Central Protein Analysis

• Applicant: Professor Dr. Franz-Georg Hanisch
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2009 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 101925924

• Final Report Year: 2017

Final Report Abstract

The CMMC-Bioanalytics (CBA, University of Cologne) and the Department of Functional Proteomics (Ruhr University, Bochum) served as a joined Z-project during the second funding period responsible for mass spectrometry-based protein analysis and for the assistance in all workflows related to the topics of the FOR 1228. We provided proteome analysis platforms and developed analysis strategies for muscle related proteome studies. Our most important achievements were: a) The establishment of a laser-microdissection mass spectrometry (LMD-MS) workflow for the proteomic analysis of aggregates in different MFM subforms and other neuromuscular diseases. The establishment of the LMD-MS enabled a specific investigation of subcellular protein aggregates in MFM. By analysis of aggregates from more than 100 patients suffering from FHL1-associated myopathy, desminopathy, myotiliopathy and filaminopathy (and the respective control tissue) we found that different MFM forms share similar pathomechanisms. The proteomics results indicate that plaques from different patients exhibit protein compositions with partial overlap, on the one hand, and mutation-dependent protein contents on the other. Additionally, the aggregate protein pattern supports differential diagnosis of MFM subtypes and filamin C ratios may be a suitable diagnostic biomarker for filaminopathy. Additionally, our proteomic data confirm the underlying gene defect with an intrafamilial variability by the ratio of the total protein content in the aggregates in FHL1-associated myopathy. b) Adaptation of the LMD-MS workflow for skeletal muscle fibre-type specific proteome analysis. As the protein distribution in skeletal muscle fiber types is unknown so far and protein aggregation myopathies predominantly show a prevalence of pathologic hallmarks in type I muscle fibers it is of interest to get insights into differences in the proteome of different fiber types. Thus, we aimed at a specific analysis of type I and type II fibers in mice. For this we adapted the LMD-MS-workflow for the specific isolation of both fiber types from WT as well as hetero- and homozygous R349P desmin knockin mice. Mitochondrial defects were identified in type I fibers of the homozygeous R349P desmin knockin mice whereas the effects were less pronounced in heterozygeous mice. Our developments open new possibilities for the molecular characterisation of skeletal muscle and will improve our knowledge about pathomechanisms of muscle diseases.

Publications

• Evaluation of standardized protein pattern of skeletal muscle fibers via laser microdissection. Neuromuscul Disord 2011; 21: 739-740
  Feldkirchner S, Mueller S, Kress W, Hanisch F-G, Schoser B, Schessl J
  
• The C224W FHL1 mutation is causing a protein aggregation disorder of muscle: Two brothers revisited. Neuromuscul Disord 2011; 21: 740-740
  Feldkirchner, S, Walter MC, Kubny C, Mueller S, Kress W, Hanisch F-G, Schoser B, Schessl J
  
• Patient-specific protein aghgregates in myofibrillar myopathies: laser microdissection and differential proteomics for identification of plaque components. Proteomics 2012; 12: 3598-3609
  Feldkirchner S, Schessl J, Müller S, Schoser B, Hanisch F-G
  
• A combined laser microdissection and mass spectrometry approach reveals new disease relevant proteins accumulating in aggregates of filaminopathy patients, Mol Cell Proteomics 2013; 12: 215-227
  Kley RA, Maerkens A, Leber Y, Theis V, Schreiner A, van der Ven PFM, Uszkoreit J, Stephan C, Eulitz S, Euler N, Kirschner J, Müller K, Meyer HE, Tegenthoff M, Fürst DO, Vorgerd M, Müller T, Marcus K
  
• Differential proteomic analysis of abnormal intramyoplasmic aggregates in desminopathy, J Proteomics 2013; 90, 14-27
  Maerkens A, Kley RA, Olivé M, Theis V, van der Ven PF, Reimann J, Milting H, Schreiner A, Uszkoreit J, Eisenacher M, Barkovits K, Güttsches AK, Tonillo J, Kuhlmann K, Meyer HE, Schröder R, Tegenthoff M, Fürst DO, Müller T, Goldfarb LG, Vorgerd M, Marcus K
  
• Proteomic characterization of aggregate components in an intrafamilial variable FHL1- associated myopathy. Neuromuscul Disord 2013; 12: 1764-1771
  Feldkirchner S, Walter MC, Müller S, Kubny C, Krause S, Kress W, Hanisch F-G, Schoser B, Schessl J
  
• Novel recessive myotilin mutation causes severe myofibrillar myopathy. Neurogenetics 2014; 15: 151-156
  Schessl J, Bach E, Rost S, Feldkirchner S, Kubny C, Müller S, Hanisch F-G, Kress W, Schoser B
  
• Myofibrillar instability exacerbated by acute exercise in filaminopathy, Hum Mol Genet 2015; 24(25), 7207-7220
  Chevessier F, Schuld J, Orfanos Z, Plank AC, Wolf L, Maerkens A, Unger A, Schlötzer-Schrehardt U, Kley RA, von Hörsten S, Marcus K, Linke WA, Vorgerd M, van der Ven PF, Fürst DO, Schröder R
  
• VCP and PSMF1: Antagonistic regulators of proteasome activity. Biochem. Biophys. Res. Commun. 2015; 7: 1210-1217
  Clemen CS, Marko M, Strucksberg KH, Behrens J, Wittig I, Gärtner L, Winter L, Chevessier F, Matthias J, Türk M, Tangavelou K, Schütz J, Arhzaouy K, Klopffleisch K, Hanisch F-G, Rottbauer W, Blümcke I, Just S, Hofmann A, Schröder R
  
• Mutant desmin substantially perturbs mitochondrial morphology, function and maintenance in skeletal muscle tissue. Acta Neuropathol 2016; 132: 453-73
  Winter L, Wittig I, Peeva, V, Eggers B, Heidler J, Chessevier F, Kley RA, Barkovits K, Strecker V, Berwanger C, Herrmann H, Marcus K, Kornblum C, Kunz WS, Schröder R, Clemen CS
  
• New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses. Acta Neuropathol Commun 2016; 4: 8
  Maerkens A, Olivé M, Schreiner A, Feldkirchner S, Schessl J, Uszkoreit J, Barkovits K, Güttsches AK, Theis V, Eisenacher M, Tegenthoff M, Goldfarb LG, Schröder R, Schoser B, van der Ven PF, Fürst DO, Vorgerd M, Marcus K, Kley RA
  
• Inclusion body myositis: Deciphering rimmed vacuoles and detection of a genetic risk factorMutant desmin substantially perturbs mitochondrial morphology, function and maintenance in skeletal muscle tissue. Ann Neurol 2017; 81: 227-239
  Güttsches AK, Brady S, Krause K, Maerkens A, Uszkoret J, Eisenacher M, Schreiner A, Galozzi S, Mertens-Rill J, Tegenthoff M, Holton JL, Harms MB, Lloyd, TE, Vorgerd M, Weihl CC, Marcus K, Kley R