Biology Reference
In-Depth Information
same time frame as the wild-type protein. Thus, it is
not necessary to first modify Cys 99 and Cys 138.
Next, the partially unfolded state was characterized
by hydrogen deuterium exchange (HDX) mass spec-
trometry.
102
In this experiment, we sought to establish
the number of backbone amide protons that exchanged
with deuterium indicating the degree to which they
are involved in hydrogen-bonding interactions. For the
þ
which both Cys 65 and Cys 93, the redox critical residues
are exposed as monitored by reaction with NEM. Thus,
this partially unfolded state of the enzyme is likely to be
its redox active form. Further, it suggests that oxidation
of the redox critical Cys residues facilitated by E3330 is
a plausible mechanism for inhibition of redox activity.
ROLE OF APE1 IN RESPONSE TO
OXIDATIVE STRESS
2 NEM adduct, the number of fast, intermediate, and
slow exchanging amide Hs are 81
1, 20
4, 43
2,
respectively; for the
þ
3 NEM adduct, the number of
amides are 81
2, 19
2, 41
1, whereas for the
þ
7
Although cells are constantly subjected to reactive
oxygen species (ROS) generated during normal meta-
bolic processes, an increase in ROS due to endogenous
or exogenous sources can lead to oxidative stress
(reviewed in
2
). APE1 is expected to play a role in
responding to oxidative stress in cells in multiple
response pathways. As an essential AP endonuclease
in the base excision repair pathway, it repairs oxidative
damage to DNA. APE1 also regulates the DNA-binding
activity of a number of transcription factors including
AP-1 through a redox mechanism, some of which are
induced in response to oxidative stress, and APE1 is
itself induced in response to oxidative stress.
NEM adduct, the numbers are 136
3, 14
1, 38
4.
þ
þ
Both
3 NEM products behave very simi-
larly to the native protein in this experiment. Clearly, the
number of fast exchanging amide Hs has increased
substantially for the
2 NEM and
7 NEM adduct, consistent with
it being a partially unfolded conformer that becomes
“locked-in” or “trapped” as a result of the reaction
with NEM. The
þ
7 NEM product has ~40 more
exchanging amide Hs than the
þ
þ
2 NEM product, or
the
3 NEM product. Approximately 51% of the amide
protons are exchanged in the wild-type protein and 67%
in the
þ
7 NEM product
represents a partially but not completely unfolded state
of the enzyme and is clearly distinct from wild-type or
the
þ
7 NEM adduct. Therefore, the
þ
Oxidative Stress Induces Specific
Transcription Factors
In contrast to bacteria, the response to ROS in higher
eukaryotes is much more complex, involving activation
of a number of signaling pathways. The role of ROS in
regulating cancer through signaling pathways invol-
ving hydroxia-inducible factors, extracellular signal-
regulated kinase (ERK1/2), and PI3K/Akt has recently
been reviewed.
1
Transcription factors known to respond
to oxidative stress include NF-
k
B and AP-1. Through
known pathways mitogen-activated protein kinase, Jun
N-terminal kinase, p38, and others, the transcriptional
activity of AP-1 is activated in response to oxidative
stress.
103,104
In a recent study, the direct effect of oxida-
tive stress induced by H
2
O
2
on transcript levels of
AP-1 transcription factors has been quantitated for the
human retinal pigment epithelium (RPE).
105
The goal
of this study was to characterize early molecular
responses to oxidative stress. Response to oxidative
stress is expected to differ in different cell types and
conditions. In RPE, significant changes in the expression
of six AP-1 familymembers, FosB, c-fos, Fra-1, c-Jun, JunB,
and ATF3, were observed. Changes observed varied
from two- to 128-fold increases in transcript levels over
the first four hours following treatment with H
2
O
2
.
Changes in the levels of transcripts for these AP-1
proteins were correlated with levels of translated protein
observed by Western blot analysis. Lang and coworkers
propose that a direct model for response to oxidative
2 NEM modified protein.
Having established that E3330 likely interacts with
a partially unfolded state of APE1, we sought to deter-
mine how it inhibits the redox activity of APE1.
102
It is
possible that E3330 simply blocks the interaction of the
redox active form of APE1 with the transcription factor
to be reduced. This assumes that the redox active form
ofAPE1 is apartiallyunfoldedstate of the enzyme, a likely
scenario given that the redox active Cys residues are
otherwise buried and not accessible. Alternatively, it
may interact directly but reversibly with the redox active
Cys residues. To examine the latter possibility, we
analyzed disulfide bond formation in APE1 resulting
from treatment with E3330 as compared to an untreated
sample. Reversible reaction of a Cys residue with E3330
would make it susceptible to nucleophilic attack by
another reduced Cys residue within APE1. There is very
little disulfide bond formation in the untreated APE1
sample, and a substantial increase in disulfide bond
formation results from treatment with E3330.
102
Disulfide
bonds were analyzed by LC-MS/MS analysis and were
identified as follows: C65-C93, C93-C99, C93-C138, and
C65-C99. Notably, all of the disulfide bonds observed
involve the two redox active Cys residues Cys 65 and
Cys 93. Thus, it is probable that E3330 recognizes a
partially unfolded state of APE1 and inhibits its redox
activity by oxidizing the redox active Cys residues.
This study provides evidence for the ability of APE1
to adopt an alternate conformation of APE1 and one in
þ
