The Biosynthesis of NAD in Humans: Isozymes and Subcellular Compartmentation

NAD is essential for energy transduction. It is also a key molecule in a variety of central signaling pathways. Nevertheless, surprisingly little is not known about its biosynthesis. Our recent research has established that NAD biosynthesis is absent from the cytosol: the three human isoforms of NMN adenylyltransferase (NMNAT), which catalyzes the final step of NAD generation, were localized to the nucleus, the Golgi complex and the mitochondria. We now wish to study two major aspects of NAD generation. First, the individual roles of the three human NMNATs will be investigated. Cell-type- and tissue-specific expression will be determined. Methods to study the impact of each isoform on important cellular processes (e.g., bioenergetics, poly-ADP-ribosylati on and DNA repair, lifespan regulation) will be developed. Second, the mechanism of mitochondrial NAD generation will be revealed. Since these organelles contain the majority of the cellular NAD, the biosynthetic pathway is of great importance. We will i dentify the cytosolic precursors required to be taken up into the organelles as well as the intramitochondrial conversions leading to NAD. A reporter system will be developed which enables the measurement of NAD formation within the organelles. This syste m includes the overexpression of a poly-ADP-ribose polymerase within the organelles, thereby converting generated NAD into protein-bound polymers. Our recent discovery of a mitochondrial NMNAT isoform has already provided valuable progress to this subproj ect. The results will unravel the mechanism and regulation of a fundamental biochemical pathway. Our results may enable the discovery of human pathologies associated with distortions of NAD biosynthetic pathways.