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Structural and Functional Analysis of Muskelin and Other Kelch-Repeat Proteins

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Structural and Functional Analysis of Muskelin and Other Kelch-Repeat Proteins structural and Functional Analysis of Muskelin by Soren Prag Hansen A Thesis submitted to the University College London for the degree of PhD September 2003 Department of Biochemistry and Molecular Biology University College London Gower Street London UK ProQuest Number: U643396 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643396 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Muskelin is an intracellular, kelch-repeat protein that is functionally involved in cell spreading on thrombospondin-1. The aim of this thesis project was to investigate the role of muskelin domains in the subcellular distribution and the regulation of the protein using cellular, biochemical, and bioinformatic approaches. Using bioinformatics to compare muskelin orthologues from Mus musculus, Homo sapiens, Rattus norvegicus, Danio rerio, Drosophila meianogaster, and Anopheles gambiae, highly conserved regions within muskelin were identified, with the combination of a discoidin domain, a LisH motif, a C-terminal to LisH motif, and six kelch repeats. A further bioinformatic analysis of the kelch repeat proteins in whole genomes from Homo sapiens, Drosophila meianogaster, and Anopheles gambiae, demonstrated that muskelin has a unique molecular architecture amongst the large family of kelch repeat proteins. EGFP-tagged forms of muskelin were prepared and used to study its cellular localisation and to explore possible regulatory mechanisms. EGFP-MK had a cytoplasmic distribution and also formed mobile, granular inclusion bodies in 25% of the transfected cells. Formation of the inclusion bodies was a specific property of EGFP-wild-type muskelin, because EGFP alone or EGFP-muskelin mutated at key residues within kelch repeat 4 (Y488AA/495A or G474S/G475S) or mutations in the discoidin domain (K132A/I133AA/134A) were equivalently expressed yet had a uniformly diffuse distribution. Because of the complex domain structure of muskelin, a rational view of domain boundaries has proven essential for successful expression of truncated forms. Truncated forms composed of the discoidin or the kelch repeats alone did not form inclusion bodies. In biochemical pull-down assays, muskelin was found to self-associate 3 by a mechanism dependent on both the discoidin domain and the fourth kelch repeat. Mutations in kelch repeat 4 altered the sensitivity to limited proteolysis by proteinase K, suggesting that the protein conformation is altered. To begin to explore physiological factors that regulate the formation of inclusion bodies, the possible roles of five putative protein kinase C (PKC) phosphorylation sites that are conserved between muskelin species orthologues were evaluated in the cellular and biochemical assays. Activation of PKC decreased the formation of the inclusion bodies and resulted in phosphorylation of muskelin. Using a site-directed mutagenesis approach, two PKC phosphorylation sites critical for this activity were identified. Both of these sites are predicted to lie on the same face of the p-propeller structure composed by the six kelch-repeats. Together, these studies have developed new evidence that muskelin is a multidomain protein with multiple protein-protein interaction sites. Acknowledgements Firstly, I would to thank the Danish Research Academy, The Wellcome Trust, and Cleveland Clinic Foundation for the financial help during this project. Also, I would to thank Dr. Ana Giannini, Nina Kureishy, Dr. N. Anilkumar, Alexia Zaromytidou, and Mark Shipman and people at the LMCB, for materials and discussions and tutoring during my stay in London. From Cleveland, I would like to thank Dr. Michael K inter and Dr. Thomas Weimbs and staff at the Lerner Research Institute, for all your help and for letting me use your equipment. Also, I would to thank Ray Monk for all your help and your friendly support during the year we stayed together in Cleveland. Also, I would like to thank Dr. Tony Stadler and Dr. Rune Hartmann for your scientific advice and friendly support in Cleveland. In addition, I very much would like to say thanks to Olga Morals for an incredible patience with me while we were apart and over the years. Furthermore, I would to thank my family for all your support over the years. And thanks to Dr. Tony Ng and the rest of his group for all your support on my return to London. Finally, a very big thanks to Dr. Josephine Adams for all the support you have given me over the years. Thanks for your patience and extraordinary focus and drive to keep this project going and everything you have taught me during this project. Table of Contents Abstract .................................................................................................................3 Acknowledgements .............................................................................................. 5 Table of Contents ................................................................................................. 6 List of Figures .....................................................................................................12 List of Tables .......................................................................................................15 Abbreviations ......................................................................................................16 Introduction............................................................................................................18 Extracellular Matrix Proteins ........................................................................... 18 Collagen and Laminin ................................................................................. 19 Fibronectin .................................................................................................. 20 Cell-Substratum Contacts ...............................................................................21 Integrins ...................................................................................................... 21 Focal Adhesions ......................................................................................... 24 Development of Cell Contractility ................................................................27 Cell-Basement Membrane Adhesion ..........................................................28 Cellular Functions of ECM proteins ................................................................29 ECM-Mediated DNA Transcription ..............................................................31 Regulation of Cell Migration ........................................................................32 Thrombospondins .............................................................................................. 37 Molecular Structures of Thrombospondins .................................................... 37 Localisation of Thrombospondins ...................................................................39 TSP-1 and TSP-2 Knock-out Mice .................................................................41 Properties of Thrombospondins .....................................................................42 Molecular Interactions of TSP-1 .....................................................................43 6 Interaction with ECM proteins .....................................................................43 TGFp ........................................................................................................... 45 MMP-2........................................................................................................ 46 Cell-Surface Receptors ...............................................................................46 Cellular Signalling mediated by TSP-1 .......................................................... 47 Formation of Lamellipodia and Microspikes ............................................... 48 Vascular Smooth Muscle Cell Proliferation and Migration .........................49 Disassembly of Focal Adhesions ................................................................50 Kelch-repeat proteins ......................................................................................... 52 Actin-Binding Kelch-Repeat proteins ..............................................................55 Kelch in Ring Canals ...................................................................................55 Spermatogenesis ........................................................................................ 58 Kelch-Repeat proteins in Neurons ............................................................. 60 Other Actin-Binding Kelch-Repeat proteins ............................................... 61 Kelch-Repeat proteins and Microtubles..........................................................62 Kelch proteins in Transcriptional Regulation ................................................. 64 Rag-2.......................................................................................................... 67 NS1-BP......................................................................................................
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