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Alternative Splicing of Bnip3 Modulates Calcium Signals to Prevent Mitochondrial-Dependent Cell Death and Regulate Gene Expression

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Alternative Splicing of Bnip3 Modulates Calcium Signals to Prevent Mitochondrial-Dependent Cell Death and Regulate Gene Expression Alternative splicing of Bnip3 modulates calcium signals to prevent mitochondrial-dependent cell death and regulate gene expression. By Jared Field A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of MASTER OF SCIENCE Department of Biological Sciences University of Manitoba Winnipeg Copyright © 2017 by Jared Field i Abstract The Bnip3 cell death gene is prominently expressed in many hypoxic-ischemic related pathologies, such as heart failure and necrotising enterocolitis in the intestine. Two isoforms of Bnip3 have been described in the literature: a pro-death full-length protein (Bnip3FL) and a pro- survival protein lacking exon3 (Bnip3∆Ex3). Bnip3∆Ex3 acts as an endogenous inhibitor of Bnip3FL function, yet how Bnip3∆Ex3 serves this function is unknown. In gain-of-function experiments, I use combinations of cell lines and primary cells to dissect the mechanism(s) of Bnip3∆Ex3 action. Herein, I report that Bnip3∆Ex3 expression orchestrates an intricate calcium signalling cascade that has two cellular outcomes. First, calcium signals avert mitochondrially- dependent cell death. Second, these calcium signals modulate transcriptional regulators and gene expression causing morphological cell changes. These data provide original evidence that Bnip3∆Ex3 has the potential to mitigate the detrimental effects of hypoxia and Bnip3FL signalling by activating a complex signalling cascade and multiple survival pathways. i Summary of Proposed Bnip3 Signalling. Left: Bnip3FL causes cell death through calcium induction of mitochondrial permeability transition pore (MPTP) dependent cell death. Middle: Bnip3∆Ex3 prevents cell death by inhibiting Bnip3FL, thereby preventing MPTP opening signalled by Bnip3FL. Bnip3∆Ex3 also regulates mitochondrial calcium levels independently of Bnip3FL, possibly through pro-survival BCL-2 proteins. Right: Bnip3∆Ex3 directs calcium to the nucleus and indirectly regulates transcriptional regulators to alter the gene expression profile of cells. The result of this, in non-proliferative cells, includes changes to cellular morphology. ii Contents Abstract ........................................................................................................................................ i List of Figures ............................................................................................................................ iv List of Abbreviations .................................................................................................................. v Acknowledgements .................................................................................................................... vi Chapter 1: Review of the Literature ................................................................................................ 1 Introduction ................................................................................................................................. 1 The BCL-2 Family of Proteins ................................................................................................... 2 BCL-2 Family Regulates Cell Death ...................................................................................... 4 BCL-2 Family and Calcium Signalling .................................................................................. 9 Bnip3 Subfamily ....................................................................................................................... 13 Bnip3 Subfamily Structure ................................................................................................... 14 Bnip3 Subfamily Regulate Cell Death .................................................................................. 17 Bnip3 Subfamily Regulation................................................................................................. 24 Bnip3 Subfamily in Cancer & Disease ................................................................................. 33 Rationale ................................................................................................................................... 41 Chapter 2: Calcium Signalling of Bnip3 Isoforms ....................................................................... 45 Introduction ............................................................................................................................... 45 Research Aims & Objectives .................................................................................................... 45 Methodology ............................................................................................................................. 46 Results ....................................................................................................................................... 49 Discussion ................................................................................................................................. 55 Chapter 3: Effects of Bnip3 Isoforms on Mitochondrial-Dependent Cell Death ......................... 57 Introduction ............................................................................................................................... 57 Research Aims & Objectives .................................................................................................... 57 Methodology ............................................................................................................................. 58 Results ....................................................................................................................................... 63 Discussion ................................................................................................................................. 65 Chapter 4: Secondary Effects of Bnip3∆Exon3............................................................................ 68 Introduction ............................................................................................................................... 68 Research Aims & Objectives .................................................................................................... 68 Methodology ............................................................................................................................. 69 iii Results ....................................................................................................................................... 73 Discussion ................................................................................................................................. 78 Chapter 5: Summary ..................................................................................................................... 82 Discussion ................................................................................................................................. 83 Conclusion ................................................................................................................................ 84 Appendices .................................................................................................................................... 86 Appendix A – Fetal Gene Activation........................................................................................ 86 Appendix B – Additional Controls & Experiments .................................................................. 90 Appendix C – Detecting Bnip3 splicing and Testing Bnip3FL & Bnip3ΔEx3 siRNAs .......... 92 Appendix D – Literature Cited ................................................................................................. 95 Appendix E – Expanded Materials and Methods ................................................................... 118 List of Figures Summary of Proposed Bnip3 Signalling......................................................................................... ii Figure 1. Bnip3 isoforms differentially regulate calcium between the mitochondrial and nucleus. ....................................................................................................................................................... 50 Figure 2. Bnip3FL-mediated mitochondrial calcium influx is dependent on calcium release from the ER............................................................................................................................................ 53 Figure 3. Bnip3∆Ex3 redirects calcium destined for the mitochondria to the nucleus independent of the ER and Bnip3FL. ................................................................................................................ 54 Figure 4. Bnip3∆Ex3 preserves cell viability through a mitochondrially-dependent pathway. ... 64 Figure 5. Bnip3∆Ex3 regulated BCL-2 translocation to the mitochondria. ................................. 75 Figure 6. Bnip3∆Ex3-mediates regulation of NFATc3 and HDAC5 transcriptional regulators and gene expression. ............................................................................................................................ 77 Figure 7. Proposed mechanisms of Bnip3∆Ex3 cell survival and gene regulation. ..................... 82 Appendix B-1. Bnip3ΔEx3 regulates the activation of NFATc3 ................................................. 90 Appendix B-2. Bnip3FL-induced MPTP opening is equally blocked by either Bnip3ΔEx3 or Cyclosporin A ............................................................................................................................... 91 Appendix C-1. Bnip3 protein
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