Abstract
Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.
Highlights
From the ‡Department of Neurology, Neuromuscular Center Ruhrgebiet, University Hospital Bergmannsheil, Ruhr-University Bochum, Germany; §Department of Functional Proteomics, Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany; ¶Institute for Cell Biology, University of Bonn, Bonn, Germany; ʈDepartment of Medical Proteomics/Bioinformatics/Biostatistics, Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany; **Department of Medical Proteomics/Bioanalytics, Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany; ‡‡Division of Neuropediatrics and Muscle Disorders, University ChildrenЈs Hospital Freiburg, Germany; §§J.W
For recombinant expression in E. coli FLNc cDNA fragments containing Ig-like domains 18 –19, 18 –21, and 20 –21 were cloned into pET23-EEF [28] whereas cDNA fragments encoding the carboxyterminus of Xin actinbinding repeat containing protein 2 (Xirp2) were cloned in pET23-T7 or pET23myc, a newly constructed vector based on pET23-T7, with a c-myc tag instead of the T7 tag
To fill this gap we applied a workflow consisting of laser microdissection and label-free spectral count based proteomics for samples of filaminopathy patients (Fig. 1)
Summary
From the ‡Department of Neurology, Neuromuscular Center Ruhrgebiet, University Hospital Bergmannsheil, Ruhr-University Bochum, Germany; §Department of Functional Proteomics, Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany; ¶Institute for Cell Biology, University of Bonn, Bonn, Germany; ʈDepartment of Medical Proteomics/Bioinformatics/Biostatistics, Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany; **Department of Medical Proteomics/Bioanalytics, Medizinisches Proteom-Center, Ruhr-University Bochum, Bochum, Germany; ‡‡Division of Neuropediatrics and Muscle Disorders, University ChildrenЈs Hospital Freiburg, Germany; §§J.W. The latter may give us important new insights in the pathogenesis of MFM. For example, abundance changes have been detected for proteins involved in metabolism, contractile activity, myofibrillar remodeling, and stress response [12]. In biomedical research global studies focused on the identification of novel panels of protein biomarker candidates for neuromuscular diseases: Analyses of muscle tissue of the dystrophin deficient mouse model of Duchenne muscular dystrophy (mdx mouse) identified altered levels of protein biomarkers involved in nucleotide metabolism, cellular stress response, energy metabolism, and ion handling (for review see [13]). Significant alterations of proteins playing an important role in various metabolic pathways were found in a mouse model for hypokalemic myopathy [14]. A comparison of protein expression in sIBM to non-IBM inflammatory myopathies indicated an impairment of detoxification, energy metabolism, and protein folding in sIBM [16]
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