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Identification and Characterisation of Novel Marker Proteins Involved in X-Linked Muscular Dystrophy

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Identification and Characterisation of Novel Marker Proteins Involved in X-Linked Muscular Dystrophy Identification and characterisation of novel marker proteins involved in X-linked muscular dystrophy Submitted to National University of Ireland Maynooth for the degree of Doctor of Philosophy. Caroline Lewis, B.Sc July 2010 Head of Department Supervisor Professor Kay Ohlendieck, Professor Kay Ohlendieck, Department of Biology, Department of Biology, NUI Maynooth, NUI Maynooth, Co. Kildare Co. Kildare Publications (peer reviewed) Lewis C. and Ohlendieck K. 2010. Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse. Biochem Biophys Res Commun; 396(4): 1024-1029 Lewis C. Jockusch, H. and Ohlendieck K. 2010. Proteomic profiling of the dystrophin- deficient mdx heart reveals drastically altered levels of key metabolic and contractile proteins. J Biomed Biotechnol; Volume 2010, article ID 648501, 20 pages Lewis C. and Ohlendieck K. 2010. Mass spectrometric identification of dystrophin isoform Dp427 by on-membrane digestion of sarcolemma from skeletal muscle. Anal Biochem. Volume 404; 197-203 Review (peer reviewed) Lewis C. Carberry, S. and Ohlendieck K. 2009. Proteomic profiling of X-linked muscular dystrophy. J Muscle Res Cell Motil.;30(7-8):267-9. Review Conferences and Presentations Poster presentation Life Sciences 2007, 8-12 July 2007 , SECC, Glasgow, UK. Poster number: PC524. Title: Optimisation of 2-D gel electrophoretic separation of dystrophic cardiac muscle Internal seminars presented to peers in NUI maynooth 2007 Identification and characterisation of novel biomarkers of dystrophic tissue 2008 Identification and characterisation of novel biomarkers of dystrophic tissue I Table of Contents Table of Contents II List of Figures VI List of Tables VIII Acknowledgements IX Declaration XI Abbreviations XII Abstract XVI 1 INTRODUCTION 1 1.1 Contractile muscle structure 1 1.2 Sliding filament mechanism 2 1.3 Skeletal muscle excitation­contraction coupling 4 1.4 The cardiac action potential 6 1.5 Cardiac excitation­contraction mechanism 6 1.6 Dystrophin 7 1.7 Dystrophinopathies 9 1.8 Dystroglycan complex 11 1.9 Animal model of Duchene muscular dystrophy 14 1.10 Proteomics 15 1.10.1 Sample preparation 15 1.10.2 2D gel electrophoresis 16 1.10.3 Western blotting 18 1.11 Difference in­gel electrophoresis 18 II 1.12 Visualisation 19 1.13 Identification 20 1.13.1 In‐gel digestion 20 1.13.2 Digestion on nitrocellulose 21 1.13.3 Electrospray ionisation mass spectrometry 21 1.14 Student’s t­test 22 1.15 Aims of proposed project 22 2 MATERIALS AND METHODS 24 2.1 Materials 24 2.1.1 General reagents 24 2.1.2 1D and 2D gel electrophoresis 24 2.1.3 Protein staining 24 2.1.4 Mass spectrometry 25 2.1.5 Western blotting 25 2.1.6 Immunofluorescence microscopy 28 2.2 Methods 29 2.2.1 Dystrophic MDX animal model 29 2.2.2 Preparation of total crude muscle extracts 29 2.2.3 Fluorescence difference in‐gel electrophoretic analysis 30 2.2.4 Protein visualization and data analysis 31 2.2.5 Mass spectrometric identification of cardiac and extraocular proteins 32 2.2.6 Immunoblot analysis 33 2.2.7 Immunofluorescence microscopy 33 2.2.8 1D gel electrophoresis for subsequent digestion 34 2.2.9 On‐membrane digestion 34 2.2.10 Mass spectrometric identification of on‐membrane digestion 35 3 PROTEOMIC PROFILING OF THE DYSTROPHIN-DEFICIENT MDX HEART REVEALS DRASTICALLY ALTERED LEVELS OF KEY METABOLIC AND CONTRACTILE PROTEINS 37 3.1 Introduction 37 III 3.1.1 Duchenne muscular dystrophy and the heart 38 3.1.2 Proteomics and the heart 39 3.1.3 Experimental design 41 3.2 Results 41 3.2.1 DIGE analysis of the dystrophin‐deficient heart 45 3.2.2 Decreased proteins in dystrophic heart muscle 56 3.2.3 Immunoblot analysis of dystrophic heart muscle 58 3.2.4 Immunofluorescence microscopy analysis of dystrophic heart muscle 62 3.3 Discussion 67 3.3.1 Drastically increased concentration of proteins by DIGE analysis 69 3.3.2 Drastically decreased concentration of protein by DIGE analysis 70 3.3.3 Decreased concentration of cytoskeletal proteins 70 3.3.4 Decreased expression level of intermediate filament proteins 71 3.3.5 Decreased concentration of mitochondrial proteins as identified by DIGE analysis 72 3.3.6 Other proteins 74 3.4 Conclusion 75 4 PROTEOMIC PROFILING OF NATURALLY PROTECTED EXTRAOCULAR MUSCLES FROM THE DYSTROPHIN-DEFICIENT MDX MOUSE 77 4.1 Introduction 77 4.1.1 Duchenne muscular dystrophy and the extraocular muscle 77 4.1.2 Properties of extraocular muscle 78 4.1.3 Proteomics and extraocular muscle 79 4.1.4 Experimental design 80 4.2 Results 81 4.2.1 Comparative proteomic analysis of MDX versus normal extraocular muscle 81 4.2.2 DIGE analysis of dystrophin‐deficient extraocular muscle 83 4.2.3 Immunoblot analysis of dystrophin‐deficient extraocular muscle 86 4.3 Discussion 93 4.3.1 Moderate expression changes of DIGE EOM analysis 93 4.3.2 Immunoblotting survey of MDX EOM tissue 94 4.3.3 Perturbed stress response in MDX EOM tissue 95 IV 4.4 Conclusion 95 5 MASS SPECTROMETRIC IDENTIFICATION OF DYSTROPHIN ISOFORM DP427 BY ‘ON-MEMBRANE’ DIGESTION OF SARCOLEMMA FROM SKELETAL MUSCLE 97 5.1 Introduction 97 5.1.1 The sarcolemmal and associated proteins 98 5.1.2 Advantages of on‐membrane digestion 99 5.2 Results 99 5.2.1 Subcellular fractionation 99 5.2.2 SDS‐PAGE fractionation 103 5.2.3 Sarcolemma‐enriched fraction 107 5.2.4 Dystrophin‐glycoprotein complex fraction 112 5.3 Discussion 116 5.3.1 Modified proteomics study 116 5.3.2 ‘On‐membrane’ digestion 117 5.4 Conclusion 117 6 GENERAL DISCUSSION 118 Bibliography 124 V List of Figures Figure 1­1 Contractile muscle structure ..................................................................................................................................................2 Figure 1­2 Sliding filament theory..............................................................................................................................................................4 Figure 1­3 Voltage­gated action potential..............................................................................................................................................5 Figure 1­4 Dystrophin and Utrophin domain structures ..................................................................................................................9 Figure 1­5 Synopsis of neuromuscular disorders with primary abnormalities in the dystrophin­glycoprotein complex................................................................................................................................................................................................................ 11 Figure 1­6 Overview of the structure of the dystrophin­glycoprotein complex form skeletal muscle and its involvement in the molecular pathogenesis of X­linked muscular dystrophy. ..................................................................... 13 Figure 1­7 DIGE image of separated cardiac muscle....................................................................................................................... 19 Figure 3­1 2­D DIGE analytical gel of normal versus MDX cardiac muscle in the pH 4­7 range. ................................ 43 Figure 3­2 2­D DIGE analytical gel of normal versus MDX cardiac muscle in the pH 6­11 range............................... 44 Figure 3­3 DIGE analysis of normal versus dystrophic MDX cardiac muscle........................................................................ 55 Figure 3­4 Decreased expression of key proteins in the dystrophic­deficient MDX heart ............................................... 57 Figure 3­5 Decreased expression of key proteins with 3D comparative images ................................................................. 57 Figure 3­6 Immunoblotting survey of equally loaded cardiac tissue ....................................................................................... 59 Figure 3­7 Immunoblot analysis of filament protein....................................................................................................................... 59 Figure 3­8 Immunoblotting survey of cardiac mitochondrial proteins in dystrophic tissue ......................................... 60 Figure 3­9 Immunoblot analysis of cardiac stress regulatory proteins .................................................................................. 61 Figure 3­10 Immunofluorescence survey of mitochondrial content and nuclei number in dystrophic cardiac tissue ..................................................................................................................................................................................................................... 64 Figure 3­11 Immunofluorescence survey of cardiac marker proteins in dystrophic cardiac tissue........................... 65 Figure 3­12 Immunofluorescence survey of cardiac mitochondrial marker proteins in dystrophic cardiac tissue ................................................................................................................................................................................................................................. 66 Figure 3­13 Overview of biological functions of DIGE­identified proteins with an altered expression in dystrophic heart muscle ............................................................................................................................................................................... 68 Figure 4­1 Diagrammatic representation of the extraocular muscles .................................................................................... 79 Figure 4­2 2­D DIGE analytical
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