NAMPT Regulation Through Post Translational Modification(2012-Present)

Investigator:  Karen Almeida, Rhode Island College

Mentor:  Robert Sobol, University of Pittsburgh

Abstract:  NAD+ is essential for life and therefore must be strictly controlled.  NAD+ is widely used as the cofactor in cell energy production and metabolism by providing high-energy electrons required for oxidation/reduction reactions. However, NAD+ concentration is diminished by its use as a substrate for enzymes such as poly (ADP-ribose) polymerases (PARPs) and Sirtuins. In each case, the ADP-ribose portion of NAD+ is cleaved from the nicotinamide (NAM) moiety. To restore NAD+, NAM is converted to nicotinamide mononucleotide (NMN) by the rate-limiting enzyme Nicotinamide phosphoribosyltransferase (NAMPT) also called NAmPRTase, Pre-B cell-enhancing factor (PBEF), or Vistafin. NAD+ is generated in the second step with concomitant hydrolysis of ATP by the enzyme NMNAT. Within the NAD+ salvage pathway, NAMPT has been shown to modulate the cellular NAD+ levels and therefore is a potential target for regulation by inhibitors. NAMPT is a 55-kDa enzyme that forms a homodimer to generate two active sites. The channel leading into the active site is primarily housed in one monomer with contributions at the active site from the opposing monomer. Although the mechanism of NAMPT activity has been established, the regulation through post-translational modifications has yet to be investigated. The goal of this proposal is to study the PTM as a potential mechanism of regulation for NAMPT activity and/or dimerization. A systematic set of point mutations encompassing the 19 known PTM sites will be generated. Each will be tested for modulation of NAMPT activity. Furthermore, a fluorescence-based protein interaction assay will be developed. The Bimolecular Fluorescence Complementation assay will fuse NAMPT mutations to truncated fragments of EYFP. Dimerization will reconstitute the EYFP fluorophore as a marker for NAMPT interaction. Finally, molecular docking programs will be developed to screen NAMPT surfaces that incorporate PTM for potential low molecular weight inhibitors. Together, these studies will provide critical information regarding NAMPT regulation to guide the development of novel low molecular weight inhibitors of NAMPT.