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CDS) Enzymes Participate in the Synthesis of Cardiolipin in Mitochondria and Phosphatidylinositol at The

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CDS) Enzymes Participate in the Synthesis of Cardiolipin in Mitochondria and Phosphatidylinositol at The Different CDP-diacylglycerol Synthase (CDS) enzymes participate in the synthesis of cardiolipin in mitochondria and phosphatidylinositol at the endoplasmic reticulum Nicholas James Blunsom A thesis submitted to University College London for the degree of Doctor of Philosophy September 2019 Department of Neuroscience, Physiology and Pharmacology UCL ‘I, Nicholas James Blunsom, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.' _______________________ Nicholas James Blunsom 2 Dedication For my Nan, Grandpa, and Babushka who always believed in me. 3 Abstract Cytidine diphosphate diacylglycerol (CDP-DG) is an essential liponucleotide that is crucial for the synthesis of the phospholipids phosphatidylinositol (PI) and cardiolipin (CL). It is the precursor for PI in the endoplasmic reticulum (ER) and two molecules are required for the synthesis of CL in mitochondria. CDP- DG is formed through the enzymes CDP-DG synthase (CDS) 1 and 2 in the ER; this is the rate-limiting step in the PIP2 cycle. CDS enzymes are critical for the maintenance of cellular structure, as depletion of the CDS enzymes causes a vast array of phenotypes. Upon constant stimulation of phospholipase C via chronic stimulation with arginine-vasopressin in H9c2 cells, there is a specific increase in CDS1 mRNA alongside a decrease in PI levels. PI levels decrease, presumably, due to the increased level of PIP and PIP2 synthesis; CDS1 mRNA increases to attempt to compensate for the drop in PI. This also shows that, contrary to previous studies, CDS1 is responsible for synthesising PI through the products of PIP2 breakdown in the PIP2 cycle, whereas CDS2 is most likely for de novo synthesis of PI. Furthermore, it appears that this increase of CDS1 mRNA due to chronic stimulation with arginine-vasopressin is regulated through protein kinase C and cFos. The two mammalian CDS enzymes are 69% similar and 73% identical, and have been shown to localise to the ER. A novel protein was recently discovered in yeast to have CDS activity which shares very little homology with the CDS enzymes, Tam41, which is a mitochondrial protein. We confirmed that a mammalian version of this enzyme, TAMM41, exists and is responsible for the supply of CDP-DG to the mitochondria for CL synthesis. 4 Impact Statement Due to the nature of this research, i.e. fundamental biology, the impact of this research will primarily affect academe. However, that is not to say that this will not have benefits further afield in years to come. In this thesis, it is shown that the enzyme responsible for CDP-DG production in mitochondria, and hence crucial for cardiolipin synthesis, is neither of the CDS proteins. Rather, it is due to a completely unrelated enzyme, TAMM41. This finding is important because knowing the actual lipid synthesis pathways in mitochondria for CL production will help the study of mitochondrial lipids and their associated diseases. Diseases such as Barth syndrome, which has multitudinous symptoms such as dilated cardiomyopathy, skeletal myopathy, neutropenia and short stature, are caused from a lack of mature CL. Furthermore, it is shown that the enzyme responsible for the rate-limiting step of PI synthesis in the PIP2 cycle is specifically CDS1. In addition, CDS1 is upregulated during chronic stimulation of H9c2 cells with arginine-vasopressin (a hormone known to induce hypertrophy), and that phosphoinositide levels are significantly decreased during this period. Furthermore, it is observed that CDS1 enzyme expression is regulated by cFos and PKC. These findings may open up new pathways of research into hypertrophy and cardiovascular disease in the future, both from a lipids perspective and a signalling perspective. Finally, it is clearly displayed that commercial antibodies should not be taken at face value. Antibodies need to be stringently examined and validated when being used in studies. One of the antibodies initially used had not been validated, thus causing major discrepancies between experimental results and published data. The lack of doing so will lead to incorrect data and analysis being published (which is a phenomenon more frequent than should be). Hopefully this data will encourage researchers to be more thorough whilst using antibodies and should lead to more accurate science. 5 Acknowledgements Firstly, I would like to thank Dr Tim Ashlin for his endless help in the lab, for teaching me a plethora of techniques, and for all those times troubleshooting them when things just wouldn’t work. I would also like to thank Professor Michael Duchen and Dr Will Kotiadis for their help, and for the training to use the Oroboros O2K machine. In addition, thanks to Prof. Justin Hsuan for doing the mass spectroscopy. On a personal note, I would like to thank my family and friends for their support and encouragement throughout my studies, and especially my Mum for her never-ending belief and support in me and my abilities. Finally, I would like to give a special thank you to my supervisor Professor Shamshad Cockcroft. Not only for her encouragement and help, but also for really making me think about the next step, the wider picture and the key experiments needed to get there. None of this would have been possible without the generous grant awarded by the British Heart Foundation, who funded this research. 6 Publications The data from this thesis has been published in the following publications: Blunsom, N. J., Gomez-Espinosa, E., Ashlin, T. G., Cockcroft, S. Mitochondrial CDP-diacylglycerol synthase activity is due to the peripheral protein, TAMM41 and not due to the integral membrane protein, CDP-diacylglycerol synthase 1. Biochim Biophys Acta Mol Cell Biol Lipids. 16 Dec 2017; 1863(3): 284-298. Blunsom, N. J., Gomez-Espinosa, E., Ashlin, T. G., Cockcroft, S. Sustained phospholipase C stimulation of H9c2 cardiomyoblasts by vasopressin induces an increase in CDP-diacylglycerol synthase 1 (CDS1) through protein kinase C and cFos. Biochim Biophys Acta Mol Cell Biol Lipids. 14 March 2019; 1864(7): 1072-1082. Blunsom, N. J., Cockcroft, S. Phosphatidylinositol synthesis at the endoplasmic reticulum. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Jan;1865(1); pii: S1388-1981(19)30089-7. 7 Table of Contents Dedication..................................................................................................... 3 Abstract ........................................................................................................ 4 Impact Statement ......................................................................................... 5 Acknowledgements ..................................................................................... 6 Publications.................................................................................................. 7 Table of Contents ......................................................................................... 8 List of Figures ............................................................................................ 12 List of Tables .............................................................................................. 14 Abbreviations ............................................................................................. 15 Chapter 1 Introduction ............................................................................ 19 Phosphatidic acid and its derivatives .............................................. 20 1.1.1 The fate of phosphatidic acid ................................................... 24 Cytidine Diphosphate Diacylglycerol Synthase ............................... 26 1.2.1 Genes and mapping ................................................................. 26 1.2.2 Structure and mechanism ........................................................ 27 1.2.3 CDS substrate selectivity and inhibition ................................... 31 1.2.4 Localisation of over-expressed protein and activity .................. 32 1.2.5 Tissue expression .................................................................... 33 1.2.6 Regulation and loss of function mutants ................................... 35 PI synthase ..................................................................................... 37 PIP2 cycle ....................................................................................... 38 Cardiac Hypertrophy ....................................................................... 44 1.5.1 Cardiomyocytes, mitochondria and phospholipids ................... 46 1.5.2 Phospholipid metabolism in cardiac physiology and pathology 47 1.5.2.1 Remodelling ....................................................................... 47 1.5.2.2 Phosphatidic acid ............................................................... 48 1.5.2.3 PI and phosphorylated derivatives ..................................... 48 1.5.2.4 Cardiolipin .......................................................................... 48 1.5.2.5 H9c2 cells: a biological system to study cardiac physiology .. ........................................................................................... 51 Thesis aims ..................................................................................... 53 Chapter 2 Methods .................................................................................. 54 Cell Culture ..................................................................................... 54 8 2.1.1 Routine growing ......................................................................
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