Functional protein networks unifying limb girdle muscular dystrophy
Morrée, A. de
Citation
Morrée, A. de. (2011, January 12). Functional protein networks unifying limb girdle muscular dystrophy. Retrieved from https://hdl.handle.net/1887/16329
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/16329
Note: To cite this publication please use the final published version (if
applicable).
Functional protein networks unifying Limb Girdle Muscular
Dystrophy
Antoine de Morrée
Cover: Winterlandschap met schaatsers, by Hendrick Avercamp (1585-1634) Reprinted with kind permission of the Rijksmuseum.
Layout by E Savelkoul, CV de Morrée and A de Morrée Printing by Off Page, www.offpage.nl
ISBN 978-94-90371-60-9
Copyright 2010 by Antoine de Morrée. All rights reserved. Copyright of the individual chapters rests with the authors, with the following exceptions:
Chapers 2 and 4: Public Library of Science Chapter 3: Oxford University Press
No part of this book may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means, without prior permission of the author.
Functional protein networks unifying Limb Girdle Muscular Dystrophy
Proefschrift ter verkrijging van
de graad van Doctor aan de Universiteit Leiden,
op gezag van Rector Magnificus prof.mr. P.F. van der Heijden, volgens besluit van het College voor Promoties
te verdedigen op woensdag 12 januari 2011 klokke 16.15 uur
door
Antoine de Morrée
geboren te Utrecht in 1982
Promotiecommissie
Promotores: Prof. Dr. Ir. Silvère M van der Maarel
1Prof. Dr. Rune R Frants
1Overige leden: Prof. Dr. Isabel Illa
2Prof. Dr. Peter ten Dijke
3Prof. Dr. Rene Toes
41 Afdeling Humane Genetica, Leids Universitair Medisch Centrum, Leiden, Nederland
2 Laboratori de Neurologia Experimental, Universitat Autònoma de Barcelona, Spanje.
3 Afdeling Moleculaire Cel Biologie, Leids Universitair Medisch Centrum, Leiden, Nederland
4 Afdeling Reumatologie, Leids Universitair Medisch Centrum, Leiden, Nederland
The studies described in this thesis have been performed at the Leiden University Medical Center, department of human genetics. This work was financially supported by the Dutch Prinses Beatrix Fonds (MAR05-0112) and the Jain Foundation.
Publication of this thesis was financially supported by the Dutch J.E. Jurriaanse Stichting and the Jain foundation.
“Avoid boring people”
James D. Watson
Table of contents
Abbreviations 11
Foreword 15
1. Protein networks unifying Limb Girdle Muscular Dystrophy 19
2. Proteomic analysis of the Dysferlin protein complex unveils its importance for sarcolemmal maintenance and
integrity 33
-Chapter 2 is currently in press, PLoS ONE-
3. Calpain 3 is a modulator of the Dysferlin protein complex in skeletal
muscle 63
-Chapter 3 has been published, HMG 2008-
4. Calpain 3 is a rapid-action, unidirectional proteolytic switch central to muscle remodeling 85
-Chapter 4 has been published, PLoS ONE 2010-
5. Self-regulated alternative splicing at
the AHNAK locus 105
6. AHNAK redistributes upon Integrin inhibition in cultured myoblasts 131
7. Crosstalk between Dysferlin and Integrin ß3 regulates cell contacts in
human monocytes 147
8. Maintenance and remodeling are central to skeletal muscle physiology and disturbed in Limb Girdle Muscular
Dystrophy 171
Supplement 189
References 213
Summary 237
Nederlandse samenvatting zonder
vakjargon 241
Curriculum Vitae 249
Publication list 251
Abbreviations
ACTN Actinin
ADP Adenosine diphosphate ANXA1 Annexin A1
ANXA2 Annexin A2 AP2 Adaptin 2
ATP Adenosine triphosphate ATP5b ATP Synthase ß Subunit AuC Area under the ROC Curve BCA Bicinchoninic acid protein
assay
BG Beta-Galatosidae-GFP fusion protein
CAPN3 CAPN3
CHAPS 3-[(3-cholamidopropyl)- dimethylammonio]- 1-propanesulfonate CLTA Clathrin alpha
CMT Charcot-Marie-Tooth disease, demyelinating
DACM Distal Anterior Compartment Myopathy
DAPI 4’,6-diamidino- 2-phenylindole
DAVID Database for Annotation, Visualization and Integrated
DGC Dystophin Glycoprotein Complex
DHPR Dihydropyridene Receptor DMD Dystrophin
DYSF Dysferlin ECL Enzymatic
Chemiluminescence ELISA Enzyme-Linked
ImmunoSorbent Assay ER Endopplasmic Reticulum FCS Fetal Calf Serum
Fer1 Ferlin Fer1L Ferlin-like FLNC Filamin C
FSHD Facioscapulohumeral muscular dystrophy GFP Green Fluorescent Protein GO Gene Ontology
GST Glutathione S-transferase HCAb Heavy Chain Antibody IGF2R Mannose-6 phosphate
receptor
IP Immunoprecipitation IPTG Isopropyl ß-D-
1-thiogalactopyranoside
KEGG Kyoto Encyclopedia of Genes and Genomes
L-AHNAK Large AHNAK isoform LGMD Limb Girdle Muscular
Dystrophy
L-PRX Large Periaxin isoform LVGCC L-Type Voltage-Gated
Calcium Channel MD Muscular Dystrophy
MG53 Matsagumin 53, also known as Trim72
MH Malignant hyperthermia MM Miyoshi Myopathy MS Mass Spectrometry MTJ Myotendinous Junctions NCBI National Center for
Biotechnology Information NES Nuclear Export Signal NLS Nuclear Localization Signal NP40 nonyl
phenoxypolyethoxylethanol OPMD Oculopharyngeal muscular
dystrophy
PABPN1 Poly(A) Binding Protein N1 PAGE Poly-Acrylamide Gel
Electrophoresis
PARVB Beta-Parvin
PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PIAS Protein Inhibitor of Activated
STATs
PKA(B)(C) Protein Kinase A(B)(C)
PRX Periaxin
PVDF Polyvinylidene difluoride ROC Receiver operating
characteristic RT Room Temperature S-AHNAK Small AHNAK isoform SC35 Spliceosome Component 35 SDS Sodium-Dodecyl-Sulphate S-PRX Small Periaxin isoform SUMO Small Unbiquitin-like Modifier TEA TriEthanolAmine
TLN Talin
TPM TropoMyosin TUBA Alpha-Tubulin
UMLS Unified Medical Language System
VHH Variable domain of a Heavy Chain Antibody
VINC Vinculin
One of the most versatile tissues of the human body is the muscle. There are three different types of muscle: cardiac muscle, smooth muscle and skeletal muscle.
Cardiac muscle cells make up the heart, which drives blood circulation. The smooth muscle cells are part of the blood vessel walls and aid blood flow by maintaining blood pressure. Finally, the skeletal muscle connects skeletal structures to allow movement of body parts in relation to one another.
Muscle tissue is highly adaptive, which is apparent in growth, exercise and degeneration and regeneration. During normal contractions it needs effective and efficient mechanisms to repair the continuous damage to the cytoskeletal anchors, the contractile apparatus, and the cellular membrane. When repair is not effective, the damaged fibers die and are rapidly cleared by immune cells. A new fiber will replace the former.
Each of these maintenance and repair processes can be affected by genetic mutations, resulting in a muscular dystrophy phenotype. Much can be learned about normal muscle function from such mutations. Such knowledge can be used in the development of treatments for these diseases. Limb Girdle Muscular Dystrophy (LGMD) is a rare heterogeneous disorder that can be caused by mutations in at least 21 different genes. These genes encode proteins with highly differing functions and yet mutations in all of them give rise to a similar clinical presentation.
In this thesis I will explore a potential molecular mechanism that unifies the different genetic defects that individually can cause a limb girdle muscular dystrophy. I will start with a thorough survey of recent literature, venturing the hypothesis that all forms of LGMD suffer from impaired muscle maintenance capacity (Chapter 1). I then describe several lines of experimental research that
Foreword
not clear which other proteins aid in its function, nor whether this is the only functionality of Dysferlin. To answer these questions a robust and reproducible method for purification of Dysferlin containing protein complexes was set up, yielding insight into which processes Dysferlin is involved (Chapter 2). The interaction with two binding partners, Calpain 3 (CAPN3) and AHNAK was further investigated, showing that CAPN3 modulates the direct interaction between Dysferlin and AHNAK through enzymatic cleavage of AHNAK (Chapter 3). CAPN3 is in addition to Dysferlin another protein to cause a LGMD phenotype. It is a proteolytic enzyme. Using the identified CAPN3 cleavage sites in AHNAK a general CAPN3 cleavage motif was uncovered, allowing for the identification of novel substrates and a functional model for this protein (Chapter 4). CAPN3 is central to muscle maintenance through cleavage of structural proteins. The other Dysferlin interaction partner, AHNAK, localizes to vesicles and binds structural proteins, and could link such two processes. As little is known about the AHNAK gene and protein family, Chapter 5 describes its evolution and transcriptional regulation in skeletal muscle. A function for AHNAK is explored by chemically inhibiting Integrins (Chapter 6). Muscle tissue strongly communicates with immune cells, which also express many skeletal muscle proteins, including Dysferlin and AHNAK. In Chapter 7 a monocyte-macrophage model system is used to indicate a role for Dysferlin and AHNAK in Integrin signaling and monocyte differentiation and phagocytosis, which are deregulated in LGMD. Finally, in Chapter 8 all the experimental conclusions are summarized, and held against the light of recent scientific literature, resulting in the conclusion that Dysferlin and CAPN3 are involved in muscle maintenance and remodeling. This Thesis ends with the description of possible experimental research lines that will help to continue this research.
