NAD+ Metabolism: aspects of biosynthesis and degradation

Increasing evidences have indicated that NAD+ and NADH play critical roles not only in energy metabolism, but also in several cellular functions including calcium homeostasis, gene expression, ageing, immunological mechanisms and cell death. The research work focused on two important aspects of NAD+ metabolism, the biosynthesis with a detailed analysis of NAD+ biosynthetic pathways in human keratinocytes and, on the other hand, the NAD+ degradation by the purification and characterization of NAD(P)+ -glycohydrolase from porcine liver. The first study is originated from the NAD+ involvement in skin cancer prevention mechanisms that include enhancing DNA repair as a substrate of poly(ADP-ribose) polymerases (PARPs), preventing photoimmune suppression, serving as a cofactor for energy generation necessary for the formation of a functional cornified epidermal barrier, and for the release of agents such as leptin known to have protective effects in skin. We report here an analysis of NAD+ biosynthetic pathways present in human keratinocytes (HaCat) cells. All our experiments were carried out with HaCat cells initially grown in NAD+ precursor free media in order to evaluate the functionality of the different biosynthetic pathways using the same starting conditions. In addition to the wellknown nicotinamide (Dieterich) and nicotinic acid (Preiss-Handler) pathways, we investigated the potential of nicotinamide riboside (NmR) to serve as a NAD precursor. Our results confirm the presence of the Preiss-Handler and Dieterich salvage pathways, but showed that HaCat cells also are able to efficiently synthesize NAD+ starting from NmR via two different pathways involving conversion to nicotinamide mononucleotide and via conversion to nicotinamide. The rates of conversion and maximum NAD content that can be achieved vary among the different precursors. The second part of the work focused on the purification and characterization of NAD(P)+-glycohydrolase from porcine liver. NAD+ glycohydrolases (NADase, EC 3.2.2.5) belong to the list of enzymes that cleave the nicotinamide-glycosyl bond of NAD+ to form nicotinamide (Nam) and adenosine diphosphoribose (ADPR), and they are highly involved in the Ca2+ cellular homeostasis. In addition to NAD(P)+ hydrolysis, this enzyme shows other catalytic activities, as transglycosidation, ADP-rybosil cyclase (also known as cADPR synthase) and cADPR hydrolysis reactions. NAD(P)+-glycohydrolase was partially purified from microsomal membranes of porcine liver after solubilization with Triton X-100. Porcine liver NADase showed a molecular mass of 64kDa. Kinetic studies were carried out in order to characterize the partially purified enzyme. The results obtained demonstrated an high stability of the purified enzyme in a wide range of temperature and pH. In order to clarify the catalytic mechanisms, NADase activity was assayed in presence of several ion species and of denaturating agents, and also utilizing NAD+ analogues as substrate. In addition to the NAD+ hydrolysis activity, the purified enzyme showed other catalytic activities, such as transglycosidase and ADP-rybosil cyclase, as reported in literature for many NAD+ glycohydrolase enzymes.