Morphological studies
Patients III-3 and III-5 of family 1 underwent a diagnostic biopsy of the deltoid muscle. Transverse cryostat sections were routinely stained for: haematoxylin-eosin, Gomori trichrome, periodic acid-Schiff, oil red O, myofibrillar ATPase, acid phosphatase, NADH dehydrogenase, cytochrome C oxidase, succinic dehydrogenase. Immunofluorescence for phosphorylated tau-protein with anti-SMI-31 antibodies (SantaCruz Biotechnology, Santa Cruz, CA, USA) and for the autophagic protein sequestosome1 (SQSTM1, p62, monoclonal sc-28359, Santa Cruz) with anti-p62 antibodies was performed. No muscle tissue was available for additional immunochemical analyses.
On the muscle biopsies from sporadic patients (quadriceps muscle in patients 1 and 3s, biceps in patient 2s, deltoid in patient 4s), in addition to the routine histochemical and immunohistochemical analyses, immunolocalization of several other proteins was performed. Muscle cryosections were incubated with one of the following antibodies: anti-DNAJB6 (rabbit polyclonal PA5-21401, Thermo Fisher Rockford, IL, USA; 1:100), anti-LC3 (rabbit polyclonal PM036, MBL, Naka-ku, Nagoya, Japan; 1:100), anti-p62 (guinea pig polyclonal GP62-c, Progene Biotechnik, Heidelberg, Germany; 1:100;), anti-transactive response element DNA-binding protein of 43 kDa (TDP-43) (rabbit polyclonal 10782-2-AP, Proteintech Chicago, IL, USA; 1:200), anti BAG3 (mouse monoclonal sc-136493, SantaCruz; 1:50) followed by incubation in Alexa 546- or Alexa 488-conjugated goat anti-mouse, anti-rabbit, or anti-guinea pig IgG (Invitrogen Life Technologies, Carlsbad, CA, USA) as appropriate, 1:2000, for 2 h. As control, sections were either incubated with rabbit non-immune serum, or with isotype-specific non-immune IgG (Dako, Copenhagen, Denmark), or the primary antibody was omitted.
For co-localization studies muscle cryosections were incubated in a mixture of primary antibodies; either DNAJB6 (monoclonal, Sigma Aldrich, St. Louis, MO, USA; 1:100) plus TDP43, or desmin (monoclonal M0760, Dako; 1:200) plus p62, or desmin (rabbit polyclonal A0611, Dako, 1:100) plus myotilin (monoclonal NCL-MYOTILIN, Novocastra, Newcastle-upon-Tyne, UK; 1:20), or p62 plus poly-ubiquitinylated proteins (monoclonal PW8810, clone FK2 from Biomol, Enzo Life Sciences, Inc. Farmingdale, USA; 1:200), or desmin plus SMI-31 (monoclonal SMI-31R, Covance Research Products, Princeton, New Jersey, USA; 1:100) followed by washes and incubation in a mixture of appropriate fluorescent dye-conjugated secondary antibodies. Muscle sections were examined under either a Zeiss Axioplan fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) or a Leica confocal microscope equipped with hybrid and argon lasers (Leica Microsystems, Wetzlar, Germany). Muscle biopsies from X-linked myopathy with excessive autophagy (XMEA), Pompe disease (PD) and GNE-mutated inclusion body myopathy patients were used as positive controls.
Western blotting
Muscle cryosections were solubilized in 25 μl lysis buffer containing 4 % SDS, 125 mM Tris–HCl pH 8.8, 40 % glycerol, and 0.5 mM PMSF; sonicated, boiled, and centrifuged at 15,000 g for 5 min. Samples of supernatant were electrophoresed on 7.5 % or 15 % SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were probed with antibodies to DNAJB6 (1:500), TDP-43 (1:250), LC3 (1:500), or p62 (1:800). Vinculin (1:1000; Sigma) was used as indicator of how much muscle protein was loaded. Blocked membranes were then incubated in biotin-conjugated secondary antibody (1:2500; Jackson ImmunoResearch Laboratories, Inc., Westgrove PA, USA), followed by peroxidase-conjugated streptavidin (1:3000, Jackson ImmunoResearch) and by detection with the ECL chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, UK).
RNA analysis
To detected alternative transcripts due to splice-site mutations, total RNA was isolated from muscle samples using TRI Reagent (Ambion, Austin, TX, USA) according to the manufacturer’s instructions, and checked for quantity and purity using a Nanodrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). Aliquots of RNA (1 μg) were reverse-transcribed in the presence of 5× first strand buffer (Life Technologies, Carlsbad, CA, USA), 1 mM each deoxynucleoside triphosphate, 8 pM random hexamers, 10 μM dithiothreitol, 1 IU/μl RNAse inhibitor (Roche Molecular Biochemicals, Basel, Switzerland), and 10 IU/μl M-MLV reverse transcriptase (Life Technologies), by incubation at 37 °C for 1 h and at 95 °C for 5 min. The resulting cDNA was amplified using appropriate primers, purified from agarose gel, using QIAquick Gel Extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and verified by sequencing.
Molecular modelling
Secondary structure prediction was made with PSIPRED [
18] and disordered regions prediction with Robetta [
19]. Both analyses suggest that the G/F domain has low structural complexity and contains a single helix that is preceded and followed by unstructured regions. The C-terminal domain of the protein is instead predicted to adopt a β structure. A search for evolutionary related homologues to DNAJB6b was performed with Phyre2 [
20] and I-Tasser [
21]. The initial homology model was obtained with Phyre2, using TtDNAJB2 as a template (pdbcode: 4 J80) and then energy minimized with Modeller version 9 [
22]. It covers residues 2–125 of DNAJB6, which comprise the J domain, the G/F domain and its junction with the C-terminal domain. Ramachandran statistics were calculated with Procheck [
23]. The geometrical quality of the model is good with 91.2 % of residues lying in the most favoured regions of the Ramachandran plot, 7.8 % in additionally allowed regions and 1 % (1 residue: Ser81) in disallowed regions. Site specific mutations were inserted with COOT [
24]. All structure representations in Fig. 8 were prepared with PyMol (The PyMOL Molecular Graphics System, Schrödinger, LLC).