Elucidation of the developmental and physiological roles of NAD+ biosynthetic pathways

Open Access
- Author:
- McReynolds, Melanie Renee
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 10, 2017
- Committee Members:
- Wendy Hanna-Rose, Dissertation Advisor/Co-Advisor
Wendy Hanna-Rose, Committee Chair/Co-Chair
Craig Eugene Cameron, Committee Member
Teh-Hui Kao, Committee Member
Andrew David Patterson, Outside Member
Lorraine C Santy, Committee Member
Pamela Hankey Giblin, Outside Member - Keywords:
- NAD+ biosynthesis
NAD+metabolism
C. elegans
Metabolic flux
Metabolomics
Carbon tracing
Deuterium tracing
Salvage NAD+ synthesis
de novo synthesis from Trp
NMRK-mediated synthesis - 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. 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.