Abstract:Maintaining an
accurate genome is critical for cellular survival. Yet the genome is
under constant assault from both endogenous and environmental agents
that cuase DNA damage. Therefore, cells have developed numerous
pathways to repair damage to the DNA. Persistent unrepaired DNA damage
has severe consequences such as global genomic instability which is a
unifying feaure of cancer cells. A marker of persistent DNA damage is
the hyper-activation of the Poly(ADP-Ribose) polymerase (PARP) family of
enzymes. PARP proteins detect single strand breaks in the DNA, bind the
ends and catalyze the transfer of poly(ADP-ribose) chains (PAR) using
NAD+ as the ADP-ribose source. These long PAR chains recruit DNA repair
proteins to the damage site, whereby initiating repair. Once the DNA is
restored, the PAR chains are degraded, allowing another ssDNA break
event to be detected. Persistent damage leads to an overactivation of
PARP, increased concentrations of PAR and a concomitant depletion of
cellular NAD+ levels. NAD+ levels can be replenished by the ADP
ribosylation of nicotinamide via the NAD+ salvage pathway. The
rate-limiting enzyme in the salvage pathway is NAMPT (aka visfatin or
PBEF). NAMPT has been implicated in numerous human disorders including
diabetes, ischemia, heart disease and cancer. NAMPT is a 55 KDa protein
that forms a homodimer and has several phosphorylation sites, one of
which (Y188) is located on the homodimerization plane. We propose to
explore the influence of NAMPT on cellular pathways by first determining
the phosphorylation status required for homodimerization.
Yeast-2-Hybrid analysis of a simulated interaction will be completed
using NAMPT, NAMPT-Y188F (a mutant representing the non-phosphorylated
protein), and NAMPT-Y188D (a mutant that mimics the phosphorylated
tryrosine residue). We further propose to identify a set of interacting
partners of NAMPT using a Y2H approach against a cDNA library.
Currently there are only six proteins known to interact with NAMPT. The
result will give insights into the possible reasons for dimerization,
identify multiple interacting partners of NAMPT and assist in the
targeting of cellular energetics for drug development.
