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reported to be differentially regulated by nucleoredoxin
and glutaredoxin in addition to thioredoxin.
42
Transcriptional activity of NF-
k
B depends on three
distinct events, degradation of I
k
B alpha in the cyto-
plasm releasing NF-
k
B from this complex, translocation
of NF-
k
B to the nucleus, and phosphorylation of p65,
one of the two components of heterodimeric NF-kB
(p50/p65).
38
E3330 was found to inhibit transcriptional
activity of NF-
k
B without directly affecting any of
these steps through suppression of its DNA-binding
activity. A nuclear factor was found to enhance the
DNA-binding activity of NF-
k
B and to be inhibited by
E3330.
38
Through incubation of E3330-NH
2
attached to
beads with Jurkat cell nuclear extracts, APE1 was found
to bind directly and with high affinity to E3330.
43
In subsequent work, APE1 was shown to enhance the
DNA-binding activity of NF-
if E3330 does indeed directly interact with APE1 as
reported later, then presumably it would inhibit
APE1's ability to reduce any of the oxidized transcrip-
tion factors that it regulates under conditions where
these transcription factors are induced.
G
ENERAL REDOX FACTOR
S
All cells contain general redox factors that sense
oxidative damage to proteins in the form of disulfide
bonds and other damage to Cys residues. They also
contain catalysts that sense and catalyze detoxification
of reactive oxygen species such as hydrogen peroxide.
Cys residues can adopt 10 different sulfur oxidation
states from
2 (the fully reduced state)
48,49
and
exist in a number of different forms in vivo including
cysteinyl radical, sulfenic acid, sulfinic acid, sulfonic
acid, cystine, and others (see Jacob et al.
48
for a recent
review of sulfur chemistry associated with Cys). Disul-
fide bonds are repaired through thiol/disulfide
exchange reactions (reviewed in
49
) by the thioredoxin
(TRX) (
Figure 11.4
) and glutaredoxin/glutathione
(GRX/GSH) systems. More recently, a nuclear redox
factor with similarity to thioredoxin was identified that
may also play an important redox role in cells.
50
Through a thiol/disulfide exchange mechanism, thiore-
doxin and glutaredoxin systems maintain a redox
cellular homeostasis (i.e. a reducing intracellular redox
รพ
6to
B p50/p50 and p50/p65
dimers but not p65/p65, and E3330 was shown to inhibit
the ability of APE1 to reduce NF-
k
B. Based on these
results, Handa and coworkers concluded that the redox
regulation must be through the p50 protein of NF-
k
B.
43
The oxidation state of cytosolic vs. nuclear NF-
k
B was
examined in addition to the role of specific Cys residues
in contributing to the redox-dependent DNA-binding
activity of NF-
k
B.
44
There are seven Cys residues in
p50 but oxidation of only Cys 62 affected the DNA-
binding properties of p50. Further, Cys 62 was found
to be highly susceptible to oxidation in the cytoplasm
but was reduced in the nucleus upon translocation. No
external stimuli are required for reduction of NF-
k
Bin
the nucleus, and APE1 was shown to reduce Cys 62,
thereby enhancing the DNA-binding activity of oxidized
p50/p50 and p50/p65 dimers.
44
Treatment with E3330
prior to phorbol 12-myristate 13-acetate (PMA) stimula-
tion of cells inhibited reduction of p50 in the nucleus but
not in the cytoplasm. However, the redox state of p65
was largely unaffected by treatment with E3330. Thus,
APE1 reduces p50 in the nucleus but not in the cyto-
plasm.
44
More recently, APE1 has been proposed to act
as a redox chaperone in stimulating the transcriptional
activity of NF-kB. This chaperone role is not dependent
on APE1's redox activity or its endonuclease activity.
45
Thus, APE1 reduces transcription factors directly and
also facilitates reduction by other redox factors such as
glutathione and thioredoxin.
45
Although Handa and coworkers reported that E3330
specifically inhibited reduction of NF-
k
B but not AP-1 or
N-FAT by APE1 under the conditions of their experi-
ments in which Jurkat cells were stimulated with PMA
and A23187,
38
others have demonstrated that E3330
inhibits reduction of AP-1 and other transcription
factors that are reduced by APE1 in cell-based transacti-
vation assays.
46,47
Under the conditions of Handa's
experiments, very low levels of transcriptional activa-
tion were observed for AP-1 and NFAT. Furthermore,
k
FIGURE 11.4
The process by which thioredoxin (TRX) reduces
a protein containing a disulfide bond involves formation of a mixed
disulfide bond between TRX and the target protein, which is then
resolved resulting in disulfide bond formation in TRX and reduction
of the target protein. The redox cascade by which TRX is reduced by
thioredoxin reductase (TR) is also shown. Reduction of thioredoxin by
thioredoxin reductase is a somewhat more complex process involving
the formation of a selenylsulfide and subsequent reduction by a pair
of Cys residues within another subunit of TR. Electron transfer reac-
tions involving the FADH
2
, a cofactor of TR, and NADPH are required
to regenerate TR. Adapted from Jacob et al.
48
