Download La nicotinammide quale segnale metabolico nella regolazione della

La nicotinammide quale segnale metabolico nella regolazione della biosintesi dei
nucleotidi piridinici.
Dottoranda: Dott.ssa Loredana Marchegiani
Docente guida: Chiar.mo Prof. Giulio Magni
Nicotinamide (NaM), o viatamin B3, represents the principal precursor of nicotinamide adenine
dinucleotide (NAD+) in the mammalia. The manitenance of intracellulars levels of
NAD+ is
fundamental for the cellular survival. NAD+ is known to play a major role as a co-enzyme in
numerous oxidation-reduction reactions. Recently a new important role in non-redox reactions
NAD-dipendent has appeared, in wich the bond -N-glicosidic of co-enzyme is divided by enzymes
involved in importants cellulars functions, such as CD38, poly(ADP-ribose)polymerases (PARPs)
and sirtuins. It is noteworthy that a common distinctive feature of these reactions is the inhibition
exerted by nicotinamide toward all the enzymes involved. Therefore, it is conceivable to hypothesize
that the fluctuations of nicotinamide concentration regulate the metabolic fluxes occurring in these
pathways, which are known to be strictly associated to fundamental events of cellular life.
The levels of NaM are untracelllarly modulated by the action of enzymes involved in NaM
salvaging, included nicotinamide riboside phosphorylase and nicotinamide deamidase. The aim of
the present work of research was to identify, in the mammalia and particularly in the human, the
genes releted to enzymes above cited.
The enzyme that catalyzes the reaction of phosphorolysis of nicotinamide riboside (NAR) has been
purified from human erytrocithes and characterized. Nevertheless the protein purified shows a
specific activity greater towards inosine. Tests of inhibition highlighted that NAR inhibits the
activity towards inosine and on the contrary, that 3-acetyl-pyridine-riboside inhibits activity towards
both substrates. Such results suggest the existence of only one enzyme that catalyzes the
phosphorolysis of NAR and of inosine, later proved by aminoacidic sequenziament of proetin
purified. Actually it reveled that it is purine nucleoside phosphorylase (PN), enzyme involved above
all in the metabolism of purine nucleosides. Nevertheless recently through tests of complementation
in cells of S. Cerevisiae and C. Glabrata, highlighted a possible physiological role of homologous
enzymes in salvage of NaM to NAD+ . Similar stidies could be done for human also enzyme using
human cells in colture and tests of RNA interference.
The gene that codes the enzyme that deamidates NaM substrate to nicotinic acid was identified in
procaryotes and eucaryotes such S. Cerevisiae and C.Elegans. Although a human homologous gene
wasn’t found in literature tell about the presence of nicotinamide deamidase activity in some
mammalian tissuesof rat, rabbit and human.
The enzyme responsible for nicotinamide deamidase activity from rabbit liver has been purified to
homogeneity. The sequenziament of purified protein has reveled that it is the carboxylesterase (rCE),
whose human homologous is carboxylesterase-1 (h-CE-1). To prove that thys enzymes is effectively
responsible for nicotinamide deamidase activity expression in mammalian cells COS-7 and in yeast
cells Pichia Pastoris was tried. In both cases enough protein wasn’t go to reveal the activity.
Recently the crystal structure of human enzyme trough expression such gene in cells of insect
(Spodoptera Frugiperda) was obtained to prove the fact that the enzyme can be expresse actively
only in expression eucaryotic systems. So, following this direction other attempts can be done.
It has to be underlined that the high concentraction ( 100 mM) used to show the activity with the
value the Km for NaM toled to be 40mM, seems specifyed a not important activity according to a
phisyological point of view, strengtheningh the hypothesis that in mammalian the pathway that
deamidates NaM substrate to Na is absent. Notwithstanding that enzymatic activity is subdmed to
regulation. In the attempt to find a possible regulator of such activity it cold be interesting going on
to investigate the hypothesis that human carboxylesterase can have also nicotinamide deamidase
activity and have an important role also in metabolism of this vitamin.