Brain Advance Access originally published online on September 19, 2006
Brain 2007 130(2):381-393; doi:10.1093/brain/awl238
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Pathological consequences of VCP mutations on human striated muscle
1 Institute of Biochemistry I University of Cologne, Cologne 2 Center for Molecular Medicine Cologne, Medical Faculty University of Cologne, Cologne 3 Department of Neurology, University Hospital Bonn Bonn 4 Department of Radiology, University Hospital Bonn Bonn 5 Institute for Neuropathology, University Hospital Bonn Bonn 6 Institute of Pathology, University Hospital Bonn Bonn 7 Department of Neuroproteomics, Max Delbrück Center for Molecular Medicine Berlin 8 Friedrich-Baur-Institut and Department of Neurology, Ludwig Maximilians University of Munich München, Germany 9 Institute of Structural and Molecular Biology, School of Biological Sciences, The University of Edinburgh Edinburgh, UK 10 Division of Genetics, Children's Hospital Boston, Harvard Medical School Boston, MA, USA
Correspondence to: Rolf Schröder, MD, Institute of Biochemistry I, Medical Faculty, University of Cologne, Joseph Stelzmann Strauss 52, 50931 Cologne, Germany E-mail: rolf.schroeder{at}uni-koeln.de
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Mutations in the valosin-containing protein (VCP, p97) gene on chromosome 9p13–p12 cause a late-onset form of autosomal dominant inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD). We report on the pathological consequences of three heterozygous VCP (R93C, R155H, R155C) mutations on human striated muscle. IBMPFD skeletal muscle pathology is characterized by degenerative changes and filamentous VCP- and ubiquitin-positive cytoplasmic and nuclear protein aggregates. Furthermore, this is the first report demonstrating that mutant VCP leads to a novel form of dilatative cardiomyopathy with inclusion bodies. In contrast to post-mitotic striated muscle cells and neurons of IBMPFD patients, evidence of protein aggregate pathology was not detected in primary IBMPFD myoblasts or in transient and stable transfected cells using wild-type-VCP and R93C-, R155H-, R155C-VCP mutants. Glutathione S-transferase pull-down experiments showed that all three VCP mutations do not affect the binding to Ufd1, Npl4 and ataxin-3. Structural analysis demonstrated that R93 and R155 are both surface-accessible residues located in the centre of cavities that may enable ligand-binding. Mutations at R93 and R155 are predicted to induce changes in the tertiary structure of the VCP protein. The search for putative ligands to the R93 and R155 cavities resulted in the identification of cyclic sugar compounds with high binding scores. The latter findings provide a novel link to VCP carbohydrate interactions in the complex pathology of IBMPFD.
Key Words: VCP; p97; myopathy; cardiomyopathy; IBMPFD
Abbreviations: GST, glutathione S-transferase; IBMPFD, inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulphate; VCP, valosin-containing protein
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Received May 10, 2006. Revised July 26, 2006. Accepted August 8, 2006.
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Introduction |
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Autosomal dominant inclusion body myopathy (IBM) associated with Paget disease of the bone (PDB) and frontotemporal dementia (FTD), or IBMPFD (OMIM 605382 [OMIM] ), is a late-onset multisystem disorder caused by mutations of the valosin-containing protein (VCP) on chromosome 9p13–p12 (Watts et al., 2004
; Haubenberger et al., 2005; Schröder et al., 2005). VCP (p97), an ubiquitously expressed member of the AAA-ATPase family, has a tripartite structure comprising an N-terminal domain (CDC48) involved in ubiquitin binding, and two central D1- and D2-domains that bind and hydrolyse ATP (DeLaBarre and Brünger, 2003). VCP assembles into functional hexamers with a central cylinder formed by the D-domains surrounded by the N-domains. Apart from R191Q and A232E mutations, which reside in the N-D1-linker region and D1-domain, respectively, all other pathogenic mutations described so far are located in exons coding for the CDC48 domain of the VCP protein (Watts et al., 2004; Haubenberger et al., 2005; Schröder et al., 2005).
VCP has been associated with a wide variety of essential cellular processes comprising nuclear envelope reconstruction, the cell cycle, post-mitotic Golgi reassembly, suppression of apoptosis, DNA damage response and the ubiquitin proteasome protein degradation system (Kondo et al., 1997; Rabouille et al., 1998; Meyer et al., 2000; Hetzer et al., 2001; Rabinovich et al., 2002). Furthermore, VCP along with its co-factors Udf1 and Npl4 as well as Derlin-1 have been implicated to play a central role in endoplasmic reticulum associated protein degradation (ERAD), a process that removes improperly folded proteins from the ER for further degradation by the 26S proteasome (Ye et al., 2001; Rabinovich et al., 2002; Ye et al., 2004; Lilley and Ploegh, 2005).
A further pathogenic link of VCP to protein degradation pathways is highlighted by the observation that VCP-positive protein aggregates have been documented in skeletal muscle and in neurons of the central nervous system of IBMPFD patients (Watt et al., 2004; Schröder et al., 2005). In neurons, these VCP-positive inclusions are exclusively present in the nucleus, whereas in skeletal muscle only cytoplasmic VCP-positive aggregates have been reported. However, VCP-positive aggregates are not specific for IBMPFD and have been documented in a wide variety of neurodegenerative disorders comprising Parkinson's disease, Lewy body disease, Huntington's disease, amyotrophic lateral sclerosis and spinocerebellar ataxia type III (SCAIII; Machado–Joseph disease) (Hirabayashi et al., 2001; Mizuno et al., 2003; Nan et al., 2005). VCP directly interacts with ataxin-3, the protein mutated in SCAIII, and recent in vivo studies using Drosophila demonstrated that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic ataxin-3 (Böddrich et al., 2006). In the present study we report on the pathological consequences of VCP mutations on human striated muscle in vivo and in vitro.
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Material and methods |
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Muscle MRI
Whole-body muscle MRI using a 1.5-T scanner, body coil (Philips Gyroscan Intera, Best, The Netherlands) was performed as described previously (Fischer et al., 2005
).
VCP mutation analysis
Isolation of DNA and VCP, desmin and 
ß-crystallin mutation analysis were performed as described previously (Vicart et al., 1998; Schröder et al., 2003; Watts et al., 2005).
Histological analysis
Cryostat sections (6 µm) from human skeletal muscle were stained by standard diagnostic techniques. Cardiac autopsy material from Patient II was taken from the left and right ventricular and septal walls at the base, mid-cavity and apical levels, and from the left atrial free wall. Sections of paraffin-embedded material were stained by standard diagnostic techniques.
Antibodies
The following primary antibodies were used: VCP rabbit antiserum (kind gift of Dr Chou-Chi Li, National Cancer Institute at Frederick, MD, USA); monoclonal mouse anti-VCP (Affinity BioReagents, USA); monoclonal anti-VCP (BD Biosciences, USA); monoclonal mouse antibody raised against ubiquitin (Novocastra, UK); rabbit anti-ubiquitin polyclonal antibody (Stressgen, Canada); rabbit anti-ubiquitin polyclonal antibody (DAKO, Denmark); monoclonal mouse anti-poly-ubiquitin, clone FK2 (Stressgen, Canada); mouse monoclonal anti-desmin antibody D33 (DAKO, Denmark); rabbit polyclonal anti-ß-crystallin antiserum (Chemicon, USA); mouse monoclonal antibody raised against Ufd1 (Transduction Laboratories, USA); mouse monoclonal antibody raised against glutathione S-transferase (GST) (Amersham Biosciences, Germany); mouse monoclonal anti-His antibody (Qiagen, Germany); mouse monoclonal anti-FLAG M2 antibody (Stratagene, USA); rabbit polyclonal enterokinase cleavage site (FLAG) antibody (Novus Biologicals, USA); TRITC-Phalloidin (Sigma, Germany); monoclonal mouse antibody specifically recognizing GFP (Noegel et al., 2004). Isotype specific secondary antibodies conjugated with fluoroisothiocyanate (FITC), Cy3 or Texas Red, and Alexa568 were applied according to the recommendations of the manufacturers (Southern Biotechnology Associates, USA; Jackson Immunoresearch Laboratories, USA; Molecular Probes, USA). Alternatively, sections incubated with anti-VCP and anti-ubiquitin antibodies were incubated with biotinylated secondary antibodies and the avidin–biotin complex. Visualization was performed with 3,3-diaminobenzidine or the APAP complex as reagent.
Indirect immunofluorescence and imaging of living cells
Indirect immunofluorescence analysis of human skeletal muscle was performed as described previously (Schröder et al., 2003). Transfected cells were seeded on coverslips, washed with phosphate-buffered saline (PBS) and subsequently fixed either in –20°C methanol for 10 min or in 4% formaldehyde for 20 min followed by treatment with 0.5% Triton X-100 and PBS/glycine 0.15% for 5 min each. All following washing steps were performed 5x for 5 min in PBS/glycine. The following staining procedures were performed as described previously (Schröder et al., 2005). Indirect immunofluorescence analysis as well as examination of living cells was done using a confocal Leica DM-IRBE microscope (Leica, Germany).
Ultrastructural analysis and immunogold electron microscopy
Electron microscopy and desmin immunogold electron microscopy of skeletal muscle were performed as described previously (Schröder et al., 2002).
Gel electrophoresis and western blotting
For one- and two-dimensional gel electrophoresis, preparation of total protein extracts, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), protein transfer and visualization of proteins were carried out as described previously (Clemen et al., 2005).
VCP-cDNA, site-directed mutagenesis and plasmids
Human wt-VCP cDNA was amplified by PCR, adding restriction sites and a FLAG-tag at the 3' end. The PCR-product was cloned in pGEMTeasy vector (Promega, Germany). The mutations R93C, R155H and R155C were introduced using the site-directed mutagenesis kit (BD Biosciences, USA). PCR products and the obtained plasmids were confirmed by direct sequencing (Perkin-Elmer Cetus, USA). For transfection, the VCP constructs were cloned into the pEGFP-N1 and pEGFP-C1 vectors (Clontech, USA). For viral transduction the cDNAs were cloned into the pBMN vector (Clemen et al., 1999).
Cell culture, transfection, transduction and cell stress experiments
HEK293 cells (ATCC: CRL-1573) and C2F3 myoblasts (Clemen et al., 1999) were grown as described. Normal and IBMPFD primary human myoblasts were grown in skeletal muscle cell growth medium (Promocell, Germany; C-23060 with supplement mix C-39365 added). For differentiation of C2F3 myoblasts the FCS was exchanged for using 1% horse serum. Transient transfection of HEK293 cells was carried out by electroporation (1 x 106 cells, 5 µg plasmid; 475 µF, 240 V, 4 mm cuvette). Stable HEK293 clones were obtained by selection with 1.2 mg/ml G418. Transient transfection of C2F3 cells using Lipofectamin was done according to the instructions of the manufacturer (Invitrogen, Germany). Viral transduction of C2F3 cells was done according to Clemen et al. (1999) and www.stanford.edu/group/nolan.
MG132 (Calbiochem, USA; 10 mM stock in DMSO; 5 µM final concentration, 16 h; Kitami et al., 2006; Weihl et al., 2006), clasto-lactacystin ß-lactone (Calbiochem; 10 mM stock in DMSO; 10 µM final concentration, 24 h; Steinhilb and Gaut, 2001; Waelter et al., 2001), wortmannin (Calbiochem, datasheet 681675; 1 mM stock in DMSO; 1 µM final concentration, 22 h), H2O2 (Sigma, Germany; 400 µM final concentration, 16 h; Ardley et al., 2003) were added to normal culturing medium. For osmotic shock experiments, cells were incubated in 150 mM urea dissolved in normal culturing medium (Sigma, Germany; D'Alessandro et al., 2002) for 5 min. The solution was changed to fresh medium and cells were subsequently fixed after 4 min. UV-B irradiation experiments were performed using opened culture dishes containing a minimal volume of PBS in a UV-cross-linker (Hoefer UVC 500, Amersham Pharmacia Biotech, Germany; Westfall et al., 2005) with a total dose of 100 J/m2 at a wavelength of 254 nm. Cells were further incubated in fresh medium and analysed after 12 h. Mitomycin C treatment (Medac, Germany; 10 µg/ml final concentration; standard protocol) was done for 3 h in normal culturing medium followed by two washing steps with PBS. Cells were subsequently analysed after 1, 7 and 14 days.
Protein-binding studies
GST-, MBP- and His-tagged fusion proteins were expressed in Escherichia coli and purified on affinity columns as described previously (Scherzinger et al., 1997) and according to manufacturer's instructions (NEB, USA; Qiagen, Germany). The plasmids pGEX-6P-VCP wt, pGEX-6P-VCP R155H, pGEX-6P-VCP R155C and pGEX-6P-VCP R93C were generated by amplifying the VCP cDNA-fragments from pGEMTeasy vector (see above) and subcloning in pGEX-6P-1 (Amersham Biosciences, Freiburg, Germany). The cDNA encoding Ufd1 was obtained from the RZPD (Deutsches Ressourcenzentrum für Genomforschung GmbH, Germany), amplified and cloned into pMAL-c2X (NEB). To produce a His-tagged Npl4 fusion protein, the Npl4 cDNA (kindly provided by H. Meyer, ETH Zürich, Switzerland) was amplified and cloned into pQE30N, a derivative of pQE30 (Qiagen, Germany). The cloning of the ataxin-3 cDNA into pQE has been described (Tait et al., 1998). For in vitro binding experiments, GST fusion proteins were bound to glutathione agarose beads and incubated with 0.1 µM MBP-Ufd1, 0.1 µM His-Npl4, 0.1 µM His-ataxin-3 Q22 or combinations of 0.1 µM MBP-Ufd1/His-Npl4 and 0.1 µM MBP-Ufd1/His-Npl4/4 µg tetra-ubiquitin (Biomol, UK) in IP-buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 20 mM NaF, 10% glycerol, 1% NP-40 and protease inhibitors) at 4°C for 1 h. After washing the beads four times with IP-buffer, bound proteins were eluted with SDS-sample buffer, boiled for 5 min and analysed by immunoblotting.
Modelling of human valosin-containing protein
The crystal structure of the ND1-domains of mouse VCP in complex with rat p47 has previously been solved at a resolution of 2.9 Å (PDB entry 1s3s
[PDB]
; Dreveny et al., 2004).
Since mouse and human VCP protein share 99.5% identity with Ile206 being Val in the mouse protein, we have taken the model from 1s3s
[PDB]
and introduced the mutation V206I using the program O (Jones et al., 1991) followed by local refinement. Mutations of interest for this study (R155C, R155H, R93C) were introduced in the same manner. The overall geometry of the models was scrutinized with PROCHECK (Laskowski et al., 1993).
Virtual screening of ligand databases
Two potential ligand-binding pockets were identified from the homology model of ND1-VCP and the automated docking programme LIDAEUS (Wu et al., 2003) was used to screen all structures of a small-molecule database (Sigma catalogue) against the potential ligand-binding pockets. First, a dummy ligand (ADP) was docked in the potential pockets by FlexX (Rarey et al., 1996) to obtain template coordinates for the generation of binding pocket site points with certain characteristic features (electrostatic potential, hydrogen donor, hydrogen acceptor, etc). On the basis of chemical and shape complementation, LIDAEUS finds ligands that complement the features of the binding pocket and generates multiple ligand-binding conformations. The programme then tries to find the best fit of the different poses by ranking the conformations according to various energy scores including hydrophobic interactions, van der Waals and H-bonding. The small-molecule database was prepared for LIDAEUS using the software EDULISS (Wu et al., 2003). As a control, the dummy ligand ADP used for site point generation was also included in the screen. The resulting ligands and poses were analysed with the graphics programme WITNOTP (A. Widmer, Novartis, Switzerland). A selection of ligands that were docked well inside the pocket was subjected to further analysis including scoring and ligand-protein contacts using the programme LIGPLOT (Wallace et al., 1995).
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Clinical phenotype
Patient I: A 74-year-old male patient was regularly seen since age 55. He has a >20-year history of slowly progressive distal muscle weakness predominantly affecting the lower extremities. Signs of progressive cognitive impairment were first noted in his late 60s. Except for Paget's disease in the patient's father, the family history was unremarkable with regard to neuromuscular or psychiatric disorders. Neurological examination in 2005 showed marked generalized weakness and atrophy of distal arms and leg muscles. In addition, axial weakness of the lumbar trunk was noticed. Repeated neuropsychiatric evaluation showed evidence of progressive personality changes and cognitive decline due to frontotemporal brain dysfunction. Axial computed tomography revealed Paget-like bone changes in the right hip. Creatine kinase (CPK) levels had been mildly elevated (115 U/l; normal <80) at the initial examination, but were in the normal range ever since.
Patient II: A 62-year-old female patient was first seen in 1993. At that time, she gave a 3-year history of progressive proximal muscle weakness. Brain MRI performed in 1995 showed frontal and temporal brain atrophy. Her last neurological examination in February 1997 showed a severely demented patient with a flaccid, predominantly proximal tetraparesis. Serum CPK levels were always within normal limits. According to information obtained by her husband in 1998, the mother of the reported patient, her mother's brother as well as one of his children suffered from similar medical conditions. She died of pneumonia and cardiac failure in 1998. Autopsy at that time showed severe generalized wasting of her skeletal muscles, but no signs of Paget's disease of the bone. The total heart weight was 480 g; left ventricular and right ventricular wall thickness was 1.7 and 0.7 cm, respectively. Neuropathological analysis of her brain has been reported previously (Schröder et al., 2005). Neurons exhibited nuclear inclusions containing VCP- and ubiquitin-containing material.
Patient III: A 54-year-old female first presented in May 2003. She gave a 30-year history of slowly progressive muscle weakness and atrophy predominantly affecting her shoulder girdle, trunk and distal leg muscles (Fig. 1A). Her family history was negative for neuropsychiatric, muscular or bone diseases. Paget's disease of the bone confined to the first lumbar vertebra was histologically diagnosed in 2002 (Fig. 1B). She presented no overt neuropsychological or behavioural abnormalities. However, a detailed neuropsychological evaluation in February 2004 revealed a performance far below average (>2 SDs below mean) in the labyrinth task testing for anticipation, and below average (>1 SD below mean) in the figural memory and naming tests, suggesting mild frontotemporal cognitive dysfunction. Brain MRI and cardiological examination gave normal results. Neurological examination showed severe weakness and atrophy of her scapular fixator muscles (deltoid, rhomboid, supra- and infraspinatus) and trunk extensors. In addition, she had slight to moderate muscle weakness of her finger extensor, hip flexor and distal leg muscles. Repeated serum CPK levels were within normal limits. Whole-body MRI demonstrated widespread muscular involvement with pronounced signal changes in her erector spinae, hamstring and calf muscles (Fig. 1C, D and E)
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Mutation analysis
Mutation analysis in Patient I revealed a novel heterozygous nucleotide substitution in exon 3 (c.277C
T) of the VCP gene (GenBank AC004472
[GenBank]
). This mutation is predicted to result in an amino acid substitution from arginine to cystein in codon 93 (R93C) and was not detected in 100 control chromosomes (data not shown). In Patient II, we previously identified a heterozygous mutation in exon 5 leading to an amino acid substitution from arginine to cystein in codon 155 (c.463C T, R155C; Schröder et al., 2005). Additionally, mutations of the desmin and ß-crystallin genes were ruled out by direct sequence analysis in this patient. VCP mutation analysis of a DNA sample from Patient III revealed a heterozygous nucleotide substitution causing an amino acid substitution in the same codon from arginine to histidine (c.464G A, R155H) (data not shown).
Skeletal muscle pathology
Morphological evaluation of a vastus lateralis and tibial anterior biopsy from Patient I showed severe degenerative changes consisting of increased fibre size variation, atrophy of both fibre types, presence of terminal atrophic and angulated fibres, hypertrophic type-1 fibres, degenerating and a few regenerating fibres, ‘myopathic grouping’ as well as marked fatty replacement of muscle fibres and broadening of connective tissue (Fig. 2A). A diagnostic muscle biopsy taken from the biceps brachii muscle of Patient II displayed the classical myopathological picture of an IBM with an abundance of rimmed vacuoles (Fig. 2B). In contrast, the biopsy from the vastus lateralis muscle in Patient III showed only mild and unspecific myopathological changes consisting of type I fibre predominance, atrophic and hypertrophic fibres (Fig. 2C). In addition, few de- and regenerating fibres could be demonstrated. It is noteworthy that in biopsies from Patient I and III only a few fibres with rimmed vacuoles could be detected. None of the three reported cases showed inflammatory infiltrates.
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Double-immunofluorescence analysis of skeletal muscle from Patient III revealed a small number of fibres (<5%) containing cytoplasmic foci of VCP- and ubiquitin-positive protein aggregates (data not shown), whereas the corresponding analysis of the muscle biopsy from Patient II showed a high number of fibres (30–40%) with single or multiple VCP- and ubiquitin-positive cytoplasmic inclusions (Fig. 3A and B). In addition, double-staining with VCP antibody and DAPI (4',6'-diamidino-2-phenylindole-dihydrochloride) revealed multiple fibres with VCP-positive nuclear inclusions (Fig. 3C and D). Further analysis revealed multiple fibres displaying subsarcolemmal and cytoplasmic areas with increased
ß-crystallin (Fig. 3E) and desmin labelling (Fig. 3F).
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A detailed ultrastructural analysis was performed on skeletal muscle from Patient II. The VCP-positive nuclear inclusions consisted of filamentous material (Fig. 4A). In analogy to the abundance of rimmed vacuoles and cytoplasmic VCP and ubiquitin-positive inclusions at the light microscopic level, many fibres contained autophagic vacuoles with haphazardly arranged filamentous material as well as large cytoplasmic areas consisting of densely or loosely packed filamentous material (Fig. 4B). Remarkably, multiple fibres also displayed areas with granulofilamentous material as seen in the group of myofibrillar myopathies (Fig. 4C). Immunogold EM showed a dense desmin-positive labelling of these areas (Fig. 4D).
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Cardiac pathology
Post-mortem analysis of the heart of Patient II revealed a marked left ventricular dilatation and thickening of the left ventricular wall (Fig. 5A). Histopathological examination showed cellular hypertrophy of myocytes and in conjunction with multiple small parenchymal scars in both ventricles. Immunostaining of formalin-fixed and paraffin-embedded cardiac tissue revealed multiple cardiomyocytes displaying ubiquitin-positive cytoplasmic and single nuclear inclusions (Fig. 5B and C).
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VCP protein expression in IBMPFD muscle
VCP immunoblotting after 1D SDS–PAGE revealed a single band corresponding in size to 97 kDa in all probes analysed without significant changes in the total amount of VCP between normal and R93C-, R155H-, R155C-IBMPFD muscle (Fig. 6). Differential centrifugation of muscle tissue lysates revealed that VCP was exclusively found in the pellet fraction of IBMPFD and normal control muscle (Fig. 6).
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VCP immunoblotting after 2D gel electrophoresis of total protein extracts from normal human skeletal muscle revealed a prominent spot at pH 5.20. In addition, a second spot with weaker signal intensity was detected at the position of pH 5.16, which corresponds well with the calculated pI 5.14. A corresponding analysis of diseased skeletal muscle (R155H, R155C) showed an identical pattern compared with control muscle (data not shown).
Analysis of normal and IBMPFD primary human myoblasts
In order to study pathological protein aggregate formation in cultured cells, we analysed normal and IBMPFD (155C VCP mutant) in primary human myoblasts. Immunostaining using FK2 (Fig. 7) and VCP antibodies (data not shown) revealed an identical reticular staining pattern in normal and IBMPFD myoblasts. In contrast to IBMPFD muscle, no pathological protein aggregate formation could be detected.
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Wild-type versus mutant VCP in transfected cells
The following transfection and transduction experiments were performed: (i) Wt- and mutant-GFP-VCP-FLAG and VCP-FLAG-GFP were transiently and stably expressed in HEK293 cells; (ii) wt- and mutant-VCP-FLAG-GFP were transiently expressed in C2F3 myoblasts; (iii) wt- and the R155C-VCP-FLAG mutant were stably expressed in C2F3 myoblasts. Transfected cells were analysed by life cell imaging and indirect immunofluorescence analysis after methanol or paraformaldehyde fixation. Expression of either N- or C-terminally tagged wt-VCP-constructs in HEK293 cells resulted in an intense labelling of the entire cytoplasm and, inconsistently, in a weaker nuclear signal of the GFP-fusion proteins (Fig. 8A, B, D and E; and data not shown). Mutant VCP showed the same localization as wt-VCP, with no evidence of abnormal cytoplasmic protein aggregate formation in HEK293 and C2F3 cells. Transfection of GFP alone yielded a strong uniform labelling of both the cytoplasm and the nucleus (Fig. 8C and F).
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Furthermore, we performed stable transfections of HEK293 and C2F3 cells. Two months after the initial transfection, cells were analysed by life cell imaging. The localization of the three VCP mutants was indistinguishable from wt-VCP, with no evidence of protein aggregate formation (Fig. 8). Even Triton X-100 treatment before or after fixation of HEK293 cells did not unmask any protein aggregates (data not shown). Additionally, we performed indirect immunofluorescence analysis of the transfected HEK293 cells using antibodies directed against VCP, FLAG and poly-ubiquitinated proteins (FK2). Here, VCP and FLAG labelling showed a pattern analogous to N- or C-terminally GFP- or FLAG-tagged wt- and mutant-VCP constructs in living and fixed cells (data not shown). The FK2 antibody, a sensitive marker for pathological aggregates containing poly-ubiquitinated proteins, showed a diffuse cytoplasmic staining with occasional small foci displaying accentuated FK2 immunolabelling in the cytoplasm and nucleus of non-transfected (Fig. 10B) as well as transfected (wt-, R93C-, R155H-, R155C-VCP) HEK293 cells (Fig. 8).
In order to rule out effects of the GFP-tag, we retrovirally transduced C2F3 (a subclone of C2C12) myoblasts using wt-VCP-FLAG and R155C-VCP-FLAG expression constructs. Anti-FLAG- (Fig. 9A–D) and anti-VCP-staining (data not shown) revealed an intense labelling of the entire cytoplasm and, inconsistently, a weaker nuclear signal. Transduced C2F3 cells differentiated into myotubes showed the same results (Fig. 9E and F). However, neither myoblasts nor up to 6-day-old myotubes showed any evidence of protein aggregates.
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For further biochemical analysis of our transfected HEK293 cells, we performed immunoblotting of total protein extracts using VCP, GFP, FLAG and FK2 antibodies. VCP immunoblotting labelled the endogenous VCP protein as well as the GFP–VCP fusion protein. The GFP and FLAG antibodies exclusively detected the respective fusion proteins (data not shown). Comparison of signal intensities indicated an endogenous VCP to wt-, R93C-, R155H and R155C–VCP fusion protein ratio of 3 : 1 (Fig. 8G). Immunoblotting after differential centrifugation of cell lysates showed that both the endogenous VCP and wt-, R93C-, R155H-, R155C–VCP fusion proteins are almost exclusively present in the soluble fraction (data not shown). In contrast to normal and IBMPFD muscle, transfections of cells with wt and mutant VCP are not associated with a detectable shift of both VCP proteins to the pellet fraction. FK2 immunoblotting revealed identical patterns of poly-ubiquitinated proteins in all samples analysed (Fig. 8G).
VCP immunoblotting after 2D gel electrophoresis of total protein extracts from wt-VCP-FLAG and R155C-VCP-FLAG transduced C2F3 myoblasts differentiated into myotubes showed an identical pattern to the one in normal and IBMPFD muscle (data not shown).
VCP response to cellular stress
Stably transfected and non-transfected HEK293 cells were treated with mitomycin C (DNA-alkylating agent), UV radiation (DNA and protein cross-linking), H2O2 (oxidative stress), osmotic shock, wortmannin (PI3-kinase inhibitor), clasto-lactacystin ß-lactone (irreversible 20S proteasome inhibitor) or MG132 (reversible 26S proteasome inhibitor). Changes in the subcellular VCP-distribution of stably transfected cells were only observed in response to MG132 treatment. In C-terminally GFP-tagged wt-VCP- or R93C-, R155H-, R155C-VCP cells treatment with this reversible 26S proteasome inhibitor resulted in the formation of a single perinuclear aggregate with marked GFP signal intensity in all cells analysed (Fig. 10G and I). However, these protein aggregates showed no labelling with the FK2 antibody (Fig. 10H and J) or phalloidin (data not shown). Furthermore, 
80% of the cells additionally displayed few small foci of intranuclear protein aggregates.
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Structural analysis of wild-type VCP versus R93 and R155 mutant VCP
All three VCP mutations identified in our IBMPFD patients concern evolutionarily highly conserved arginine residues in the CDC48 domain of the VCP protein (Fig. 11A). We introduced the R93C, R155H and R155C mutations into a human VCP protein model derived from the murine VCP crystal structure (see Material and methods). Our analysis indicates that R93 and R155 are surface-accessible residues located in the centre of cavities that may enable ligand binding (Fig. 11B). Both R93 and R155 are also surface-accessible in the hexameric state of VCP (data not shown). While the cleft around R155 is larger and predominantly negatively charged (Fig. 11C), the cavity around R93 appears smaller and rather positively charged (Fig. 11D). The particular shape and charge distribution within the clefts around R93 and R155 indicate that these may be putative ligand-binding sites.
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barrel (amino acids 25–106, orange) and the four-stranded ß barrel (amino acids 112–186, cyan), connected by a short linker region (amino acids 107–111, green). The CDC48 domain connects the D1-AAA-ATPase domain (amino acids 208–459, blue) by a linker region (amino acids 187–208, yellow). Linker region L2 (dark grey), second AAA-ATPase domain (amino acids 481–761, D2, dark blue) and C-domain (amino acids 762–806, grey) are indicated. Mutations detected in our three German IBMPFD patients affect evolutionarily highly conserved arginine residues in codon 93 and codon 155 of the CDC48 domain. (B) Transparent Connolly surface with ribbon backbone of the human VCP model. The bound ADP in the D1-nucleotide-binding site is shown as Corey, Pauling, Koltun colouring scheme. The locations of the clefts around R93 and R155 are indicated by blue colouring of the surface of the two arginine residues; prepared with InsightII. (C and D) GRASP (Nicholls et al., 1993Further analysis revealed that R93 maintains interactions with amino acid residues E194 and R65, as well as with the backbone carbonyl group of N90. These interactions are all within the cleft around R93. The R93C mutation leads to a loss of these contacts due to the shorter side chain of cystein. In contrast, R155 interacts with amino acid residue N387 residing in the D1-domain, which binds and hydrolyses ATP. The N- and D1-domains are spatially separated and form only three direct contacts, R155-N387, R89-E261 and E30-K217. The mutations R155C and R155H lack the interaction with N387 owing to the shorter amino acid side chains and thus may alter the relative orientation of the N- and D1-domains of VCP.
Protein-binding studies
We studied the binding of recombinant VCP to various ERAD-VCP-co-factors and other known ligands. Our GST pull-down assays demonstrated that wt-VCP as well as all three VCP mutants showed identical binding to Ufd1- (Fig. 12), Npl4- and ataxin-3 (data not shown). Furthermore, we tested whether purified wild-type and mutant VCP lacking the GST-tag can form insoluble aggregates in vitro. The formation of SDS-insoluble VCP aggregates was monitored by the filter retardation assay (Wanker et al., 1999). These experiments showed that neither mutant VCP nor wild-type VCP formed SDS-insoluble aggregates in vitro (data not shown).
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Virtual screening of ligand databases
We used the automated docking programme LIDAEUS to screen a small-molecule database for potential ligands for the two putative R93 and R155 ligand-binding pockets. The virtual screening approach considering docking and chemical interaction indicated that ADP/ATP are unlikely binding partners for the R155 site, but identified a steroid (16
-hydroxypregnenolone; Sigma H8252; SPH1-005-061) as well as a hexose-like compound (N-acetyl--D-glucosamine-1-phosphate disodium salt; Sigma A2142; SPH1-000-376) as ligands with significantly higher binding scores than all other target screened compounds (Fig. 13A). Although the R155 pocket is not a typical steroid binding pocket (Tanenbaum et al., 1998; Williams and Sigler, 1998; Bledsoe et al., 2002; Li et al., 2005), some of the VCP:SPH1-005-061 interacting residues are analogous to the amino acid–steroid contacts made by the steroid receptors.
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For the R93 pocket screening yielded a number of putative target molecules. The highest scoring compounds were the cyclic sugars (
-D-glucose-1-phosphate disodium salt; Sigma G7000; SPH1-004-510 and -D-galacturonic acid-1-phosphate lithium salt; Sigma G4884; SPH1-004-402) (Fig. 13B). |
Discussion |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
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VCP mutation analysis in our three German IBMPFD patients revealed a novel heterozygous R93C mutation in Patient I and R155C and R155H mutations in Patients II and III, respectively. Our histopathological analysis revealed a broad spectrum of pathological changes in muscle reflecting different stages of disease progression in our three IBMPFD patients. Immunostaining using VCP antibodies demonstrated the presence of VCP-positive cytoplasmic aggregates, a phenomenon described previously (Watts et al., 2004
). Our analysis demonstrated that these VCP-positive aggregates also display positive ubiquitin staining. As a further novel finding we demonstrated the presence of VCP- and ubiquitin-positive nuclear inclusions in muscle. This aspect mirrors the brain pathology in IBMPFD, which is characterized by the presence of VCP- and ubiquitin-positive nuclear inclusions in neurons (Schröder et al., 2005). Our ultrastructural analysis demonstrated that both the cytoplasmic and the nuclear inclusions in IBMPFD muscle were composed of haphazardly arranged filaments.
In analogy to the aberrant desmin immunofluorescence staining, our ultrastructural studies revealed the presence of desmin-positive granulofilamentous material, the characteristic ultrastructural hallmark of primary desminopathies and myofibrillar myopathies (Schröder et al., 2003; Selcen et al., 2004; Bär et al. 2005). This finding indicates that, at least in advanced degenerative stages of IBMPFD, VCP mutations induce secondary alterations of the extrasarcomeric desmin cytoskeleton.
The post-mortem analysis of Patient II revealed a novel clinical aspect in IBMPFD. VCP mutations not only affect skeletal muscle but may also lead to a dilatative cardiomyopathy characterized by ubiquitin-positive cytoplasmic aggregates and nuclear inclusions. This novel aspect clearly warrants appropriate clinical awareness and repeated cardiological work-up in IBMPFD patients.
Our immunoblotting analyses revealed no significant differences in the total amount, subcellular distribution and post-translational modifications of VCP protein between normal and diseased muscle. In contrast to IBMPFD muscle, where endogenous VCP is solely present in the insoluble fraction, western blot analysis of transfected cells (wt and mutant VCP) demonstrated that endogenous and transfected VCP is predominately present in the soluble protein fraction.
In contrast to IBMPFD muscle our transient and stable transfection experiments using mutant and wt VCP did not lead to cytoplasmic or nuclear protein aggregate formation. In line with previous studies (Meriin et al. 1998), aggresome formation could be elicited using the proteasome inhibitor MG132 in HEK293 cells, but no apparent differences in the extent or subcellular localization of aggresomes were noted in cells either transfected with wt or mutant VCP. All other stress experiments as well as differentiation of C2F3 myoblasts into myotubes did not provide any evidence suggesting abnormal protein aggregate formation.
These findings are in contrast to the recently published study by Weihl et al. (2006), who described large perinuclear aggregates in up to 33% of cells transfected with R155H-VCP-GFP and R95G-VCP-GFP and in 7% of cells transfected with wt-VCP-GFP. These aggregates were reported to contain poly-ubiquitinated proteins; a subgroup also mutated VCP protein. A potential cause for aggregates in such transient transfection experiments may be strong overexpression of the transfected gene. This is strongly supported by the following reasons. (i) A previous study by Ye et al. (2004) demonstrated strikingly similar perinuclear aggregates in double transient transfection studies using His-wt-VCP and Myc-wt-VIMP, a membrane protein that recruits the VCP-ATPase. (ii) In our immunoblot-controlled stable transfections a ratio of 1:3 of mutant VCP to wt VCP did not lead to protein aggregate formation. (iii) In primary human myoblasts derived from IBMPFD skeletal muscle, which most closely represent the physiological situation with one wt and one mutated VCP allele, no abnormal protein aggregation was detected by VCP and FK2 immunostaining. (iv) In the vast majority of IBMPFD patients, it takes at least 40–50 years until the disease manifests and protein aggregates are exclusively found in post-mitotic cells (neurons, striated muscle cells). Taken together, these findings strongly implicate that these cell culture models are of very limited value in studying IBMPFD-associated protein aggregate formation.
VCP and its co-factors Ufd1 and Npl4 are part of the ERAD pathway, which has a crucial role in removing misfolded proteins from the endoplasmic reticulum (Lederkremer and Glickman, 2005). In GST pull-down experiments no detectable changes in the binding of recombinant Ufd1 and Npl4 to wt and mutant VCP could be observed. Moreover, binding analysis of wt and mutant VCP to Udf1, Npl4 and ataxin-3, well-established VCP-ligands, showed identical results. Furthermore, in a previous study, the ATPase-activity of purified R155H-VCP was similar to the one reported for wt-VCP (Weihl et al., 2006). These results imply that the various VCP mutants analysed so far are not associated with gross alterations in VCP binding to known co-factors and its intrinsic ATPase enzyme activity. This favours the hypothesis that IBMPFD pathology is due to a toxic gain of VCP function.
All three VCP mutations identified in our IBMPFD patients affect evolutionarily highly conserved arginine residues in the CDC48 domain of the VCP protein. R93 and R155 are both surface-accessible residues (in monomeric and hexameric VCP) located in the centre of cavities that may enable ligand binding. R155 interacts with amino acid residue N387 residing in the D1-domain, which binds and hydrolyses ATP. The mutations R155C and R155H lack the interaction with N387 and thus may alter the relative orientation of the N- and D1-domains of VCP. DeLaBarre and Brünger (2003) proposed a model where the mobile state of the N-domain is triggered by release of a latch provided by the D1-domain. Three pairs of residues can be identified that could act as latch. Importantly, one of these pairs is R155-N387. A VCP mutant with an impaired ability to lock the N-domain is very likely to have lost its regulatory properties. R93 maintains interactions with amino acid residues E194 and R65, as well as with the backbone carbonyl group of N90. The R93C mutation leads to a loss of these contacts due to the shorter side chain of cystein.
Our screening of a small-molecule database identified a steroid (16-hydroxypregnenolone) as well as a hexose-like compound (N-acetyl--D-glucosamine-1-phosphate) and the cyclic sugar compounds -D-glucose-1-phosphate and -D-galacturonic acid-1-phosphate for the R155 and R93 binding pockets, respectively. The appearance of sugar-like compounds for the R93 site establishes a link to the reported interactions between SCF(Fbs1,2), a cytosolic ubiquitinase for glycoproteins and VCP (Yoshida et al., 2005). Here, it is tempting to speculate that VCP mutations interfere with the binding to carbohydrates from misfolded glycoproteins in the endoplasmic reticulum and cytosol (Spiro, 2002).
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Footnotes |
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*These authors have contributed equally to this work.
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Acknowledgements |
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We thank Wolfram Kress (Institute of Human Genetics, University of Würzburg, Germany), for desmin and
ß-crystalline mutation analysis in Patient II. Furthermore, the excellent technical assistance of Ms K. Kappes-Horn, Ms M. Stepien-Mering, Ms S. Plassmann, Ms M. Schmuck and Ms E. Schmidtmeyer is gratefully acknowledged. R. Schröder, H. Lochmüller and B. Schoser are members of the German network on muscular dystrophies (MD-NET; 01GM0302) funded by the German ministry of education and research (BMBF, Bonn, Germany). Human myoblast cultures were obtained from the Muscle Tissue Culture Collection (Friedrich-Baur-Institute, Munich, Germany), part of the MD-NET (service structure S1), partner of EuroBioBank (www.eurobiobank.org). We thank P. Taylor and M. Walkinshaw (Edinburgh) for access to the virtual screening software. |
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