Hydrolase Regulates NAD+ Metabolites and Modulates Cellular
Redox |
| |
Authors: | Lei Tong Susan Lee and John M Denu |
| |
Institution: | Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706 |
| |
Abstract: | Although the classical redox functions of co-enzyme NAD+ are
firmly established in metabolism, there are numerous enzymes that catalyze
cleavage of NAD+ to yield free ADP-ribose (ADPr) or related
metabolites, whose functions remain largely unknown. Here we show that the
Nudix (nucleoside diphosphate linked to another moiety
X) hydrolase Ysa1 from Saccharomyces cerevisiae
is a major regulator of cellular ADPr and O-acetyl-ADP-ribose
(OAADPr). OAADPr is the direct product of
NAD+-dependent protein deacetylases (sirtuins) and is readily
converted to ADPr. Ysa1 cleaves ADPr/OAADPr into ribose
phosphate/acetyl-ribose phosphate and AMP. In cells lacking Ysa1
(Δysa1), ADPr and OAADPr levels increased ∼50%,
with a corresponding decrease in AMP. Strikingly, Δysa1 cells
display higher resistance to exogenous reactive oxygen species (ROS) and 40%
lower basal levels of endogenous ROS, compared with wild type. The biochemical
basis for these differences in ROS-related phenotypes was investigated, and
the results provide evidence that increased ADPr/OAADPr levels
protect cells via the following two pathways: (i) lower ROS production through
inhibition of complex I of the mitochondrial electron transport chain, and
(ii) generation of higher levels of NADPH to suppress ROS damage. The latter
occurs through diverting glucose into the pentose phosphate pathway by ADPr
inhibition of glyceraldehyde-3-phosphate dehydrogenase, a central enzyme of
glycolysis.NAD+ is well known for its role as a hydride-transferring
co-enzyme in many oxidation-reduction reactions of metabolism. However,
NAD+ is also a substrate for NAD+ glycohydrolases,
ADP-ribose transferases, poly(ADP-ribose) polymerases
(PARPs),2 cyclic
ADP-ribose synthases (1,
2), and sirtuins
(3,
4), all of which cleave the
glycosidic bond of NAD+ to produce nicotinamide and an ADP-ribosyl
product. Notably, sirtuins catalyze NAD+-dependent lysine
deacetylation to generate nicotinamide, deacetylated lysine, and
OAADPr (5,
6). OAADPr has been
proposed to act as a second messenger, signaling to other processes that
NAD+-dependent protein deacetylation has occurred
(7–9).
The biological functions and in vivo metabolism of OAADPr
and free ADPr are largely unknown.Through a quantitative microinjection assay of starfish oocytes, both ADPr
and OAADPr caused a delay/block in oocyte maturation, suggesting
ADPr/OAADPr may have specific biological activity
(10). In mammalian cells,
intracellular ADPr/OAADPr can activate the TRPM2 (transient receptor
melastatin-related ion channel 2) nonselective cationic channel
(11–13).
TRPM2 contains a conserved intracellular Nudix hydrolase domain (referred to
as NudT9H) that directly binds ADPr/OAADPr, but it is incapable of
cleaving the ligand because a major catalytic residue is missing
(11,
14). Although still disputed,
ADPr binding to NudT9H appears to be required for the well known oxidative
stress activation of the channel
(13,
15). Cell stress via puromycin
treatment led to TRPM2-mediated cell death that was dependent on sirtuin
deacetylases, presumably from the production of OAADPr
(12).Increasing evidence suggests that free ADPr may function as a cellular
signal. ADPr can be produced from the coordinate actions of PARPs and
poly(ADP-ribose) glycohydrolase (PARG), which cleave ADPr polymers to free
ADPr (16,
17). Under massive genotoxic
stress, hyper-stimulation of the NAD+-dependent PARPs depletes
cellular NAD+, which is linked to catastrophic ATP loss and cell
death (18,
19). The mechanism by which
PARP1 hyperactivity in the nucleus impairs ATP production in mitochondria is
unclear. The fact that PARP1 and poly(ADP-ribose) are localized in the nucleus
adds a perplexing aspect. However, recent data suggest that PARP1-induced loss
of ATP requires PARG (20).
Under conditions of PARP1 hyperactivation, it has been suggested that the
PARG-dependent production of ADPr can exit the nucleus and interfere with ATP
production in mitochondria
(21,
22). Thus ADPr could be the
molecular signal released from the nucleus of cells undergoing massive
poly(ADP-ribosyl)ation and rapidly triggers mitochondrial dysfunction.In support for ADPr/OAADPr as potential signaling molecules, the
existence of enzymes capable of metabolizing these compounds suggests that
their cellular concentrations may be subject to tight regulation
(23,
24). To understand the
biological roles played by ADPr/OAADPr, it is essential to elucidate
the degradation pathways that can modulate their levels. Previously we
described the ability of several conserved members of the Nudix hydrolase
family to hydrolyze in vitro the diphosphate linkage in
ADPr/OAADPr, generating ribose phosphate or acetyl-ribose phosphate
and AMP (10,
24). Here we examine the
biochemical and cellular functions of the Nudix hydrolase Ysa1
(14) from Saccharomyces
cerevisiae. We determined that Ysa1 is the major ADPr Nudix hydrolase and
an important regulator of cellular ADPr/OAADPr levels. A
Δysa1 strain displays increased resistance to both exogenously
and endogenously generated ROS. Basal level of ROS decreased by 40% in the
Ysa1 deletion strain. We provide biochemical evidence that increased
ADPr/OAADPr levels protect cells via the following two pathways: (i)
lower ROS production through the inhibition of complex I of the electron
transport chain, and (ii) generation of higher NADPH levels to suppress ROS
damage. The latter occurs by diverting glucose into the pentose phosphate
pathway by ADPr inhibition of glycolysis. |
| |
Keywords: | |
|
|