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A New Model for the Dystrophin Associated Protein Complex in Striated A new model for the dystrophin associated protein complex in striated muscles. DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Eric K. Johnson Graduate Program, The Ohio State Biochemistry Program The Ohio State University 2012 Dissertation Committee: Federica Montanaro , PhD, Advisor Denis C. Guttridge, PhD Muthu Periasamy, PhD Jill A. Rafael-Fortney, PhD Copyright by Eric K. Johnson 2012 Abstract Dystrophin is a large, cytoskeletal protein localized to the intracellular side of the muscle membrane, and is the central organizer of a large protein complex known as the dystrophin associated protein complex (DAPC). Through the DAPC, dystrophin links the actin cytoskeleton to the extracellular matrix and functions to stabilize the membrane from the forces generated by muscle contraction. However, further studies have also shown that dystrophin functions in intracellular signaling mediated through DAPC proteins. Absence of dystrophin destabilized the DAPC disrupting membrane integrity and muscle function ultimately leading to muscle damage and necrosis. Clinically, mutations in the dystrophin gene give rise a group of muscular dystrophies, termed the dystrophinopathies which represent the most common form of all muscular dystrophy. The functions of dystrophin are generally believed to be similar for all striated muscles. However, it has become clear that loss of dystrophin does not affect all muscles types equally, particularly the heart. Clinically, studies have shown that there is no correlation in disease severity or age of onset between cardiac and skeletal muscles. Coupled with additional lines of evidence it appears that dystrophin has unique tissue specific functions yet to be elucidated. Because the majority of dystrophin functions are facilitated though the DAPC, we ii have hypothesized that the tissue specific functions of dystrophin are mediated by unique protein interactions. In order to identify dystrophin associated proteins, we first developed a high throughput proteomics approach that combines dystrophin immunoprecipitation with downstream protein identification by shotgun mass spectrometry. Using this approach we identified major differences in the protein interactions of dystrophin between cardiac and skeletal muscle, including differences in the composition of known DAPC proteins. Furthermore, we identified novel cardiac-specific dystrophin associated proteins known to regulate cardiac contraction and to be involved in cardiac disease. From our studies in the heart, we next extended our approach to the study of dystrophin associated proteins in the diaphragm. In the mdx mouse the diaphragm is the most affected muscle and is the only muscle that closely resembles pathology seen in humans. Because of this, the diaphragm has been intensely studied in an attempt to understand this unique pathology. However the reasons for the more dramatic phenotype are not fully understood. Using our immunoprecipitation approach we show that in the diaphragm, dystrophin has a unique set of protein interactions compared to limb muscles. A subset of these proteins are involved in membrane repair. Therefore, this finding suggests for the first time a possible molecular basis for the more severe phenotype observed in the diaphragm. An important aspect of our approach is that it can easily be adapted to the study of different proteins. Specifically, we chose to study β-dystroglycan, the iii DAPC protein that anchors dystrophin to the membrane. Our current model of the DAPC is that in the absence of dystrophin, the DAPC is destabilized and lost from the sarcolemmal membrane in skeletal muscle. However, mutations affecting the functions of dystroglycan give rise to a much more severe pathology than loss of dystrophin, and in the absence of dystrophin, β-dystroglycan and some other DAPC proteins are still expressed at the membrane. Together this suggests that dystroglycan must have additional functions not mediated directly by the DAPC and may be mediated by yet unidentified protein complexes. Using a similar approach as to dystrophin we show that in skeletal muscle multiple complexes of β-dystroglycan exist and interact with a unique set of proteins compared to dystrophin. Importantly, although these complexes are distinct from the DAPC, a sub-set is disrupted upon loss of dystrophin. These findings suggest that three distinct β-dystroglycan complexes exist in skeletal muscle, and we propose a new model of β-dystroglycan function and organization in striated muscle. The studies presented here provide clear evidence that the DAPC is a dynamic protein complex with unique differences between individual muscle tissues. These novel differences suggest tissue specific functions of dystrophin in the heart and diaphragm. Furthermore, our studies on β-dystroglycan add additional complexity our understanding of components of the DAPC and highlight new possible functions of both β-dystroglycan and dystrophin. Importantly the technique described here overcomes a significant challenge in the field and offers a new method for studying dystrophin protein interactions. iv The generation of the method and the novel findings described here will likely prove vital to our understanding of the mechanisms leading to muscle disease for patients with dystrophinopathies. v Acknowledgements First and foremost I would like to thank my advisor, Dr. Federica Montanaro for her support and guidance over the time I have been in her lab. She has been an outstanding mentor and teacher, and has always provided me with an environment that promoted my academic achievements and scientific success. I would also like to thank my former and current lab mates. Christopher Penton has been a good friend and outstanding lab partner, and I thank him for all of his help and thoughtful discussion. Thanks to former lab members including Jesse Gibbons for teaching me several techniques used throughout all of my studies, and Eva Partida for her assistance with the studies presented in chapter III. I am also grateful for the positive experience and helpful discussions provided by other current and former lab members, Robert Orellana, Allison Macke, Dr. Jennifer Thomas-Ahner, Nick Beastrom, Nelson Salgado, Stephanie Klatil, and Dr. Rita Kaspar. I would also like to personally thank my graduate committee members, Drs. Denis Guttridge, Muthu Periasamy, and Jill Rafael-Fortney, for their guidance, suggestions, and time. I am extremely grateful to have worked alongside several collaborators and thank them their help and thoughtful discussion including Dr. Paul Martin and vi Dr. Jung Hae Yoon at the Research Institute at Nationwide Children’s Hospital, Dr. Kari Green-Church and Liwen Zang at The Ohio State University Proteomics Core, Dr. Stanley Froehner and Dr. Marvin Adams at the University of Washington, Dr. Michael Freitas at the The Ohio State University, Dr. James Ervasti at the Univeristy of Minnesota, Allistar Phillips at Cincinnati Children’s Hospital, and Dr. Dongsheng Duan at the University of Missouri. Finally I would like to thank my parents, Diane and Keith Johnson, my sister Kristen Johnson, and all of my family and friends for their love and support over the years. vii Vita 2007………………...………………………………………………..Bachelor of Arts Biochemistry/Molecular Biology Illinois State University 2007-Present……………………………………….Graduate Research Associate, OSBP Graduate Program The Ohio State University Publications Johnson, E.K., Zhang, L., Adams, M.E., Phillips, A., Freitas, M.A., Froehner, S.C., Green-Church, K.B., Montanaro, F. (2012) Proteomic analysis reveals differences in the cell signaling components of the dystrophin-associated protein complex between cardiac and skeletal muscle. PLOS ONE. 7(8): e43515. Yoon, Jung; Johnson, Eric; Xu, Rui; Martin, Laura; Martin, Paul; Montanaro, Federica. (2012) Comparative proteomic profiling of dystroglycan-associated proteins in wild type, mdx and Galgt2 transgenic mouse skeletal muscle. J Proteome Res 11(9): 4413-442. Beastrom, N., Lu, H., Macke, A., Canan, B. D., Johnson, E. K., Penton, C. M., Kaspar, B. K., Rodino-Klapac, L. R., Zhou, L., Janssen, P. M., and Montanaro, F. (2011) mdx(cv) mice manifest more severe muscle dysfunction and diaphragm force deficits than do mdx Mice. Am J Pathol 179, 2464-2474. Lash, T. D., Lamm, T. R., Schaber, J. A., Chung, W. H., Johnson, E. K., and Jones, M. A. (2011) Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase. Bioorg Med Chem 19, 1492-1504. viii Field of Study Major Field: Biochemistry ix Table of Contents Abstract .................................................................................................................ii Acknowledgements ..............................................................................................vi Vita ..................................................................................................................... viii Field of study .................................................................................................... viiix Publications ........................................................................................................ viii Table of Contents ................................................................................................
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