A Combined Laser Microdissection and Mass Spectrometry Approach Reveals New Disease Relevant Proteins Accumulating in Aggregates of Filaminopathy Patients*

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.

[1]  Baogang J. Xu Combining laser capture microdissection and proteomics: Methodologies and clinical applications , 2010, Proteomics. Clinical applications.

[2]  D. Selcen Myofibrillar myopathies , 2011, Neuromuscular Disorders.

[3]  C. Faul,et al.  Protein Kinase A, Ca2+/Calmodulin-Dependent Kinase II, and Calcineurin Regulate the Intracellular Trafficking of Myopodin between the Z-Disc and the Nucleus of Cardiac Myocytes , 2007, Molecular and Cellular Biology.

[4]  Keith Dudley Short protocols in molecular biology , 1990 .

[5]  S. Simon,et al.  Myopathy-associated αB-crystallin Mutants , 2007, Journal of Biological Chemistry.

[6]  Mike P. Wattjes,et al.  Neuromuscular imaging in inherited muscle diseases , 2010, European Radiology.

[7]  D. Parry,et al.  A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin. , 1999, The Journal of biological chemistry.

[8]  K. Marcus,et al.  The AICD interacting protein DAB1 is up-regulated in Alzheimer frontal cortex brain samples and causes deregulation of proteins involved in gene expression changes. , 2011, Current Alzheimer research.

[9]  B. Tang,et al.  Rab35 – A vesicular traffic‐regulating small GTPase with actin modulating roles , 2010, FEBS letters.

[10]  K. Ohlendieck,et al.  Proteomic profiling of x-linked muscular dystrophy , 2009, Journal of Muscle Research and Cell Motility.

[11]  U. Moens,et al.  Heat shock protein 27 phosphorylation: kinases, phosphatases, functions and pathology , 2009, Cellular and Molecular Life Sciences.

[12]  I. Ferrer,et al.  In-frame deletion in the seventh immunoglobulin-like repeat of filamin C in a family with myofibrillar myopathy , 2009, European Journal of Human Genetics.

[13]  D. Parry,et al.  A High Molecular Weight Intermediate Filament-associated Protein in BHK-21 Cells Is Nestin, a Type VI Intermediate Filament Protein , 1999, The Journal of Biological Chemistry.

[14]  J. Duarte,et al.  Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle , 2010, Proteomics.

[15]  S. Kempa,et al.  Indications for a Novel Muscular Dystrophy Pathway , 2000, Journal of Cell Biology.

[16]  B. Sitek,et al.  Identification of Proteomic Differences between Squamous Cell Carcinoma of the Lung and Bronchial Epithelium*S , 2009, Molecular & Cellular Proteomics.

[17]  Hsuan-Ting Huang,et al.  Myomaxin Is a Novel Transcriptional Target of MEF2A That Encodes a Xin-related α-Actinin-interacting Protein* , 2006, Journal of Biological Chemistry.

[18]  Helmut E Meyer,et al.  Sense and nonsense of pathway analysis software in proteomics. , 2011, Journal of proteome research.

[19]  M. Gautel,et al.  The structure of the sarcomeric M band: localization of defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy , 1996, The Journal of cell biology.

[20]  A. Engel,et al.  Myofibrillar myopathy caused by novel dominant negative alpha B-crystallin mutations. , 2003, Annals of neurology.

[21]  N. Marceau,et al.  Distinct chaperone mechanisms can delay the formation of aggresomes by the myopathy-causing R120G alphaB-crystallin mutant. , 2003, Human molecular genetics.

[22]  Andrew G Engel,et al.  Mutations in myotilin cause myofibrillar myopathy , 2004, Neurology.

[23]  C. Garrido,et al.  Heat Shock Proteins: Cell Protection through Protein Triage , 2010, TheScientificWorldJournal.

[24]  I. Ferrer,et al.  Molecular pathology of myofibrillar myopathies , 2008, Expert Reviews in Molecular Medicine.

[25]  C. Glabe,et al.  Protective Effect of Geranylgeranylacetone via Enhancement of HSPB8 Induction in Desmin-Related Cardiomyopathy , 2009, PloS one.

[26]  Visith Thongboonkerd,et al.  Proteomic identification of altered proteins in skeletal muscle during chronic potassium depletion: Implications for hypokalemic myopathy. , 2006, Journal of proteome research.

[27]  K. Blennow,et al.  cNEUPRO: Novel Biomarkers for Neurodegenerative Diseases , 2010, International journal of Alzheimer's disease.

[28]  J. Hartwig,et al.  The small GTPase RalA targets filamin to induce filopodia. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Kai A Reidegeld,et al.  An easy‐to‐use Decoy Database Builder software tool, implementing different decoy strategies for false discovery rate calculation in automated MS/MS protein identifications , 2008, Proteomics.

[30]  G. Klöppel,et al.  Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma , 2005, Proteomics.

[31]  M. Olivé Extralysosomal Protein Degradation in Myofibrillar Myopathies , 2009, Brain pathology.

[32]  Stefan Eulitz,et al.  Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VASP. , 2006, Experimental cell research.

[33]  A. Vortmeyer,et al.  Proteomic Analysis of Inclusion Body Myositis , 2006, Journal of neuropathology and experimental neurology.

[34]  U. Vinkemeier,et al.  The globular head domain of titin extends into the center of the sarcomeric M band. cDNA cloning, epitope mapping and immunoelectron microscopy of two titin-associated proteins. , 1993, Journal of cell science.

[35]  A. Engel,et al.  Myofibrillar myopathy caused by novel dominant negative αB‐crystallin mutations , 2003 .

[36]  K. Ohlendieck Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques , 2011, Skeletal Muscle.

[37]  C. Heyer,et al.  Clinical and morphological phenotype of the filamin myopathy: a study of 31 German patients. , 2007, Brain : a journal of neurology.

[38]  W. Rottbauer,et al.  Nexilin mutations destabilize cardiac Z-disks and lead to dilated cardiomyopathy , 2009, Nature Medicine.

[39]  B. Ozanne,et al.  New N-RAP-binding partners α-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly , 2003, Journal of Cell Science.

[40]  M. Chiesi,et al.  Alpha B-crystallin in cardiac tissue. Association with actin and desmin filaments. , 1992, Circulation research.

[41]  Hanns Lochmüller,et al.  A mutation in the dimerization domain of filamin c causes a novel type of autosomal dominant myofibrillar myopathy. , 2005, American journal of human genetics.

[42]  K. Kato,et al.  Purification and characterization of a 20-kDa protein that is highly homologous to alpha B crystallin. , 1994, The Journal of biological chemistry.

[43]  Cecilia Gelfi,et al.  Comparative proteomic profile of rat sciatic nerve and gastrocnemius muscle tissues in ageing by 2‐D DIGE , 2009, Proteomics.

[44]  K. Claeys,et al.  Differential involvement of sarcomeric proteins in myofibrillar myopathies: a morphological and immunohistochemical study , 2009, Acta Neuropathologica.

[45]  J. Golden,et al.  Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy. , 2008, The Journal of clinical investigation.

[46]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[47]  S. Simon,et al.  Myopathy-associated alphaB-crystallin mutants: abnormal phosphorylation, intracellular location, and interactions with other small heat shock proteins. , 2007, Journal of Biological Chemistry.

[48]  P. Cerretelli,et al.  Proteomic investigation of the molecular pathophysiology of dysferlinopathy , 2006, Proteomics.

[49]  K. Davies,et al.  Association of syncollin and desmin: Linking intermediate filament proteins to the dystrophin-associated protein complex. , 2002 .

[50]  R. Kley,et al.  The sarcomeric Z-disc component myopodin is a multiadapter protein that interacts with filamin and alpha-actinin. , 2010, European journal of cell biology.

[51]  M. Vorgerd,et al.  The pathomechanism of filaminopathy: altered biochemical properties explain the cellular phenotype of a protein aggregation myopathy. , 2007, Human molecular genetics.

[52]  Wei Zhang,et al.  A novel heterozygous deletion–insertion mutation (2695–2712 del/GTTTGT ins) in exon 18 of the filamin C gene causes filaminopathy in a large Chinese family , 2010, Neuromuscular Disorders.

[53]  Y. Capetanaki,et al.  Desmin in muscle formation and maintenance: knockouts and consequences. , 1997, Cell structure and function.

[54]  M. Prevost,et al.  A missense mutation in the αB-crystallin chaperone gene causes a desmin-related myopathy , 1998, Nature Genetics.

[55]  M. Willingham,et al.  A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. II. Effects on cell movement, organization of microtubules, and intermediate filaments, and arrangement of Golgi elements , 1983, The Journal of cell biology.

[56]  C. Heyer,et al.  Distinct muscle imaging patterns in myofibrillar myopathies , 2008, Neurology.