The Pennsylvania State University The Graduate School Department of Biochemistry and Molecular Biology ELUCIDATION OF THE DEVELOPMENTAL AND PHYSIOLOGICAL ROLES OF NAD+ BIOSYNTHETIC PATHWAYS A Dissertation in Biochemistry, Microbiology and Molecular Biology by Melanie R. McReynolds © 2017 Melanie R. McReynolds Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2017 The dissertation of Melanie R. McReynolds was reviewed and approved* by the following: Wendy Hanna-Rose Associate Professor of Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee Craig E. Cameron Professor of Biochemistry and Molecular Biology Eberly Chair in Biochemistry and Molecular Biology Teh-hui Kao Distinguished Professor of Biochemistry and Molecular Biology Chair of Plant Biology Graduate Program Lorraine Santy Associate Professor of Biochemistry and Molecular Biology Pamela A. Hankey-Giblin Professor of Immunology Andrew D. Patterson Associate Professor of Molecular Toxicology Special Signatory David Gilmour Professor of Molecular and Cell Biology Co-Director for Graduate Education in BMMB *Signatures are on file in the Graduate School iii ABSTRACT NAD+ biosynthesis has proven to be an attractive and promising therapeutic target for influencing healthspan and obesity-related phenotypes as well as tumor growth. However, NAD+ is a key metabolite that impacts the entire metabolome. Therefore, it is necessary to elucidate exactly how manipulating NAD+ biosynthetic pathways can lead to therapeutic benefits. Also, it is imperative to characterize the unexpected adverse reactions to manipulating the biosynthetic pathways to fully utilize this target for drug discovery. The goal of our research is to understand how NAD+ homeostasis is maintained to support its core metabolic roles and its signaling and regulatory roles involving NAD+ consumers. In this work, I investigate the developmental and physiological role of NAD+ biosynthetic pathways in C. elegans, their homeostatic interactions, and I reveal a biosynthetic pathway involving an enzyme outside of NAD+ biosynthesis. NAD+ is synthesized via distinct routes including de novo synthesis from tryptophan, salvage synthesis from nicotinamide, which feeds into the Preiss-Handler pathway from nicotinic acid in C. elegans, and via the phosphorylation of nicotinamide ribosides or nicotinic acid ribosides using nicotinamide riboside kinase (NMRK). We previously discovered that NAD+ salvage synthesis through the nicotinamidase PNC-1 is required for normal progression of gonad development in C. elegans. Global metabolic profiling suggested that glycolysis was perturbed in our pnc-1 mutants, which have lower global levels of NAD+. Furthermore, we were able to link compromised glycolysis to gonad delay in our loss of salvage NAD+ synthesis mutants. I investigated this model and demonstrated using metabolic carbon tracing that glycolysis is compromised in our pnc-1 mutants. iv It’s been reported in the literature that C. elegans lack the de novo NAD+ biosynthetic pathway because quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in the genome. However, all genes coding for the key enzymes required for production of quinolinic acid (QA) from tryptophan are present in the C. elegans genome. Using metabolic deuterium tracing I revealed that de novo NAD+ synthesis from tryptophan is active. I also demonstrated that UMPS-1 as the enzyme responsible required for converting QA into NAD+ during de novo biosynthesis. In addition to this, I discovered a novel role for NMRK-mediated synthesis in embryonic hatching in C. elegans. Finally, I uncovered a compensatory network amongst the biosynthetic pathways that maintains NAD+ homeostasis. In summary, this work has expanded our knowledge of the developmental and physiological roles of NAD+ biosynthetic pathways. Metabolic carbon tracing was implemented as a tool to examine metabolic flux in C. elegans. Also, this work suggests that an underground metabolic mechanism may contribute to NAD+ biosynthesis. The conserved enzyme UMPS-1 is substituting for the missing QPRTase, raising questions about the relevance of similar underground metabolic activity in higher organisms. This work associates a novel C. elegans’ hatching phenotype to NAD+ biosynthesis. Finally, this work deciphers the impact of manipulating NAD+ biosynthesis for therapeutics. v TABLE OF CONTENTS List of Figures………………………………………………………………………………...…viii List of Tables…………………………………………………………...………………...………xi List of Abbreviations…………………………………………………………………………….xii Acknowledgments………………………………………………………………………………xiv Chapter 1: Introduction……………………………………………………………………………1 Part I: NAD+ is a central hub in cellular metabolism……………………………………..1 + Historical context of Vitamin B3 as a precursor for NAD ……………………….1 Role of NAD+ in redox reactions …………………………………………………2 Role of NAD+ as a substrate ……………………………………………………...3 Part II: Eukaryotic NAD+ biosynthesis…………………………………………………... 4 Overview of NAD+ biosynthesis………………………………………………… 4 Salvage NAD+ synthesis…………………………………………………. 4 Preiss-Handler Pathway………………………………………………….. 5 de novo NAD+ synthesis…………………………………………………..5 Riboside synthesis………………………………………………………... 6 Part III: Targets for NAD+ biosynthesis and metabolism based drug discovery………….8 Cancer inhibition……………………………………………………………..……8 Health and lifespan benefits.……………………………………………………..10 Neurological disorders…………………………………………………………...11 Novel antibiotics………………………………………………………………... 12 Part IV: Role of NAD+ biosynthesis in metabolic homeostasis………………………... 13 Chapter 2: Metabolic carbon tracing reveals disrupted glycolysis due to a loss of salvage NAD+ synthesis in C. elegans………………………………………………………………….. 15 Introduction………………………………………………………………………………15 Importance of NAD+ in energy-producing redox reactions……………………...15 Relationship between glucose metabolism and cellular NAD+ pools…………... 16 Loss of NAD+ salvage synthesis disrupts glycolysis leading to developmental reproductive delay in C. elegans………………………………………………... 17 Results…………………………………………………………………………………... 19 Development of metabolic tracing protocol to model compromised glycolysis in pnc-1 mutants………………………………………………………………… 19 Metabolic tracing supports compromised glycolysis in pnc-1 mutants………… 20 Glucose storage in pnc-1 mutants………………………………………………. 24 Discussion………………………………………………………………………………. 26 Compromised NAD+ biosynthesis leads to disrupted glycolysis………………. 26 Requirement to maintain a supply of carbon for oxidative phosphorylation by the mitochondria…………………..……………………………………………. 27 vi Disruption of NAD+ metabolism leading to developmental delay can model tumor growth and progression………………………….....................…………. 29 Materials and Methods…………………………………………………………………...31 C. elegans Culture and Strains…………………………………………………...31 Targeted Metabolomics………………………………………………………….31 Metabolic Tracing with Stable Isotopes………………………………………....32 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)……………….32 Chapter 3:Eukaryotic de novo NAD+ biosynthesis from tryptophan in the absence of a QPRTase homolog………………………………………………………………………………… 34 Introduction……………………………………………………………………………... 34 Results…………………………………………………………………………………... 37 NAD+ de novo synthesis contributes to NAD+ biosynthetic capacity………….. 37 Supplementation with NAD+ de novo precursors reverses NAD+-dependent phenotypes……………………………………………………………………….40 UMPS-1 is required for QA label to be incorporated into NAD+ biosynthesis….43 Loss of kyneurine pathway affects reproductive development…………………..47 Discussion………………………………………………………………………………..48 Intact de novo NAD+ biosynthesis in the absence of QPRTase homolog……….48 Requirement for NAD+ de novo biosynthesis for normal reproduction….……. 49 Materials and Methods………………………………………………………………….. 50 C. elegans Culture and Strains…………………………………………………. 50 Metabolite Supplementation……………………………………………………. 50 Phenotypic Analysis…………………………………………………………….. 51 Targeted Metabolomics………………………………………………………… 51 Metabolic Tracing with Stable Isotopes………………………………………... 52 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)……………… 53 Chapter 4: Nicotinamide Riboside contributes to NAD+ biosynthesis and embryonic hatching in C. elegans……………………………………………………………………………..….54 Introduction……………………………………………………………………………... 54 Nicotinamide riboside as a precursor for NAD+ biosynthesis…………..……… 54 Results…………………………………………………………………………………... 56 Supplementation with NR reverses NAD+-dependent phenotypes……………...56 NR contributes to NAD+ biosynthesis………………………………………….. 58 NR contributes to embryonic hatching during development………………….... 60 Discussion………………………………………………………………………………. 63 NR contribution to the cellular NAD+ pool……………………………………...63 NR contribution to C. elegans’ embryogenesis………………………………….64 Materials and Methods…………………………………………………………………...66 C. elegans strains and culture……………………………………………………66 Metabolite supplementation…………………………………………….………..66 Targeted metabolomics…………………………………………………………..67 Phenotypic Analysis……………………………………………………………...68 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)……………….68 vii Chapter 5: Compensatory roles for NAD+ biosynthetic pathways and consumers in C. elegans………………………………………………………………………………..70 Introduction………………………………………………………………………………70 Critical nature of NAD+ pool in cellular metabolism……………………………70 Results……………………………………………………………………………………72 NAD/NADH ratio is not impacted in loss of salvage NAD+ synthesis mutants.......................................................................................................72