Investigating the Biological Role of O-Acyl ADP Ribose by Elyse Blazosky A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science Baltimore, Maryland November, 2018 © Elyse Blazosky All Rights Reserved Abstract Sirtuins are an ancient family of deacetylase enzymes found in all three domains of life, where they have diverse biological roles. These widely studied enzymes are popular drug targets for treating diseases associated with aging, neurological disorders, cardiovascular disorders, metabolic disorders and even cancer. Unlike most deacetylase enzymes which use water to hydrolyze the amide bond linking the acetyl group to a lysine side chain, sirtuins catalyze a unique NAD+-dependent reaction that yields O-acetyl ADP ribose, nicotinamide and the deacetylate lysine. This seemingly wasteful use of NAD+ has led some to hypothesize that sirtuin activity is coupled to NAD+ levels in the cell. While sirtuin activity does rely on NAD+ biosynthesis and salvage pathways, it is unclear whether NAD+ levels fluctuate to a level that could affect sirtuin activity in-vivo. More recent studies have revealed new roles for sirtuins which suggests a more complex role of the sirtuin and a re-evaluation of the current hypothesis for why sirtuins uses NAD+. It has been shown that some sirtuins preferentially remove a variety of acyl lysine groups such as malonyl, succinyl, and butyryl, forming the corresponding O-acyl ADP ribose product. Mass spectrometry studies have revealed an abundance of these acyl modifications on cellular proteins, some of which are thought to result from non-enzymatic reaction with metabolites such as acyl-CoAs. I hypothesize that sirtuins use NAD+ to generate a carrier molecule (ADP ribose) to shuttle the leaving acyl chain to downstream metabolic pathways. This thesis focuses on progress made testing this hypothesis including the synthesis, purification and stability of 13C-labeled O-succinyl ADP ribose, as well as preliminary mass spectrometry metabolomics studies. ii Thesis Advisor: Dr. Cynthia Wolberger, Ph.D Professor Department of Biophysics and Biophysical Chemistry Thesis Reader: Dr. Namandje Bumpus, Ph.D Associate Professor; Associate Dean for Basic Science Research Division of Clinical Pharmacology iii Acknowledgments To Dr. Cynthia Wolberger, for your time and the many lessons I learned from you. To the members of the Wolberger Lab, for your support of both me and this project. To Dr. Namandje Bumpus, for inviting me into your lab, and for your mentorship. To the members of the Bumpus Lab, in particular Ms. Carley Heck, for treating me as a member of the lab, and for your guidance with mass spectrometry. To Dr. Ananya Majumdar, for your unconditional friendship. To Mr. Anthony Veltri, for tea walks. To Mr. Brendan Long, for not eating my bear claws too many times while I was writing this thesis, and for your love. To Dr. Don Wilmes, for helping me remember who I really am. To Mrs. Elizabeth Harrison, for your wisdom. iv Table of Contents Title ………………………………………………………………………………………………. i Abstract .......................................................................................................................................... ii Acknowledgements ....................................................................................................................... iv Table of Contents ………………………………………………………………………………... v List of Figures ............................................................................................................................. viii List of Abbreviations .................................................................................................................... ix Chapter 1: Introduction and Background 1.1 Sirtuins ………………………………………………………………………………. 1 1.2 Sirtuin Structure ……………………………………………………………………... 2 1.3 Sirtuin Reaction Mechanism ………………………………………………………… 6 1.4 NAD+-Dependent Deacetylation ……………………………………………….…… 8 1.5 Substrates of Sirtuins………………………………………………………..……… 10 1.6 Non-enzymatic Acylation and the Hypothesized role of Sirtuins………………….. 13 1.7 Hypothesis: NAD+ Provides a Nucleotide Carrier to Clean up Carbon Stress ……...15 1.8 Objectives…………………………………………………………………………... 15 1.9 References ………………………………………………………………………….. 17 Chapter 2: Synthesis and Purification of 13C-labeled O-succinyl ADP ribose 2.1 Introduction ………………………………………………………………………… 22 2.2 Methods …………………………………………………………………………….. 26 Chemical Succinylation of Histone H2B SIRT5 Growth and Purification SIRT5 Activity Assay v Characterization of Succinylated Histone H2B by MALDI-TOF Enzymatic Synthesis of O-succinyl ADPr Liquid Chromatography – Mass Spectrometry HPLC Purification of O-succinyl ADPr 1H-NMR 2.3 Results………………..…...………………………………………………………… 29 Succinylation of H2B Activity of Sirtuins and Synthesis of O-succinyl ADPr Detection of O-succinyl ADPr by LC-MS HPLC Purification and Large-Scale Synthesis of O-succinyl ADPr Purity of O-succinyl ADPr by 1H-NMR 2.4 References ………………………………………………………………………….. 41 Chapter 3: O-succinyl ADPr Stability and Metabolomics Studies 3.1 Introduction ………………………………………………………………………… 43 3.2 Methods …………………………………………………………………………….. 44 Stability of O-succinyl ADPr in water Time Course Studies Liquid Chromatography – Mass Spectrometry (LC-MS) Liquid Chromatography tandem mass spectrometry (LC-MS/MS) Metabolomics 3.3 Results………………….…………………………………………………………… 46 Stability of O-succinyl ADPr Timecourse Analysis of O-succinyl ADPr in Phosphate Buffered Saline vi Timecourse Analysis of O-succinyl ADPr in E.coli cell lysates Metabolomics Analysis of O-succinyl ADPr in E.coli cell lysates 3.4 References ………………………………………………………………………….. 57 Chapter 4: Conclusion and Future Directions 4.1 Conclusion and Future Directions …………………………………………………. 59 4.2 References ………………………………………………………………………….. 61 vii List of Figures Figure 1.1. Bacterial sirtuin, Sir2Tm, bound to NAD+ and acetylated peptide…………………. 4 Figure 1.2. Sirtuin binding interaction with acetylated lysine peptide ………………………….. 5 Figure 1.3. Sirtuin catalyzed deacetylation reaction mechanism ….……………….……………. 7 Figure 1.4. Substrates of sirtuins ……………………………………………………………….. 12 Figure 1.5. Model of sirtuin-mediated protein quality control ………………………………… 14 Figure 1.6. Sirtuins provide a carrier molecule to clean up carbon stress ……………………... 16 Figure 2.1. 13C-labeled O-succinyl ADP Ribose ………………………………………………. 23 Figure 2.2. Synthesis method of O-succinyl ADPr …..……………………………………...… 25 Figure 2.3. MALDI-TOF Spectra of unmodified and succinylated histone H2B………….…... 31 Figure 2.4. Purification and activity of SIRT5…………………….…………………………… 34 Figure 2.5. Extracted ion chromatogram and mass spectra of unlabeled and 13C-labeled O- succinyl ADPr ………………………………………………………………………………….. 36 Figure 2.6. HPLC purification of O-succinyl ADPr ……..............….............…......................... 38 Fig 2.7. 1H-spectra of 13C and 12C O-succinyl ADPr …………...……………………………… 40 Fig 3.1. Timecourse analysis of O-succinyl ADPr in water……………...…………………….. 48 Fig 3.2. Timecourse of O-succinyl ADPr in PBS, pH 7.4……………...………………...…….. 50 Fig 3.3. Second timecourse of O-succinyl ADPr in PBS, pH 7.4………...…………………….. 51 Fig 3.4. Consumption of O-succinyl ADPr in PBS, Tris-HCl and E. coli cell lysates……...….. 53 Fig 3.5. Consumption of O-succinyl ADPr and accumulation of ADPr in E. coli cell lysates and PBS measured by LC-MS………………………………………………………………………. 56 Fig 3.6. Chromatogram of initial dosing of 13C and unlabeled O-succinyl ADPr…………….... 57 viii List of Abbreviations Adenosine monophosphate (AMP) Adenine dinucleotide (ADP) Adenine dinucleotide ribose (ADPr) Coenzyme A (CoA) Histone deacetylase (HDAC) Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) Mass to charge ratio (m/z) Michalis constant (KM) Nicotinamide adenine dinucleotide (NAD+) O-acyl ADP ribose (O-acyl ADPr) O-succinyl ADP Ribose (O-succinyl ADPr) Retention Time (RT) Liquid chromatography mass spectrometry (LC-MS) Liquid chromatography tandem mass spectrometry (LC-MS/MS) ix Chapter 1 Introduction and Background 1.1 Sirtuins Sirtuins are a universally conserved family of nicotinamide adenine dinucleotide (NAD+) - dependent deacetylase enzymes. These enzymes are widely studied because of their diverse roles in biology and disease (Guarente 2018). Sirtuins are key players in a vast array of biological processes such as lipogenesis, glucose metabolism, circadian clock regulation, transcriptional regulation, cell autophagy, and DNA damage response (Sebastian, Satterstrom et al. 2012). For example, the mammalian sirtuin, SIRT1, deacetylates forkhead box O proteins (FOXO), which activates genes involved in stress response (Xiong, Salazar et al. 2011), while the mammalian sirtuin, SIRT2, can deacetylate alpha-tubulin thereby signaling the recruitment of transport complexes such as kinesin and dynein (Reed, Cai et al. 2006). Because of their diverse roles, sirtuins are attractive drug targets (Kane and Sinclair 2018) and are implicated in many diseases including age-related diseases such as neurodegeneration (Szegő, Outeiro et al. 2018), mood and metabolic disorders (Alageel, Tomasi et al. 2018), and cardiovascular disease (Ma and Li 2015), as well as pathological contexts including the inflammatory response (Vachharajani, Liu et al. 2016), bacterial infection (Eskandarian, Impens et al. 2013) and HIV-1 transcription (Pinzone, Cacopardo et al. 2013). Sirtuins comprise an ancient class of enzymes found in all three domains of life.