspects of protein folding and quality control. Science 353, aac4354, https://doi.org/10.1126/science.aac4354 (2014) Gloge, F., Becker, A. H., Kramer, G. & Bukau, B. Co-translational mechanisms of protein maturation. Curr Opin Struct Biol 24, 24–33, https://doi.org/10.1016/j.sbi.2013.11.004 (2014) Takayama, S. & Reed, J. C. Molecular chaperone targeting and regulation by BAG family proteins. Nat Cell Biol 3, E237–41, https://doi.org/10.1038/ncb1001-e237 (2001) Behl, C. Breaking BAG: the co-chaperone BAG3 in health and disease. Trends Pharmacol Sci 37, 672–88, https://doi.org/10.1016/j.tips.2016.04.007 (2016) Takayama, S. et al. Cloning and functional analysis of BAG-1: a novel Bcl-2 binding protein with anti-cell death activity. Cell 80, 279–84, https://doi.org/10.1016/0092-8674(95)90410-7 (1995) Takayama, S. et al. BAG-1 modulates the chaperone activity of Hsp70/Hsc70. EMBO J 16, 4887–96, https://doi.org/10.1093/emboj/16.16.4887 (1997) Takayama, S., Xie, Z. & Reed, J. C. An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators. J Biol Chem 274, 781–6, https://doi.org/10.1074/jbc.274.2.781 (1999) Fuchs, M. et al. Identification of the key structural motifs involved in HspB8/HspB6–Bag3 interaction. Biochem J 425, 245–57, https://doi.org/10.1042/BJ20090907 (2010) Meriin, A. B. et al. Hsp70-Bag3 complex is a hub for proteotoxicity-induced signaling that controls protein aggregation. Proc. Natl. Acad. Sci 115, E7043–E7052, https://doi.org/10.1073/pnas.1803130115 (2018) Doong, H. et al. CAIR-1/BAG-3 forms an EGF-regulated ternary complex with phospholipase C-γ and Hsp70/Hsc70. Oncogene 19, 4385–95, https://doi.org/10.1038/sj.onc.1203797 (2000) Gamerdinger, M., Kaya, A. M., Wolfrum, U., Clement, A. M. & Behl, C. BAG3 mediates chaperone-based aggresome-targeting and selective autopaghy of misfolded proteins. EMBO Rep 12, 149–56, https://doi.org/10.1038/embor.2010.203 (2011) Morelli, F. F. et al. An interaction study in mammalian cells demonstrates weak binding of HSPB2 to BAG3, which is regulated by HSPB3 and abrogated by HSPB8. Cell Stress Chaperones 22, 531–40, https://doi.org/10.1007/s12192-017-0769-x (2017) Rauch, J. N. et al. BAG3 is a modular, scaffolding protein that physically links heat shock protein 70 (Hsp70) to the small heat shock proteins. J Mol Biol 429, 128–41, https://doi.org/10.1016/j.jmb.2016.11.013 (2017) Carra, S., Seguin, S. J., Lambert, H. & Landry, J. HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy. J Biol Chem 283, 1437–44, https://doi.org/10.1074/jbc.M706304200 (2008) Arndt, V. et al. Chaperone-Assisted Selective Autophagy is essential for muscle maintenance. Curr Biol 20, 143–148, https://doi.org/10.1016/j.cub.2009.11.022 (2010) Carra, S. et al. Small heat shock proteins: multifaceted proteins with important implications for life. Cell Stress Chaperones 24, 295–308, https://doi.org/10.1007/s12192-019-00979-z (2019) Jiang, J. et al. CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation. J Biol Chem 276, 42938–44, https://doi.org/10.1074/jbc.M101968200 (2001) Murata, S., Minami, Y., Minami, M., Chiba, T. & Tanaka, K. CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein. EMBO Rep 2, 1133–8, https://doi.org/10.1093/embo-reports/kve246 (2001) CHIP: a link between the chaperone and proteasome systems 10.1379/1466-1268(2003)008<0303:calbtc>2.0co;2 (2003) Lamark, T. et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 171, 603–14, https://doi.org/10.1083/jcb.200507002 (2005) Ciuffa, R. et al. The selective autophagy receptor p62 forms a flexible filamentous helical scaffold. Cell Rep 11, 748–58, https://doi.org/10.1016/j.celrep.2015.03.062 (2015) Cha-Molstad, H. et al. p62/SQSTM1/Sequestosome-1 is an N-recognin of the N-end rule pathway which modulates autophagosome biogenesis. Nat Commun 8, 102, https://doi.org/10.1038/s41467-017-00085-7 (2017) Rusmini, P. et al. The role of the heat shock protein B8 (HSPB8) in motoneuron diseases. Front Mol Neurosci 10, 1–9, https://doi.org/10.3389/fnmol.2017.00176 (2017) Selcen, D. et al. Mutation in BAG3 causes severe dominant childhood muscular dystrophy. Ann Neurol 65, 83–9, https://doi.org/10.1002/ana/21553 (2009) Semmler, A. L. et al. Unusual multisystemic involvement and a novel BAG3 mutation revealed by NGS screening in a large cohort of myofibrillar myopathies. Orphanet J Rare Dis 1, 121, https://doi.org/10.1186/s13023-014-0121-9 (2014) Shy, M. et al. Mutations in BAG3 cause adult-onset Charcot-Marie-Tooth disease. J Neurol Neurosurg Psychiatry 3, 313–5, https://doi.org/10.1136/jnnp-2017-315929 (2018) Meister-Boekema, M. et al. Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks. Nat Commun 9, 5342, https://doi.org/10.1038/s41467-018-07718-5 (2018) Weihl, C. C., Udd, B. & Hanna, M. ENMC workshop study group 234th ENMC international workshop: chaperone dysfunction in muscle disease December 8-10th 2017, Naarden Netherlands. Neuromuscul Disord 28, 1022–1030, https://doi.org/10.1016/j.nmd.2018.09.004 (2018) Villard, E. et al. A genome-wide association study identifies two loci associated with heart failure due to dilated cardiomyopathy. Eur Heart J 32, 1065–76, https://doi.org/10.1093/eurheartj/ehr105 (2011) Fang, X. et al. Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy. J Clin Invest 127, 3189–200, https://doi.org/10.1172/JCI94310 (2017) Whiten, D. et al. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking. Sci Rep 6, 1–9, https://doi.org/10.1038/srep31138 (2016) The CamSol method of rational design of protein mutants with enhanced solubility Fernandez-Escamilla, A. M., Rousseau, F., Schymkowitz, J. & Serrano, L. Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nat Biotechnol 22, 1302–1306, https://doi.org/10.1038/nbt1012 (2004) Johnston, J. A., Ward, C. L. & Kopito, R. R. Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143, 1883–1898, https://doi.org/10.1083/jcb.143/7/1883 (1998) Kawaguchi, Y. et al. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115, 727–738, https://doi.org/10.1016/s0092-8674(03)00939-5 (2003) Rauch, J. N., Zuiderweg, E. R. P. & Gestwicki, J. E. Non-canonical Interactions between heat shock cognate protein 70 (Hsc70) and Bcl2-associated anthanogene (BAG) co-chaperones are important for client release. J Biol Chem 291, 19848–57, https://doi.org/10.1074/jbc.M116.742502 (2016) Guilbert, S. M. et al. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency. FASEB J 32, 3518–3535, https://doi.org/10.1096/fj.201700558RR (2018) Fujita, K., Maeda, D., Xiao, Q. & Srinivasula, S. M. Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degradation. Proc Natl Acad Sci 108, 1427–1432, https://doi.org/10.1073/pnas.1014156108 (2011) Crippa, V. et al. The small heat shock protein B8 (HspB8) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS). Hum Mol Genet 19, 3440–3456, https://doi.org/10.1093/hmg/ddq257 (2010) Cristofani, R. et al. The small heat shock protein B8 (HSPB8) efficiently removes aggregating species of dipeptides produced in C9ORF72-related neurodegenerative diseases. Cell Stress Chaperones 23, 1–12, https://doi.org/10.1007/s12192-017-0806-9 (2018) d’Ydewalle, C. et al. HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-induced Charcot-Marie-Tooth disease. Nat Med 17, 968–974, https://doi.org/10.1038/nm.2396 (2011) Guo, W. et al. HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients. Nat Commun 8, 861, https://doi.org/10.1038/s41467-017-00911-y (2017) Benoy, V. et al. HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth Disease. Brain 141, 673–687, https://doi.org/10.1093/brain/awx375 (2018) Inhibition of histone deacetylase 6 (HDAC6) protects against vincristine-induced peripheral neuropathies and inhibits tumor growth Prior, R., Helleputte, L., Van Kling, Y. E. & Van den Bosch, L. HDAC6 as a potential therapeutic target for peripheral nerve disorders. Expert Opin Ther Targets 22, 993–1007, https://doi.org/10.1080/14728222.2018.1541235 (2018) Hubbert, C. et al. HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458, https://doi.org/10.1038/417455a (2002) Matsuyama, A. et al. In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J 21, 6820–6831, https://doi.org/10.1093/emboj/cdf682 (2002) Adriaenssens, E., Geuens, T., Baets, J., Echaniz-Laguna, A. & Timmerman, V. Novel insights in the disease biology of mutant small heat shock proteins in neuromuscular diseases. Brain 140, 2541–2549, https://doi.org/10.1093/brain/awx187 (2017) Andersen, A. G. et al. BAG3 myopathy is not always associated with cardiomyopathy. Neuromuscul Disord 28, 798–801, https://doi.org/10.1016/n.nmd.2018.06.019 (2018) P209L mutation in BAG3 does not cause cardiomyopathy in mice Long, M. et al. Multifunctional p62 Effects Underlie Diverse Metabolic Diseases. Trends Endocrinol Metab 28, 818–30, https://doi.org/10.1016/j.tem.2017.09.001 (2017) Carra, S., Sivilotti, M., Chavez Zobel, A. T., Lambert, H. & Landry, J. HSPB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells. Hum Mol Genet 14, 1659–1669, https://doi.org/10.1039/hmg/ddi174 (2005) Minoia, M. et al. BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta. Autophagy 10, 1603–1621, https://doi.org/10.4161/auto.29409 (2014) Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–82, https://doi.org/10.1038/nmeth.2019 (2012) Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH image to ImageJ: 25 years of image analysis. Nat Methods 9, 671–5, https://doi.org/10.1038/nmeth.2089 (2012) Fischle, W. et al. A new family of human histone deacetylases related to Saccharomyces cerevisiae HDA1p. J Biol Chem 274, 11713–11720, https://doi.org/10.1074/jbc.274.17.11713 (1999) Download references These authors contributed equally: Elias Adriaenssens Elias Adriaenssens & Vincent Timmerman Dipartimento di Scienze Farmacologiche e Biomolecolari Centro di Eccellenza sulle Malattie Neurodegenerative and Center for Neuroscience and Neurotechnology VIB-UAntwerp Center for Molecular Neurology wrote the paper with assistance from all authors The authors declare no competing interests Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-020-65664-z Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science