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Regulation of Pyruvate Dehydrogenase Kinase 4 (PDK4) Through Protein-Protein Interaction and Its Role in Apoptosis

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Regulation of Pyruvate Dehydrogenase Kinase 4 (PDK4) Through Protein-Protein Interaction and Its Role in Apoptosis View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Wits Institutional Repository on DSPACE Regulation of pyruvate dehydrogenase kinase 4 (PDK4) through protein-protein interaction and its role in apoptosis Patricia Coliwe Ntlabati A dissertation submitted to the faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2008 Declaration I, Patricia Coliwe Ntlabati, declare that this dissertation is my own work and any added information from other sources has been carefully referenced. The dissertation “The regulation of pyruvate dehydrogenase kinase 4 through protein-protein interaction and its role in apoptosis” is being submitted for the degree of Master of Science at the University of the Witwatersrand, South Africa. ………………………………… Patricia Coliwe Ntlabati Date:…………………………… i Dedication I would like to dedicate this work to my family at large, my father and mother who always believed, supported and loved me, my two sisters whom I have inspired throughout the years and not forgetting my two brothers (Thapelo and Eddie) who encouraged me when things were difficult. For my uncle who has been there for me ever since I started with my degree until today. Lastly and foremost I dedicate this thesis to my life partner Makhosini Dlamini who has supported, encouraged and loved me throughout and unconditionally. Your love kept me strong and enabled me to conquer anything including this difficult challenge. ii Abstract Pyruvate dehydrogenase kinase 4 (PDK4), a mammalian mitochondrial serine kinase has emerged as an interesting candidate for diabetes therapy. Due to the high prevalence of this disease especially type 2 diabetes (T2D) and the health complications associated with it, there is extensive effort to find the appropriate treatment. Understanding the regulation of PDK4 would therefore contribute significantly to the development of therapeutic agents. This research outlines the bioinformatics analysis of PDK4, using tools such as Interweaver, ClustalW and Protein Structure Visualiser. These programs were used to determine potentially interacting partners for PDK4. Interweaver database identified 96 proteins which have possible interaction sites for PDK4. Protein p100/p49, containing a death domain that is known to have a role in suppressing apoptosis, was identified as a potential partner for PDK4. The alignment between p100/p49 primary sequence and that of PDK4 using Gene explorer/ClustalW demonstrated sequence similarity between the two proteins. Swiss PDB Viewer then located the positions of the amino acids that are in the hypothetical protein binding motif of p100/p49 within the 3D structure of hPDK4. These amino acids were found to be located in the region of PDK4 which is known to bind protein substrates of PDK4 and may be accessible to other proteins as well. These findings were very interesting as PDK4 have not previously been associated with apoptosis and this could be the link between apoptosis and insulin resistance. iii Cell biology studies were then performed to verify the relationship between PDK4 and apoptosis. In this regard, HeLa and HepG2 cells were treated with apoptosis inducing agents such as TNFα, C2-Ceramide, and Linoleic acid. These cells were then monitored for apoptosis and PDK4 mRNA expression using a DNA laddering assay as well as Real Time PCR. The results showed that these factors to induce apoptosis in a concentration dependent manner. This induction of apoptosis by these three factors indicated to suppress PDK4 mRNA levels. These findings suggested a regulatory role for PDK4 in apoptosis. iv Acknowledgements My biggest thank you goes to the Council for Scientific and Industrial Research (CSIR), Biosciences unit in Modderfontein for granting me this opportunity to further my studies in terms of funding and allowing me access to their premises and facilities. To Dr Lungile Shoba who has been the driving force to the completion of my degree as being the best supervisor ever. I thank you for the patience, encouragement, guidance, support, understanding and advice you gave me throughout. I would also like to thank my other supervisor Dr Zodwa Dlamini from the University of the Witwatersrand Medical School, Molecular and Cellular Pathology Group Leader for her undivided support, guidance and advice as well as using her laboratory to conduct some of my experiments. To the Enzyme Technology group, I would like to thank Dr Dean Brady and Dr Justin Jordaan, for understanding and allowing me the opportunity to successfully complete my degree. I would also like to thank everyone in the group for the advice and support in particular Phiyane and Fritha for the contribution they made towards my thesis. To my colleagues from the PDK project Thandeka and Lindiwe, thanks for support, good times, assistance and true friendship. Lastly I want to acknowledge the University of the Witwatersrand, National Research Foundation (NRF), Wits Medical School Molecular and Cellular Pathology group in particular Zukile, Lindiwe and Lebogang for assisting with experiments and others for being there as friends (Mfundo, Raymond and Makgotso). v Publications Meetings: • SASBMB XXth Conference Ntlabati P.C. Lungile S. and Dlamini Z. Regulation of pyruvate dehydrogenase kinase 4 (PDK4) through protein-protein interaction and its role in apoptosis. 2006. University of KwaZulu Natal. P120. • Bio-08 Conference Ntlabati P.C. Lungile S. and Dlamini Z. Regulation of pyruvate dehydrogenase kinase 4 (PDK4) through protein-protein interaction and its role in apoptosis. 2008. University of Rhodes. P188. vi Table of Contents Declaration ...................................................................................................................... i Dedication ...................................................................................................................... ii Abstract ......................................................................................................................... iii Acknowledgements ........................................................................................................ v Publications ................................................................................................................... vi Table of Contents ......................................................................................................... vii List of Figures ............................................................................................................... xi List of Tables .............................................................................................................. xiv Abbreviations ............................................................................................................... xv 1 Introduction ................................................................................................................. 1 1.1 Physiological role of PDK4 ................................................................................. 2 1.2 Regulation of PDK4 ............................................................................................. 4 1.2.1 Regulation of PDK enzyme activity ............................................................. 5 1.2.2 Regulation of PDK gene expression ............................................................. 6 1.2.2.1 Signalling pathways activated by insulin ................................................ 7 1.2.2.2 Signalling pathways activated by Fatty acids ....................................... 10 1.2.2.3 Signalling pathways activated by Glucocorticoids ............................... 13 1.3 Regulation of PDK4 by protein-protein interaction ........................................... 14 1.4 The role of NFκB and p100 in apoptosis and T2D ............................................ 15 1.4.1 Signal transduction pathway involved in induction of apoptosis………….15 1.4.2 NFκB and the death receptor pathway…………………………………….17 1.4.3 Signal transduction through NFκB ………………………………………..20 1.5 NFκB and PDK4: Roles in apoptosis and Type II diabetes…………………... 21 vii 1.6 Objectives of the study ....................................................................................... 23 2 Materials and Methods .............................................................................................. 24 2.1 Bioinformatics analysis ...................................................................................... 24 2.1.1 Analysis of protein-protein interaction ....................................................... 24 2.1.2 Consensus sequence identification ............................................................. 25 2.1.3 Structural localization of the hypothetical protein binding motif in hPDK4 .............................................................................................................................. 25 2.1.3.1 Modelling of hPDK4................................................................................ 25 2.1.3.2 Localization of the hypothetical protein binding motif in hPDK4 structure .............................................................................................................................. 25 2.2 Cellular and Molecular Analysis ....................................................................... 26 2.2.1 Culturing of cells......................................................................................... 26 2.2.2 Cell Viability
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