Biology Reference
In-Depth Information
within the vicinity of Thr 58, the structure and chemical
environment is highly conserved.
47
In contrast, residues
in the vicinity of Val 4 in the E. coli enzyme exonuclease
III are not similar to those found in the vertebrate APEs.
A second Cys residue implicated in the redox activity is
Cys 93. Compared to the wt APE1, C93A has reduced
redox activity in both EMSA redox assays and transacti-
vation assays.
47
This finding suggests that it may also
play a role in the redox activity. Unless APE1 functions
as a dimeric redox factor like peroxiredoxin, then it
must have at least two redox active Cys residues.
Presumably, Cys 65 serves as the nucleophilic Cys and
potentially Cys 93 as the resolving Cys residue.
The report of a viable C64A knock-in mouse and data
within challenged the validity of a role for Cys 65 in the
redox activity of APE1.
97
This study suggests that Cys 65
or its equivalent Cys 64 in mouse is not essential for the
development of the mouse, potentially confirming the
presence of redundant redox systems. However, it
does not directly address the role of Cys 65 in APE1's
redox activity. In direct contradiction to the findings of
this study are the results of our own study in which
we substituted Thr 58 with Cys in zAPE thereby confer-
ring redox activity to the enzyme as measured both in
EMSA redox and transactivation redox assays.
47
To
date, the preponderance of evidence from a number of
different laboratories supports a role for Cys 65 in the
redox activity of hAPE1. We conclude that evolution of
redox activity in hAPE1 is coincident with the appear-
ance of Cys 65 in mammalian sequences.
the protein would be required in order for a disulfide
bond to form between these residues.
It is possible that, like peroxiredoxin, APE1
undergoes a conformation change that positions two
Cys residues in a more favorable position for disulfide
bond formation. An interesting similarity between
APE1 and peroxiredoxin is the proximity of a Cys
residue to a terminus. In the case of hAPE1, Cys 65 is
located relatively close to the N-terminus of the protein
and located within the first secondary structural element
(a beta strand) in the fold. In summary, hAPE1 is unique
as a redox factor having evolved this additional function
while maintaining its essential base excision repair
activity.
Following reduction of an oxidized protein, redox
factors are oxidized and must be reduced. Unlike the
TRX and GRX systems that include thioredoxin reduc-
tase and glutathione reductase, respectively, to ensure
reduction of the oxidized factors, APE1 does not have
an associated enzyme dedicated to its reduction. Two
proteins to date have been reported to reduce APE1,
TRX and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). Numerous reports have shown that TRX
reduces APE1 thereby stimulating its enhancement of
the reduction of transcription factors.
67
e
70
Glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH) is evolu-
tionarily conserved and potentially plays a key role in
redox sensing through its active site cysteine sulfhy-
dryl. GAPDH was shown to interact with APE1
through the active site cysteine 152 and convert the
“oxidized” form of APE1 to its reduced form reestab-
lishing its ability to cleave AP sites.
100
Oxidation of
APE1 significantly decreases its endonuclease
activity.
101
Reduction of APE1 enhanced detection by
anti-APE1 antibodies, suggesting a structural change,
and siRNA knockdown of GAPDH in HCT116 cells
enhanced sensitivity to alkylating DNA damaging
agent methyl methane sulfonate (MMS), which
produces AP sites, and increased the level of sponta-
neous AP sites in the genomic DNA. Thus, GAPDH
may play an important role in promoting BER activity
by maintaining APE1, a key AP endonuclease, in an
active reduced state.
Comparison of APE1 with Other Redox Factors
During the redox reaction, the redox factor is itself
oxidized, resulting in disulfide bond formation. Both,
TRX and GRX have Cys residues positioned appropri-
ately to form a disulfide bond. However, in the crystal
structures reported to date of APE1, there are no disul-
fide bonds present.
47,96,98,99
The Cys residues positioned
closest to one another are 93 and 208, but their respective
S atoms are too far apart to form a disulfide bond.
Further, these residues are buried in the core of the
protein and are not accessible. The critical redox residue
Cys 65 is also a buried residue. There are only two
solvent-accessible Cys residues, 99 and 138, but they
are not in close proximity to one another and would
not be expected to interact to form an intramolecular
disulfide bond. Furthermore, substitution of Ala for
either Cys 99 or 138 has no effect on redox activity.
95
Prior to the determination of the crystal structure of
APE1, it was proposed that Cys 65 and 93 form a disul-
fide bond.
95
Given the respective locations of Cys 65 and
Cys 93 in the protein, more than 8
˚
apart and posi-
tioned on opposite sides of the beta sheet (
Figure 11.6
),
a substantial conformational change in the structure of
Mechanism of Redox Regulation by APE1
Although roles for Cys 65 and Cys 93 in the redox
activity of hAPE1 have been established,
47,95
a detailed
mechanism has yet to be elucidated. One approach to
interrogating the mechanism by which APE1 reduces
transcription factors is to determine how E3330 inhibits
APE1's redox activity. To further characterize the redox
properties of APE1, biochemical and mass spectrometric
studies on the interaction of E3330 with APE1 were
performed.
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