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Investigating Polyphosphate Biology: from Post-Translational Modification to Rare Disease

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Investigating Polyphosphate Biology: from Post-Translational Modification to Rare Disease Investigating polyphosphate biology: From post-translational modification to rare disease Amanda Bentley-DeSousa A thesis submitted to the Faulty of Graduate and Postdoctoral Studies in partial fulfillment of requirements for the Doctorate in Philosophy degree in Cellular and Molecular Medicine Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa Ó Amanda Bentley-DeSousa, Ottawa, Canada, 2021 Authorizations Manuscript #1 Bentley-DeSousa, A., Holinier, C., Moteshareie, H., Tseng, Y-C., Kajjo, S., Nwosu, C., Amodeo, G.F., Bondy-Chorney, E., Sai, Y., Rudner A., Golshani, A., Davey, N.E., and Downey, M. (2018). A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis. Cell Reports 22, 3427-3439. This is an open access article distributed under the terms of the Creative Commons CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Manuscript #2 McCarthy, L.*, Bentley-DeSousa, A.*, Denoncourt, A., Tseng, Y-C., Gabriel, M., and Downey M. (2020). Proteins required for vacuolar function are targets of lysine polyphosphorylation in yeast. FEBS Letters 594, 21-30. *Authors contributed equally License number: 5011081359570 License date: February 16, 2021 License content published: John Wiley and Sons License content publication: FEBS Letters Manuscript #3 Bentley-DeSousa, A., and Downey, M. Vtc5 is localized to the vacuole membrane by the conserved AP-3 complex to regulate polyphosphate synthesis in budding yeast. Manuscript submitted to BioRxiv and in review at mBio Manuscript #4 (Review – Appendix C) Bentley-DeSousa, A., and Downey, M. (2019). From underlying chemistry to therapeutic potential: open questions in the new field of lysine polyphosphorylation. Current Genetics 65, 57-64. License number: 5011081197927 License date: February 16, 2021 License content publisher: Springer Nature License content publication: Current Genetics ii Abstract The first report of polyphosphates (polyP) was in 1890 by L. Liberman and since then, polyP’s role in biology has been explored. PolyPs are chains of phosphoanhydride-linked inorganic phosphates ranging from 3-1000s of units in length. These chains are implicated in many cellular pathways including blood clotting, bacterial virulence, and neuroproteotoxic disease. Given the diversity of polyP, they make an excellent candidate in the development of novel therapeutics. In yeast, polyP is synthesized by the vacuolar transporter chaperone (VTC) complex as a translocation event into the vacuole lumen. In 2015, polyP chains were found to act as a post-translational modification termed polyphosphorylation on yeast proteins (Nsr1 and Top1). This modification occurs non-enzymatically on lysine residues within poly-acidic, serine, and lysine (PASK) motifs and can only be detected via electrophoretic mobility shift on NuPAGE gels. We have since expanded the pool of yeast polyphosphorylated substrates to 25, with an enrichment of proteins with roles related to RNA biology. Additionally, we were the first group to demonstrate polyphosphorylation of 6 human proteins by expressing E. coli PPK1 in HEK293T cells. We next focused on elaborating how polyP is being regulated via the VTC complex by assessing which protein trafficking pathways are critical for VTC localization at the vacuole membrane. We found the adaptor protein 3 (AP-3) complex is responsible for localizing Vtc5 subunit to the vacuole membrane and in AP-3 mutants, Vtc5 becomes mislocalized to the vacuole lumen and degraded. Vtc5 degradation, upon AP-3 mutation, is mediated by the endosomal sorting complex required for transport (ESCRT) complex. The loss of polyP in AP-3 mutants is imparted by Vtc5 mislocalization. In humans, mutations in AP-3 cause a rare genetic disorder termed Hermansky-Pudlak Syndrome (HPS) which has a wide range of symptoms. These include defects in polyP accumulation in platelets, likely related to a loss of polyP. We expect that our work using yeast will provide a framework for understanding fundamental aspects of polyP biology related to HPS and other health conditions. iii Contents List of tables vii List of figures viii Abbreviations ix Acknowledgements xi Chapter 1 – Introduction 1 1.1: Polyphosphate: an ancient molecule with wide applications 1 1.2: PolyP chains are biochemically versatile molecules 2 1.3: The many methods to assess polyP qualitatively and quantitatively 3 1.4: PolyP chains are synthesized by polyP synthetases and degraded by 4 polyphosphatases 1.5: Saccaromyces cerevisiae is an ideal model system to study polyP biology 6 1.6: PolyP is implicated in many aspects of human health 8 1.7: Polyphosphorylation as a new function for polyP at the protein level 11 1.8: Intracellular regulation of polyP through localization and polyphosphatases 12 1.9: Trafficking pathways regulate protein localization and function 14 1.10: Many pathways can be activated to degrade proteins 16 1.11: Open questions in the field of polyP biology 20 1.12: Description of rationales and hypotheses 20 1.12.1: A Candidate Screen for Novel Polyphosphorylated Proteins 21 1.12.1.1: Rationale 21 1.12.1.2: Hypotheses 21 1.12.2: A Follow Up Yeast Screen to Complete a PASK Protein-Wide Set of 21 Substrates 1.12.2.1: Rationale 21 1.12.2.2: Hypotheses 21 1.12.3: Assessing the Role of Protein Trafficking Pathways in VTC Complex 22 Regulation 1.12.3.1: Rationale 22 1.12.3.2: Hypotheses 22 iv Chapter 2 – Manuscript #1 23 A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis 2.1: Abstract 25 2.2: Introduction 26 2.3: Results 29 2.4: Discussion 51 2.5: Experimental Procedures 56 2.6: Acknowledgements 58 2.7: Supplemental Experimental Procedures 59 Chapter 3 – Manuscript #2 69 Proteins required for vacuolar function are targets of lysine polyphosphorylation in yeast 3.1: Abstract 71 3.2: Introduction 72 3.3: Methods 75 3.4: Results and Discussion 79 3.5: Acknowledgements 90 Chapter 4 – Manuscript #3 91 Vtc5 is localized to the vacuole membrane by the conserved AP-3 complex to regulate polyphosphate synthesis in budding yeast 4.1: Abstract 93 4.2: Introduction 94 4.3: Results 98 4.4: Discussion 112 4.5: Methods 117 4.6: Acknowledgements 122 Chapter 5 – General Discussion 123 5.1: Polyphosphorylation: a novel PTM in yeast and human cells 123 5.2: Polyphosphorylation modifies 25 yeast substrates 123 5.3: Towards developing more experimental methods to detect 125 polyphosphorylation v 5.4: EcPPK1: a novel system to study human polyP biology and 126 polyphosphorylation 5.5: Polyphosphorylation may be implicated in human disease 127 5.6: The regulation of polyP as seen through the trafficking of VTC 128 5.7: Vtc5 as a means to regulate VTC complex function 129 5.8: In AP-3 mutants, Vtc5 is localized to the vacuole lumen in an ESCRT- 130 dependent manner 5.9: Subcellular localization of other VTC subunits may use other pathways 131 5.10: The AP-3 approach: A means to uncover the elusive mammalian polyP 132 synthetase? Chapter 6 – References 133 Chapter 7 – Appendices 151 7.1: Appendix A 151 PASK-like proteins in Saccharomyces cerevisiae (table 1) 151 PASK-like proteins in Homo sapiens (table 2) 164 7.2: Appendix B 205 Supplemental tables from manuscripts (tables 3-10) 7.3: Appendix C: Review 221 Bentley-DeSousa, A., & Downey, M. (2018). Curr Genet. 65, 57-64 vi List of Tables Table 1. PASK-like proteins in Saccharomyces cerevisiae (Bentley-DeSousa et al., 2018) Table 2. PASK-like proteins in Homo sapiens (Bentley-DeSousa et al., 2018) Table 3. Antibodies used in this work (Bentley-DeSousa et al., 2018) Table 4. Synthetic DNA constructs (ATUM) (Bentley-DeSousa et al., 2018) Table 5. Strains used in this study (Bentley-DeSousa et al., 2018) Table 6. Candidates screened (McCarthy et al., 2020) Table 7. Yeast strains used in this study (McCarthy et al., 2020) Table 8. Antibodies used in this work (McCarthy et al., 2020) Table 9. Yeast strains used in this study (Bentley-DeSousa & Downey, in review) Table 10. Antibodies used in this work (Bentley-DeSousa & Downey, in review) *Note: due to the size of these tables, they will be included in Appendices A and B. vii List of Figures Figure 1. PolyP is Comprised of Phosphate Units Linked via Phosphoanhydride Bonds Figure 2. PolyP can be Assessed using Qualitative and Quantitative Methods Figure 3. PolyP is Synthesized by PolyP Synthetases and Degraded by Polyphosphatases Figure 4. PolyP is Synthesized by the VTC Complex in Yeast Figure 5. PolyP is Implicated in Many Aspects of Human Health Figure 6. Polyphosphorylation is Detected via Electrophoretic Mobility Shift on NuPAGE Gels Figure 7. Yeast Proteins are Localized from the TGN to the Vacuole Primarily Via the AP-3 and CPY Pathways Figure 8. Proteins Destined for Degradation can be Targeted by Many Pathways Figure 9. Analysis of PASK-Containing Proteins in Yeast Figure 10. Identification of Polyphosphorylated Proteins Figure 11. Identification of Polyphosphorylated Candidates and In Vitro Analysis Figure 12. Molecular Characterization of Polyphosphorylation Figure 13. Characterization of Rpa34 and Chz1 PASK Motifs Figure 14. PASK Serine and Acidic Amino Acid Content Do Not Impact Shift Size Figure 15. Analysis of Polyphosphate Induced Electrophoretic Mobility Shift Figure 16. Regulation of Polyphosphorylation Figure 17. Analyzing the Regulation of Polyphosphorylation Figure 18. Vtc4 Impacts Ribosome Function Figure 19. Polyphosphorylation of Human Proteins Figure 20. Hps90 and HIP are Not Polyphosphorylated Figure 21. An Updated Model for PolyP Function in Yeast Figure 22. Strategy for TAG Checking Figure 23. Identification of New Polyphosphorylated Targets in Yeast Figure 24. Collapse of Polyphosphate-Induced Shifts on SDS-PAGE Gels Figure 25. Polyphosphorylated Targets are Connected by Genetic and Physical Interactions Figure 26. Analysis of Prb1 Polyphosphorylation Figure 27.
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