92 6 ART for Antiaging
In the context of signaling, it is clear that AMPK (encoded by Snf1 in yeast) and
SIRT1 (encoded by Sir2 in yeast) are responsible for the coactivation of PGC-1α,
which leads to the initiation of transcription and translation (protein biosynthesis)
essential for mitochondrial biogenesis. However, no significant fluctuations of both
Snf1 and Sir2 were observed in our study, so we had to alternatively consider the
expression alterations of other signal transducers. Consequently, we did notice a
marked downregulation of Kss1 and Fus3 by CR or mimetics. Both of which belong
to MAPK pathway genes, and their encoding products serve as critical checkpoints
for protein biosynthesis (Gustin et al. 1998 ; Pearson et al. 2001 ). In other words, if
MAPK is inactivated, protein biosynthesis should be suppressed (Son et al. 2011 ).
After exposure of PME yeast cells to CR or mimetics, we observed the downregula-
tions of MAPK pathway genes and ribosome genes, indicating protein biosynthesis
being hampered in PME yeast cells. In similar, a previous report has also described
that inhibition of mRNA translation extends nematode lifespan (Pan et al. 2007 ).
Why MAPK pathway genes are downregulated in PME cells? To answer this
question, we should remember that ROS is almost thoroughly scavenged due to
potent antioxidative responses provoked by CR and mimetics. While Sod1 and
Sod2 are induced in ME cells, Cta2 and Gpx1 are induced in PME cells. Also, it is
noted that ROS is a powerful activator of all MAPK subfamilies (ERK, JNKs, and
p38-MAPK) (Gaitanaki et al. 2003 ). Indeed, we did not observed the upregulation
of Sod1, Sod2, and other antioxidant genes (except for Cta2 and Gpx1) in PME
cells, suggesting a low ROS level due to potent antioxidation. Therefore, it seems
that MAPK may not be necessarily activated due to ROS deprival. However, why
inactivation of MAPK leads to suppression of MAPK pathway genes remains
unexplained, it is likely a consequence of nonphosphorylated MAPK being more
than phosphorylated MAPK. Although other protein kinases such as Tor1 and
Sch9 are also implicated in yeast longevity (Fabrizio et al. 2001 ; Thomson et al.
2008 ; Pan et al. 2011 ; Leprivier et al. 2013 ), we only observed the significant
upregulation of Tor1 in PME cells, as same as the case described in fission yeast
(Rollis et al. 2013 ). It is unclear whether the upregulation of Tor1 is due to inhibi-
tion of Tor per se in yeast underlying the condition of CR and mimetics.
Numerous investigations on CR paradigms have revealed markedly elevated
NO levels although no NOS homologues are found in yeast until now (Kig and
Temizkan 2009 ). However, COX is proposed to produce NO from nitrite under
hypoxic conditions (Castello et al. 2006 ; Taylor and Moncada 2010 ). We really
detected potent NO burst from yeast during CR, but no elevation of NO levels
were detected when the NOS substrate ARG was added in yeast cultures (data
not shown). On the other hand, it is shown that the mitochondrial uncoupler
DNP significantly increases the expression of mitochondrial biomarkers, indicat-
ing that DNP-mediated mitochondrial uncoupling promotes mitochondrial bio-
genesis (Cerqueira et al. 2011 ). Our results describing the inducible expression of
Cox1 in yeast cells upon exposure to ART seem to support such a preposition that
NO-COX interaction can be mimicked by ART-COX conjugation. The covalent
binding of ART to COX was monitored by the time-course assay of ART-heme
adducts, along with the synchronous measurement of increased COX activity.