Structure, Function and Regulation of NAD Kinases

The availability of NADP represents a key prerequisite for cellular functions. Besides its role in bioenergetic conversions, this nucleotide also serves in signalling pathways. The reduced form, NADPH, plays a pivotal role for the maintenance of cellular integrity, because it provides the reducing power to all known oxidative defence systems. Therefore, the biosynthesis of this dinucleotide has a critical impact on vital processes. NADP is generated by phosphorylation of NAD which is catalysed by NAD kina ses. Based on our previous identification and kinetic characterisation of the only known human isoform, we will conduct a comprehensive functional and structural analysis of this enzyme. Particular attention will be paid to a Ca2+/calmodulin-dependent act ivation of NAD kinase which is indirect, but has otherwise remained unexplored. Our preliminary evidence suggests that it might be mediated by a Ca2+/calmodulin-dependent phosphorylation, but other possibilities will be considered as well. We will exploit our recently established cell lines (stable knock-down or overexpression of human NAD kinase) to assess the physiological roles and its modulation during oxidative stress. As an additional experimental system we include the sea urchin egg and sea urchin NAD kinases, which we have identified in preliminary studies. Structural studies will include crystallization and mass-spectrometric analyses to reveal phosphorylation-dependent structural changes and associations with other proteins. The results of this study will greatly extend the knowledge about a fundamental cellular process and provide novel tools to modulate the cellular response to oxidative stress. Several collaborations with recognized experts, for example, in the field of structural analyses, will consolidate the broad methodological spectrum required to establish a comprehensive characterisation of human NAD kinase and the molecular mechanisms of its regulation