DETERMINING THE EXTRACELLULAR FUNCTION AND ROLE IN FAT METABOLISM OF THE PROTEIN PNC-1 IN CAENORHABDITIS ELEGANS

Open Access
Author:
Krebs, Kelsey
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Wendy Hanna Rose, Thesis Supervisor
  • Joseph C. Reese, Honors Advisor
Keywords:
  • NAD+
  • PNC-1
  • fat metabolism
  • Caenorhabditis elegans
  • biology
Abstract:
The nicotinamidase PNC-1 is responsible for converting nicotinamide (NAM) to nicotinic acid (NA) in the NAD+ salvage pathway, which ultimately recycles NA back into NAD+. NAD+ levels are essential for the control of multiple cellular processes, so the mutation of pnc-1(pk9605) can indirectly affect these processes by regulating NAD+ levels in the cell. Sirtuins are a group of NAD+ consumers that use NAD+ and produce NAM as a byproduct. In mammals, sirtuins are known to be involved in fat metabolism, in particular with fat mobilization, so I hypothesized that the NAD+ salvage pathway may be involved in the regulation of fat metabolism via sirtuins. I hypothesized that a mutation in pnc-1(pk9605) would cause the down-regulation of sirtuins, resulting in increased fat storage. I fixed and stained worms and quantified their fat storage. I found that pnc-1(pk9605) mutant worms stored more fat than wild type, but sirtuin mutant worms did not phenocopy the pnc-1(pk9605) fat storage and conversely stored consistently less fat than wild type. I concluded that sirtuins function to assist in fat storage in C. elegans, and that the increased fat stores in pnc-1(pk9605) worms are mediated by some other mechanism. Through a starvation assay I was able to demonstrate that there is no decrease in fat mobilization in pnc-1(pk9605) worms. Therefore, I concluded that the excess fat stored in pnc-1(pk9605) worms was due to an increase in fat storage. PNC-1 has an isoform that is predicted to be secreted and to primarily exist outside of the cell. Therefore, I hypothesized that the nicotinamidase PNC-1 functions extracellularly. In order to test this hypothesis, I injected PNC-1 protein into a pnc-1(pk9605) mutant worm’s extracellular environment and tested its ability to rescue specific developmental defects that are present in all pnc-1(pk9605) mutant worms. The results were not statistically different from the percentages of defects normally expected in pnc-1(pk9605) mutants. However, I was unable to demonstrate that PNC-1 definitely does not function outside of the cell, so the results of this experiment were inconclusive.