1 Nature Genetics 2012 Vol: 44(4):450-455. DOI: 10.1038/ng.1103

Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy

Bjarne Udd and colleagues show that mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 result in limb-girdle muscular dystrophy. Their studies suggest that the mutations reduce the protective anti-aggregation effects of DNAJB6, leading to protein accumulation and autophagic pathology.

Mentions
Figures
Figure 1: Microscopy of muscle biopsies from Finnish individuals with LGMD1D. (a) Confocal microscopy of LGMD1D muscle showed that the predominant localization of DNAJB6 in the Z-disc was preserved in the regions with unaltered sarcomeric structure. Scale bar, 10 μm. (b) Transmission electron microscopy showed early disruption of Z-disks (arrows; left) and autophagic pathology (right) in LGMD1D. For comparison, see region with normal ultrastructure (boxed at left). Scale bars, ~1 μm. (c) The autophagic pathology was highlighted by areas of LC3 accumulation within myofibers, revealed by brown precipitate in LC3 immunohistochemistry (IHC; left) and by the presence of rimmed vacuoles in the Herovici staining (arrows; right). Scale bars, ~50 μm. (d) DNAJB6, HSPA8, MLF1, desmin, myotilin, filamin and KRT18 were found in protein accumulations. For individual channels, see Supplementary  Confocal images show single optical sections. Scale bars, 10 μm. Figure 2: Muscle disintegration in DNAJB6b mutant and morphant zebrafish. (a–i) Lateral views of fish embryos 2 d.p.f. subjected to whole-mount immunofluorescence staining of slow myosin heavy chain. Injected embryos expressing wild-type (wt) DNAJB6a or DNAJB6b showed slow myofibers spanning the somite normally between adjacent myosepta (c,f) and were indistinguishable from control embryos. Injection of DNAJB6b p.Phe93Leu and p.Phe89Ile mutant mRNAs resulted in detachment of fibers from the vertical myoseptum (a,b), whereas injection of DNAJB6a mutants resulted in a normal appearance (d,e). Similar muscular disintegration was observed in dnajb6b morphant embryos injected with sb-MO against the zebrafish ortholog of DNAJB6 (g) and in embryos expressing DNAJB6b p.Phe93Ala or p.Phe93Gly (h,i). The detachment of myofibers from the myoseptum can be partial (a,b,g,h; white arrowheads) or complete (i, white asterisk). Scale bar, ~50 μm. (j) Embryos injected with the indicated constructs were categorized phenotypically based on the presence of muscle fiber detachment affecting 1–2 somites (class I, mild) or multiple somites (class II, severe; see Supplementary for an example). The phenotype in dnajb6b MO-injected embryos was rescued efficiently by wt human DNAJB6b mRNA (χ2 test). Figure 3: Dominant effect of mutant DNAJB6 proteins. (a) Co-injection of wt DNAJB6b mRNA with p.Phe93Leu or p.Phe89Ile mutant mRNA in zebrafish embryos led to a more severe muscle phenotype, with a statistically significant increase in the number of class II embryos (χ2 test). (b) DNAJB6b constructs were expressed in 293FT cells, and protein synthesis was blocked by cycloheximide (CHX). Whole-cell extracts were obtained at the indicated time points and analyzed by protein blotting. DNAJB6 band intensities were quantified and normalized to α-tubulin (single transfections) or HSP90 (co-transfections) intensity to obtain the relative DNAJB6 levels. In single transfections (solid lines), CHX treatment rapidly decreased wt DNAJB6b protein levels, whereas p.Phe89Ile and p.Phe93Leu mutant proteins showed reduced turnover. In co-transfections of HA- and GFP-tagged wt and p.Phe93Leu mutant constructs (dashed lines), the wt DNAJB6b level remained stable after 4 h of CHX treatment. Data from three independent experiments are presented as mean ± s.d. Relative DNAJB6 level at t = 0 h was set to 100 for each construct. Representative protein blots are shown in Supplementary Figure 4: Impaired anti-aggregation activity of mutant DNAJB6b. (a,b) GFP-tagged 120Q-huntingtin was expressed in T-REx 293 cells, with co-expression of V5-tagged wt or mutant (p.Phe89Ile, p.Phe93Leu) DNAJB6b or wt DNAJB6a, induced (+) or uninduced (−). In a, aggregated (aggr.) huntingtin was detected in a filter-trap assay, and SDS-soluble (sol.) huntingtin, DNAJB6 (V5) and α-tubulin (α-tub.) were analyzed by protein blotting. Filter trap and protein blot show representative samples. In b, aggregated and soluble huntingtin were quantified, and anti-aggregation activity of DNAJB6 constructs was determined as the ratio of aggr./sol. in induced to that in uninduced cells (aggregation score). Both mutant DNAJB6b constructs showed significantly impaired anti-aggregation activity (Mann-Whitney U-test; P values adjusted for multiple testing with the Bonferroni correction). The nuclear DNAJB6a construct, used as a negative control, showed only weak activity. For each construct, n = 12 (three experiments performed in quadruplicate). For supporting information, see Supplementary Figure 5: Association of DNAJB6 with the CASA complex. (a) Co-immunoprecipitation. Tested constructs were expressed in COS-1 cells, cross-linked and immunoprecipitated (IP) with anti-V5 beads. V5-tagged wt and p.Phe93Leu mutant DNAJB6b pulled down endogenous BAG3 and HSPB8. No co-immunoprecipitation was observed with an untagged DNAJB6b construct or an unrelated bait (V5-tagged titin is6–M9). (b) Proximity ligation assay (PLA) on rat muscle sections. PLA signals (red dots) indicated spatial proximity of DNAJB6 with its known interaction partner HSPA8, and with the CASA complex proteins BAG3, HSPB8 and STUB1. Negative control experiments were performed with DNAJB6 or BAG3 antibodies alone. Each image shows a representative maximum-intensity projection through a z stack of 6.4 μm, with the PLA signal (red) superimposed on the phalloidin counterstain (gray). Scale bar, 5 μm. (c) Co-injection of DNAJB6 and BAG3 in zebrafish. Zebrafish embryos were injected with DNAJB6b p.Phe93Leu mRNA and wild-type (wt) or p.Pro209Leu mutant BAG3 mRNA in indicated combinations. Co-injection of wild-type BAG3 with mutant DNAJB6 resulted in a more severe phenotype than mutant DNAJB6 alone, reflected by a significant increase in class II embryos. In contrast, co-injection of p.Pro209Leu BAG3 with mutant DNAJB6b did not alter the phenotype distribution. Statistical significance (χ2 test) is denoted by P value or n.s. (not significant). Scale bar, ~50 μm.
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References
  1. Speer, M.C. Identification of a new autosomal dominant limb-girdle muscular dystrophy locus on chromosome 7 Am. J. Hum. Genet. 64, 556-562 (1999) .
    • . . . Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to chromosome 7q36 over a decade ago1, but its genetic cause has remained elusive . . .
    • . . . Here we studied the molecular cause of a dominant LGMD in five previously reported Finnish (FF1–5)3, 4 and two US families (DUK1047 and DUK1701)1, 5, as well as two previously unreported Italian families (IT1 and IT2) identified on the basis of clinical phenotypes and of the pattern of muscle involvement by magnetic resonance imaging . . .
    • . . . The disorder in the two US families was originally linked to 7q36 and classified as LGMD1D1 . . .
    • . . . The Finnish (FF1–5) and US families (DUK1047 and DUK1701) have been described1, 3, 4, 5 . . .
  2. Gordon, E.; Pegoraro, E.; Hoffman, E.P. Limb-girdle muscular dystrophy overview Link GeneReviews , (2000) .
    • . . . Limb-girdle muscular dystrophies (LGMDs) are a genetically and mechanistically heterogeneous group of disorders caused by dominant or recessive mutations in a number of sarcolemmal, sarcomeric, cytoplasmic and nuclear proteins2 . . .
  3. Sandell, S. The enigma of 7q36 linked autosomal dominant limb girdle muscular dystrophy J. Neurol. Neurosurg. Psychiatry 81, 834-839 (2010) .
    • . . . Here we studied the molecular cause of a dominant LGMD in five previously reported Finnish (FF1–5)3, 4 and two US families (DUK1047 and DUK1701)1, 5, as well as two previously unreported Italian families (IT1 and IT2) identified on the basis of clinical phenotypes and of the pattern of muscle involvement by magnetic resonance imaging . . .
    • . . . Clinical and genetic characterization of the Finnish families established linkage to the same locus and refined LGMD1D to a 3.4-Mb region containing 12 genes3, 4 . . .
    • . . . Notably, the DNAJB6 mutations we identified cause a tissue-specific disease (muscular dystrophy); despite high expression of DNAJB6 in brain, there are no indications of neurological involvement3, 4, 5. . . .
    • . . . The Finnish (FF1–5) and US families (DUK1047 and DUK1701) have been described1, 3, 4, 5 . . .
  4. Hackman, P. Four new Finnish families with LGMD1D; refinement of the clinical phenotype and the linked 7q36 locus Neuromuscul. Disord. 21, 338-344 (2011) .
    • . . . Here we studied the molecular cause of a dominant LGMD in five previously reported Finnish (FF1–5)3, 4 and two US families (DUK1047 and DUK1701)1, 5, as well as two previously unreported Italian families (IT1 and IT2) identified on the basis of clinical phenotypes and of the pattern of muscle involvement by magnetic resonance imaging . . .
    • . . . Clinical and genetic characterization of the Finnish families established linkage to the same locus and refined LGMD1D to a 3.4-Mb region containing 12 genes3, 4 . . .
    • . . . Notably, the DNAJB6 mutations we identified cause a tissue-specific disease (muscular dystrophy); despite high expression of DNAJB6 in brain, there are no indications of neurological involvement3, 4, 5. . . .
    • . . . The Finnish (FF1–5) and US families (DUK1047 and DUK1701) have been described1, 3, 4, 5 . . .
  5. Speer, M.C. Evidence for locus heterogeneity in autosomal dominant limb-girdle muscular dystrophy Am. J. Hum. Genet. 57, 1371-1376 (1995) .
    • . . . Here we studied the molecular cause of a dominant LGMD in five previously reported Finnish (FF1–5)3, 4 and two US families (DUK1047 and DUK1701)1, 5, as well as two previously unreported Italian families (IT1 and IT2) identified on the basis of clinical phenotypes and of the pattern of muscle involvement by magnetic resonance imaging . . .
    • . . . Notably, the DNAJB6 mutations we identified cause a tissue-specific disease (muscular dystrophy); despite high expression of DNAJB6 in brain, there are no indications of neurological involvement3, 4, 5. . . .
    • . . . The Finnish (FF1–5) and US families (DUK1047 and DUK1701) have been described1, 3, 4, 5 . . .
  6. Kampinga, H.H.; Craig, E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity Nat. Rev. Mol. Cell Biol. 11, 579-592 (2010) .
    • . . . These co-chaperones interact with chaperones of the HSPA (Hsp70) family, increasing and modulating their activity6 . . .
    • . . . Some J proteins, including DNAJB6, also have HSPA-independent functions6, 7. . . .
  7. Hageman, J. A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation Mol. Cell 37, 355-369 (2010) .
    • . . . Some J proteins, including DNAJB6, also have HSPA-independent functions6, 7. . . .
    • . . . DNAJB6 is composed of a conserved N-terminal J domain, a G/F domain rich in glycine and phenylalanine residues and a C-terminal domain containing a serine-rich (“SSF-SST”) region7, 8 (Supplementary Fig. 2a) . . .
    • . . . The SSF-SST region has been shown to be important for interactions with the transcription factor NFATc3 (ref. 10) and histone deacetylases (HDACs)7 and for DNAJB6 oligomerization7 . . .
    • . . . DNAJB6 has been reported to suppress aggregation and toxicity of aggregation-prone proteins such as polyglutamine-containing huntingtin and α-synuclein7, 8, 17 and to participate in autophagic and proteasomal turnover of proteins and organelles18, 19, as well as in regulation of gene expression10 and cell cycle20 . . .
    • . . . On the basis of evidence from the closely related DNAJB8 protein, DNAJB6 has been suggested to form oligomeric complexes7 . . .
    • . . . DNAJB6 has been shown to suppress aggregation of various proteins in cell culture models7, 8, 28 . . .
    • . . . The nuclear DNAJB6a isoform, known to be inefficient in inhibiting cytoplasmic huntingtin aggregation7, served as negative control . . .
    • . . . The density gradient centrifugation protocol was adapted from Hageman et al.7 . . .
    • . . . The gradients were fractionated and proteins precipitated with TCA essentially as described7. . . .
    • . . . Each transfection was done in duplicate, and after 7 h, DNAJB6 expression was induced in one of the duplicates with 1 μg/ml tetracycline. 48 h after transfection, FTA was performed according to a protocol adapted from Hageman et al.7 . . .
  8. Chuang, J.Z. Characterization of a brain-enriched chaperone, MRJ, that inhibits Huntingtin aggregation and toxicity independently J. Biol. Chem. 277, 19831-19838 (2002) .
    • . . . DNAJB6 is composed of a conserved N-terminal J domain, a G/F domain rich in glycine and phenylalanine residues and a C-terminal domain containing a serine-rich (“SSF-SST”) region7, 8 (Supplementary Fig. 2a) . . .
    • . . . DNAJB6 has been reported to suppress aggregation and toxicity of aggregation-prone proteins such as polyglutamine-containing huntingtin and α-synuclein7, 8, 17 and to participate in autophagic and proteasomal turnover of proteins and organelles18, 19, as well as in regulation of gene expression10 and cell cycle20 . . .
    • . . . DNAJB6 is expressed in all tissues22, with the highest expression in brain8, but its expression and localization in skeletal muscle have not been characterized previously . . .
    • . . . DNAJB6 has been shown to suppress aggregation of various proteins in cell culture models7, 8, 28 . . .
    • . . . First, DNAJB6 is a known co-chaperone of HSPA8 (ref. 8) . . .
    • . . . This protein has been implicated in a variety of degenerative disorders such as Parkinson's17 and Huntington's8 diseases, as well as cancer16 . . .
  9. Izawa, I. Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein J. Biol. Chem. 275, 34521-34527 (2000) .
    • . . . The J domain interacts with the constitutively expressed chaperone HSPA8 (also known as Hsc70 or Hsp73)9 . . .
    • . . . Immunofluorescence microscopy of patient muscle showed DNAJB6 in protein accumulations together with its known ligands MLF1 (ref. 21) and HSPA8 (ref. 9) (Fig. 1d and Supplementary Fig. 4) . . .
  10. Dai, Y.S.; Xu, J.; Molkentin, J.D. The DnaJ-related factor Mrj interacts with nuclear factor of activated T cells c3 and mediates transcriptional repression through class II histone deacetylase recruitment Mol. Cell. Biol. 25, 9936-9948 (2005) .
    • . . . The SSF-SST region has been shown to be important for interactions with the transcription factor NFATc3 (ref. 10) and histone deacetylases (HDACs)7 and for DNAJB6 oligomerization7 . . .
    • . . . DNAJB6 has been reported to suppress aggregation and toxicity of aggregation-prone proteins such as polyglutamine-containing huntingtin and α-synuclein7, 8, 17 and to participate in autophagic and proteasomal turnover of proteins and organelles18, 19, as well as in regulation of gene expression10 and cell cycle20 . . .
  11. Perales-Calvo, J.; Muga, A.; Moro, F. Role of DnaJ G/F-rich domain in conformational recognition and binding of protein substrates J. Biol. Chem. 285, 34231-34239 (2010) .
    • . . . The G/F domain has been suggested to participate in recognition of partially unfolded client proteins in bacterial DnaJ11, 12, 13 and yeast Sis1 (ref. 14) . . .
  12. Cajo, G.C. The role of the DIF motif of the DnaJ (Hsp40) co-chaperone in the regulation of the DnaK (Hsp70) chaperone cycle J. Biol. Chem. 281, 12436-12444 (2006) .
    • . . . The G/F domain has been suggested to participate in recognition of partially unfolded client proteins in bacterial DnaJ11, 12, 13 and yeast Sis1 (ref. 14) . . .
  13. Wall, D.; Zylicz, M.; Georgopoulos, C. The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone J. Biol. Chem. 270, 2139-2144 (1995) .
    • . . . The G/F domain has been suggested to participate in recognition of partially unfolded client proteins in bacterial DnaJ11, 12, 13 and yeast Sis1 (ref. 14) . . .
  14. Yan, W.; Craig, E.A. The glycine-phenylalanine-rich region determines the specificity of the yeast Hsp40 Sis1 Mol. Cell. Biol. 19, 7751-7758 (1999) .
    • . . . The G/F domain has been suggested to participate in recognition of partially unfolded client proteins in bacterial DnaJ11, 12, 13 and yeast Sis1 (ref. 14) . . .
  15. Hanai, R.; Mashima, K. Characterization of two isoforms of a human DnaJ homologue, HSJ2 Mol. Biol. Rep. 30, 149-153 (2003) .
    • . . . Notably, human DNAJB6 has two known isoforms characterized by alternative C termini15 . . .
    • . . . The long isoform DNAJB6a (36 kDa) localizes to the nucleus, whereas the short isoform DNAJB6b (27 kDa) is cytoplasmic15, 16. . . .
  16. Mitra, A. Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer Breast Cancer Res. 10, R22 (2008) .
    • . . . The long isoform DNAJB6a (36 kDa) localizes to the nucleus, whereas the short isoform DNAJB6b (27 kDa) is cytoplasmic15, 16. . . .
    • . . . This protein has been implicated in a variety of degenerative disorders such as Parkinson's17 and Huntington's8 diseases, as well as cancer16 . . .
  17. Durrenberger, P.F. DnaJB6 is present in the core of Lewy bodies and is highly up-regulated in parkinsonian astrocytes J. Neurosci. Res. 87, 238-245 (2009) .
    • . . . DNAJB6 has been reported to suppress aggregation and toxicity of aggregation-prone proteins such as polyglutamine-containing huntingtin and α-synuclein7, 8, 17 and to participate in autophagic and proteasomal turnover of proteins and organelles18, 19, as well as in regulation of gene expression10 and cell cycle20 . . .
    • . . . This protein has been implicated in a variety of degenerative disorders such as Parkinson's17 and Huntington's8 diseases, as well as cancer16 . . .
  18. Watson, E.D.; Geary-Joo, C.; Hughes, M.; Cross, J.C. The Mrj co-chaperone mediates keratin turnover and prevents the formation of toxic inclusion bodies in trophoblast cells of the placenta Development 134, 1809-1817 (2007) .
    • . . . DNAJB6 has been reported to suppress aggregation and toxicity of aggregation-prone proteins such as polyglutamine-containing huntingtin and α-synuclein7, 8, 17 and to participate in autophagic and proteasomal turnover of proteins and organelles18, 19, as well as in regulation of gene expression10 and cell cycle20 . . .
  19. Rose, J.M.; Novoselov, S.S.; Robinson, P.A.; Cheetham, M.E. Molecular chaperone-mediated rescue of mitophagy by a Parkin RING1 domain mutant Hum. Mol. Genet. 20, 16-27 (2011) .
    • . . . DNAJB6 has been reported to suppress aggregation and toxicity of aggregation-prone proteins such as polyglutamine-containing huntingtin and α-synuclein7, 8, 17 and to participate in autophagic and proteasomal turnover of proteins and organelles18, 19, as well as in regulation of gene expression10 and cell cycle20 . . .
  20. Zhang, Y. The Hsp40 family chaperone protein DnaJB6 enhances Schlafen1 nuclear localization which is critical for promotion of cell-cycle arrest in T-cells Biochem. J. 413, 239-250 (2008) .
    • . . . DNAJB6 has been reported to suppress aggregation and toxicity of aggregation-prone proteins such as polyglutamine-containing huntingtin and α-synuclein7, 8, 17 and to participate in autophagic and proteasomal turnover of proteins and organelles18, 19, as well as in regulation of gene expression10 and cell cycle20 . . .
  21. Li, Z.F. Non-pathogenic protein aggregates in skeletal muscle in MLF1 transgenic mice J. Neurol. Sci. 264, 77-86 (2008) .
    • . . . The inhibition of aggregate-induced cytotoxicity may involve myeloid leukemia factor 1 (MLF1), which interacts with DNAJB6 and colocalizes with it in aggregates21. . . .
    • . . . Immunofluorescence microscopy of patient muscle showed DNAJB6 in protein accumulations together with its known ligands MLF1 (ref. 21) and HSPA8 (ref. 9) (Fig. 1d and Supplementary Fig. 4) . . .
  22. Seki, N. Cloning, tissue expression, and chromosomal assignment of human MRJ gene for a member of the DNAJ protein family J. Hum. Genet. 44, 185-189 (1999) .
    • . . . DNAJB6 is expressed in all tissues22, with the highest expression in brain8, but its expression and localization in skeletal muscle have not been characterized previously . . .
  23. Selcen, D.; Engel, A.G. Mutations in myotilin cause myofibrillar myopathy Neurology 62, 1363-1371 (2004) .
    • . . . For comparison, we examined muscle tissue from an individual with myofibrillar myopathy caused by a p.Ser60Cys alteration in myotilin, characterized by aggregation of myotilin and other proteins23 . . .
  24. Kimmel, C.B.; Ballard, W.W.; Kimmel, S.R.; Ullmann, B.; Schilling, T.F. Stages of embryonic development of the zebrafish Dev. Dyn. 203, 253-310 (1995) .
    • . . . Detachment of slow fibers from their insertion sites at the vertical myoseptum was evident as early as 2 d after fertilization (d.p.f.) (Fig. 2), suggesting that the fiber termini in morphants are prone to adhesion failure soon after mechanical load is applied by the onset of strong contraction24 . . .
  25. Ding, W.X. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability Am. J. Pathol. 171, 513-524 (2007) .
    • . . . This is also consistent with the observation that DNAJB6 abundance was reduced in the presence of both lactacystin and CHX (compared to CHX alone), as suppression of the proteasome has been reported to enhance autophagy25, 26, 27. . . .
  26. Pandey, U.B. HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS Nature 447, 860-864 (2007) .
    • . . . This is also consistent with the observation that DNAJB6 abundance was reduced in the presence of both lactacystin and CHX (compared to CHX alone), as suppression of the proteasome has been reported to enhance autophagy25, 26, 27. . . .
  27. Zhu, K.; Dunner, K. Jr.; McConkey, D.J. Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells Oncogene 29, 451-462 (2010) .
    • . . . This is also consistent with the observation that DNAJB6 abundance was reduced in the presence of both lactacystin and CHX (compared to CHX alone), as suppression of the proteasome has been reported to enhance autophagy25, 26, 27. . . .
  28. Hageman, J.; van Waarde-Verhagen, M.; Zylicz, A.; Walerych, D.; Kampinga, H.H. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities Biochem. J. 435, 127-142 (2011) .
    • . . . DNAJB6 has been shown to suppress aggregation of various proteins in cell culture models7, 8, 28 . . .
  29. Arndt, V. Chaperone-assisted selective autophagy is essential for muscle maintenance Curr. Biol. 20, 143-148 (2010) .
    • . . . Chaperone-assisted selective autophagy (CASA), mediated by a complex containing HSPA8, HSPB8 (also known as Hsp22), and the co-chaperones BAG3 and STUB1 (also known as C terminus of Hsc70-interacting protein (CHIP)), is important for Z-disk maintenance29, 30 . . .
  30. Kettern, N.; Dreiseidler, M.; Tawo, R.; Höhfeld, J. Chaperone-assisted degradation: multiple paths to destruction Biol. Chem. 391, 481-489 (2010) .
    • . . . Chaperone-assisted selective autophagy (CASA), mediated by a complex containing HSPA8, HSPB8 (also known as Hsp22), and the co-chaperones BAG3 and STUB1 (also known as C terminus of Hsc70-interacting protein (CHIP)), is important for Z-disk maintenance29, 30 . . .
  31. Selcen, D. Mutation in BAG3 causes severe dominant childhood muscular dystrophy Ann. Neurol. 65, 83-89 (2009) .
    • . . . Finally, mutations in BAG3 cause a myofibrillar myopathy with protein accumulations and autophagic pathology31 . . .
    • . . . In addition, injection of the BAG3 myopathy mutant (p.Pro209Leu)31 alone led to a phenotype comparable to that of DNAJB6 mutants, whereas its co-injection with DNAJB6b p.Phe93Leu mRNA did not have an additive effect . . .
  32. Ianzano, L. Loss of function of the cytoplasmic isoform of the protein laforin (EPM2A) causes Lafora progressive myoclonus epilepsy Hum. Mutat. 23, 170-176 (2004) .
    • . . . This is reminiscent of the mechanism proposed for Lafora progressive myoclonus epilepsy, in which mutations in EPM2A are expressed in both the nucleus and the cytoplasm but seem to interfere specifically with its cytoplasmic phosphatase activity32 . . .
  33. Wang, E.T. Alternative isoform regulation in human tissue transcriptomes Nature 456, 470-476 (2008) .
    • . . . The literature contains few other such examples, despite the documented prevalence of alternative splicing33 . . .
  34. Mologni, L.; Moza, M.; Lalowski, M.M.; Carpén, O. Characterization of mouse myotilin and its promoter Biochem. Biophys. Res. Commun. 329, 1001-1009 (2005) .
    • . . . Myotilin antibody34 was a gift from O . . .
  35. Hasholt, L. Antisense downregulation of mutant huntingtin in a cell model J. Gene Med. 5, 528-538 (2003) .
    • . . . The huntingtin construct pEGFP/HD-120Q, STUB1 construct in pcDNA3.1/Myc-His and titin construct is6–M9-V5 have been described35, 36, 37. . . .
  36. Qian, S.B.; McDonough, H.; Boellmann, F.; Cyr, D.M.; Patterson, C. CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70 Nature 440, 551-555 (2006) .
    • . . . The huntingtin construct pEGFP/HD-120Q, STUB1 construct in pcDNA3.1/Myc-His and titin construct is6–M9-V5 have been described35, 36, 37. . . .
  37. Sarparanta, J. Interactions with M-band titin and calpain 3 link myospryn (CMYA5) to tibial and limb-girdle muscular dystrophies J. Biol. Chem. 285, 30304-30315 (2010) .
    • . . . The huntingtin construct pEGFP/HD-120Q, STUB1 construct in pcDNA3.1/Myc-His and titin construct is6–M9-V5 have been described35, 36, 37. . . .
    • . . . The samples were prepared and PLA performed essentially as described37. . . .
  38. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio) , (1995) .
    • . . . Zebrafish (Danio rerio) embryos were raised and fish were maintained as described38 . . .
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