Protein modification by adenine propenal

Article abstract of DOI:10.1021/tx500218g

Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. N^ε-Oxopropenyllysine, a lysine-lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is N^ε-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modi fication of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modi fi cation by adenine propenal dramatically reduces XPA's ability to bind to a DNA substrate. (Chemical Equation Presented).

Full text of DOI:10.1021/tx500218g

Article
pubs.acs.org/crt
Protein Modification by Adenine Propenal
Sarah C. Shuck,† Orrette R. Wauchope,† Kristie L. Rose,∥ Philip J. Kingsley,† Carol A. Rouzer,†
Steven M. Shell,†,# Norie Sugitani,‡,# Walter J. Chazin,†,‡,# Irene Zagol-Ikapitte,§ Olivier Boutaud,§
John A. Oates,§,∇ James J. Galligan,† William N. Beavers,‡ and Lawrence J. Marnett*,†,‡,§,⊥
†A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, ‡Chemistry, and §Pharmacology, ∥Mass
⊥
#
Spectrometry Research Center, Center in Molecular Toxicology, Center for Structural Biology, ∇Department of Medicine,
Vanderbilt Institute of Chemical Biology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-0146, United States
S
* Supporting Information
ABSTRACT: Base propenals are products of the reaction of DNA with oxidants such as
peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is
mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the
reaction of adenine propenal with protein has not yet been investigated. A survey of the
reaction of adenine propenal with amino acids revealed that lysine and cysteine form
adducts, whereas histidine and arginine do not. Nε-Oxopropenyllysine, a lysine−lysine
cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of
the reaction of adenine propenal with human serum albumin and the DNA repair protein,
XPA, revealed that the only stable adduct is Nε-oxopropenyllysine. The most reactive sites
for modification in human albumin are K190 and K351. Three sites of modification of
XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal
dramatically reduces XPA’s ability to bind to a DNA substrate.
lysine residues.12,13 XPA coordinates recruitment and assembly
of the enzymes that repair the damaged DNA, and mutations in
INTRODUCTION
■
The major reactive aldehyde produced during peroxidation of
polyunsaturated fatty acids, malondialdehyde (MDA), reacts
with nucleophilic sites of DNA and protein to form oxopropenyl
adducts. Reaction of MDA with DNA produces primarily 3-(2-
its DNA-binding domain are associated with the most severe
clinical XP phenotypes.11
Oxopropenyl adducts to DNA and protein can also be formed
through reaction with base propenal, a reactive aldehyde
produced by oxidant-mediated hydrogen abstraction from the
4′-position of DNA bases (Figure 1).14−17 Base propenals are
approximately 100-fold more reactive to DNA than MDA, and
experiments in which Escherichia coli strains of varying
membrane composition were exposed to oxidants demonstrated
that base propenals are more important than MDA as a source of
M1dG.18 Currently, very little is known about potential protein
targets of base propenal adduction; however, as a highly reactive
form of DNA damage, base propenals are ideally situated to
oxopropenylate DNA-binding proteins such as XPA. This has led
us to hypothesize that base propenal-mediated protein damage
contributes to toxicity and mutagenicity associated with oxidative
or electrophile stress. To test this hypothesis, a comprehensive
evaluation of base propenal’s ability to directly damage proteins
is required. Adenine propenal is known to react with glutathione
via Michael addition to form glutathionylpropenal and a
glutathione−adenine propenal cross-link,19 but, to date, no
detailed investigation of the reaction of any base propenal with
amino acids or proteins has been reported.
deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purine-
10(3H)-one (M1dG) and lesser amounts of 2′-oxopropenyl-
deoxyadenosine (Figure 1).1 M1dG is mutagenic in vitro and in
vivo, and 2′-oxopropenyl-deoxyadenosine alters DNA polymer-
ase activity.2−4
The reaction of MDA with protein occurs predominantly at
lysine residues under physiological conditions.5−9 Our under-
standing of the detailed chemistry and physiological con-
sequences of MDA−protein adduct formation is rudimentary,
as the exact targets and sites of adduction are largely unknown.
However, a particularly interesting effect of MDA exposure in
human cells is the inhibition of nucleotide excision repair (NER),
which is associated with increased sensitivity to the mutagenicity
of ultraviolet light and benzo[a]pyrene dihydrodiolepoxide.10
NER is the primary process for repair of bulky DNA adducts, and
in humans, NER deficiencies result in xeroderma pigmentosum, a
spectrum of disorders characterized by hypersensitivity to
sunlight, neurological degeneration, and a greatly increased
incidence of cancer.11 Although not verified experimentally,
inhibition of NER by MDA was proposed to arise from loss of
function of MDA-modified NER proteins.10 A potential MDA
target is the NER scaffold protein, xeroderma pigmentosum
complementation group A (XPA), which binds to the damaged
DNA substrate via a C-terminal basic cleft comprising multiple
Received: June 3, 2014
© XXXX American Chemical Society
A
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Figure 1. Schematic of base propenal and MDA production. Both species can react with dA or dG, resulting in oxypropenylation of the base. M1dG is
subsequently formed by ring closure of the oxypropenylated dG.
One approach to evaluate the protein adduction potential of
an electrophile is to identify all sites of modification and the
structures of those modifications using a model protein. A
frequently used target for this purpose is human serum albumin
(HSA).20,21 Albumin is highly abundant and has affinity for a
broad range of ligands, a relatively long half-life, and the potential
for noninvasive sampling. Thus, it is an ideal candidate for the
global qualitative and quantitative evaluation of exposure to
electrophiles of endogenous or exogenous origin (the
exposome). In fact, in a large multilaboratory comparison of
biomarkers of in vivo oxidant generation, the level of
carbonylated plasma protein (i.e., albumin) was one of only
two markers that correlated with oxidant exposure and tissue
pathology, and it was the only marker that was effective longer
than 6 h after exposure.22 To exploit albumin’s potential as a
biomarker, however, requires a more complete understanding of
its major sites of adduction and determinants of their reactivity to
a range of electrophiles. For example, whereas much attention
has been directed to HSA’s highly reactive Cys-34 as a sensor of
oxidant damage,23 the major site of oxopropenylation by MDA is
Lys-525.7
Here, we report the application of a recently described mass
spectrometric approach24 to identify adenine propenal mod-
ifications of HSA and XPA. Our data demonstrate that, like
MDA, adenine propenal primarily reacts at protein lysine
residues; however, the major site of attack on HSA is distinct
from that of MDA. We also report that adenine propenal
modifies lysine residues of XPA, including residues within its
DNA-binding domain and residues reported to be sites of
regulation via SIRT1-mediated deacetylation.12,13,25 Biophysical
analysis confirms that modification of XPA by adenine propenal
decreases its affinity for damaged DNA.
EXPERIMENTAL PROCEDURES
■
Materials. HSA was obtained from Abcam (Cambridge, MA). N-α-
Acetyllysine, trichloroacetic acid (TCA), dithiothreitol, iodoacetate
(Sigma Ultra), and NaCNBH3 were obtained from Sigma-Aldrich (St.
Louis, MO). Trypsin (gold) was obtained from Promega (Madison,
B
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WI). 2,2,2-Trifluoroethanol was obtained from Acros Organics
(Pittsburgh, PA).
δ (ppm) 1.30 (m, 4H), 1.59−1.61 (m, 6H), 1.73 (m, 2H), 1.88 (s, 6H),
3.22 (m, 2H), 3.30 (t, J = 7.3 Hz, 2H), 4.08 (m, 2H), 5.45 (m, 1H), 7.49
(d, J = 11.5 Hz, 1H), 7.56 (d, J = 11.5 Hz, 1H). 13C NMR (D2O): δ
(ppm) 21.3, 21.8, 25.7, 30.2, 42.0, 53.7, 88.5, 158.9, 160.5, 177.5, 178.9.
ESI-MS: [MH]+, m/z 413.3. (See Supporting Information Figures S8−
S10.)
Characterization of Compounds. NMR experiments were
acquired using a 14.0 T Bruker magnet equipped with a Bruker AV-III
console operating at 600.13 MHz. All spectra were acquired in 3 mm
NMR tubes using a Bruker 5 mm TCI cryogenically cooled NMR probe.
Chemical shifts were referenced internally to D2O (4.70 ppm), which
also served as the 2H lock solvent. For 1D 1H NMR, experiments were
acquired with presaturation in order to suppress the residual H2O signal,
and typical experimental conditions included 32K data points, 13 ppm
HPLC Analysis of N-α-Acetyllysine and Adenine Propenal
Reactions. N-α-Acetyllysine (various concentrations) was mixed with
10 mM adenine propenal in 50 μL reaction mixtures containing 10 mM
NaCl and 10 mM 4-morpholinepropanesulfonic acid (MOPS) (pH
6.5). At 1 h intervals, aliquots of each reaction mixture were injected
onto a Waters HPLC system equipped with a 717 Plus autosampler,
1525 Binary HPLC pump, and 2996 photodiode array detector.
Analytes were separated using a Phenomenex Polar-RP Synergi HPLC
column, 75 cm × 2 mm with 4 μm particle size, at a flow rate of 0.6 mL/
min. The mobile phase solvents were H2O + 0.05% acetic acid (v/v,
solvent A) and 3:1 MeOH/ACN + 0.05% acetic acid (v/v, solvent B)
delivered in a 19 min gradient consisting of the following: 0−10.5 min,
5−20% B; 10.5−12.5 min, 20−80% B; 12.5−16 min, 80% B; 16−19
min, 5% B. Data were analyzed using Empower software, and peak areas
were manually integrated. Eluting peaks were collected and analyzed by
MS (see below).
MS Analysis of the Reaction Products of Adenine Propenal
and N-α-Acetyllysine. Reactions were performed as described above
and analyzed using a Thermo Accela HPLC pump (Thermo Fisher
Scientific) and HTC PAL autosampler (LEAP Technologies) in-line
with a Thermo Quantum triple quadrupole mass spectrometer (Thermo
Fisher Scientific). The Quantum was equipped with an electrospray
source operated in Q1 full scan, positive ion mode. Q1 was scanned from
100−600 amu in 0.45 s. Analytes were chromatographically separated
on a Synergi Polar-RP HPLC column (Phenomenex, 7.5 × 0.2 cm, 4 μm
particle size) using the following gradient: 0−10.5 min, 5−20% B; 10.5−
12.5 min, 20−80% B; 12.5−16 min, 80% B; 16−17 min, 5% B. The
mobile phase solvents were H2O + 0.05% acetic acid (v/v, solvent A)
and acetonitrile + 0.05% acetic acid (v/v, solvent B), and they were
delivered at a flow rate of 0.6 mL/min. All data were acquired and
analyzed using Thermo Xcalibur software.
1
sweep width, a recycle delay of 1.5 s, and 32 scans. For 2D H−1H
COSY, presaturation was also performed, and experimental conditions
included a 2048 × 1024 data matrix, 13 ppm sweep width, recycle delay
of 1.5 s, and 8 scans per increment. The data were processed using
squared sinebell window function, symmetrized, and displayed in
magnitude mode. Multiplicity-edited 1H−13C HSQC experiments were
acquired using a 1024 × 128 data matrix, a J(C−H) value of 145 Hz,
which resulted in a multiplicity selection delay of 34 ms, a recycle delay
of 1.5 s, and 80 scans per increment along with GARP decoupling on 13C
during the acquisition time (150 ms). The data were processed using a
p/2 shifted squared sine window function and displayed with CH/CH3
signals phased positive and CH2 signals phased negative. J1(C−H)
filtered 1H−13C HMBC experiments were acquired using a 2048 × 128
data matrix, a J(C−H) value of 9 Hz for detection of long-range
couplings resulting in an evolution delay of 55 ms, J1(C−H) filter delay
of 145 Hz (34 ms) for the suppression of one-bond couplings, a recycle
delay of 1.5 s, and 144 scans per increment. The HMBC data were
processed using a p/2 shifted squared sine window function and
displayed in magnitude mode. Compounds were greater than 90% pure,
and mass spectral analysis was accomplished on an Applied Biosystems
3200 Q trap mass spectrometer (MDS Sciex). The mass spectrometer
was equipped with an electrospray source and operated in positive ion
mode.
Synthesis of Adenine Propenal. Adenine propenal was
synthesized as previously described.23,26 NMR data were consistent
with literature values. 1H NMR (DMSO-d6): δ (ppm) 7.17 (dd, J1 = 7.88
Hz, J2 = 14.4 Hz, 1H), 7.55 (br s, 2H), 8.27 (s, 1H), 8.35 (d, J = 14.4 Hz,
1H), 8.63 (s, 1H), 9.67 (d, J = 7.88 Hz, 1H). 13C NMR (DMSO-d6): δ
(ppm) 119.2, 120.5, 136.4, 142.8, 147.6, 151.4, 158.2, 190.8.
Spectrophotometric Quantification of the Lysine−Lysine
Cross-Link (4) in Reaction Mixtures of N-α-Acetyllysine and
Adenine Propenal. Solutions containing known concentrations of
synthetic 3 and 4 were used to measure extinction coefficients of each
compound. The value obtained for both compounds (33 600 200
M−1cm−1) was the same within experimental error; however, the
wavelengths of maximal absorbance were different (280 nm for 3 and
300 nm for 4) (Figure S1).
Preparation of the Sodium Salt of Malondialdehyde (MDA).
Sodium malondialdehyde was synthesized as previously described.28
1
NMR data were consistent with literature values. H NMR (D2O): δ
(ppm) 9.15 (d, J = 10.0 Hz, 2H), 5.73 (t, J = 10.0 Hz, 1H). 13C NMR
(D2O): δ (ppm) 193.3, 110.7.
Preparation of Nε-Oxopropenyl-N-α-acetyllysine (3). The
sodium salt of malondialdehyde (MDA) (100 mg, 1.06 mmol) was
added to a solution of N-α-acetyllysine (200 mg, 1.06 mmol) in 5 mL of
ammonium acetate buffer (pH 4.5). The reaction was then stirred at
room temperature for 15 h. The reaction mixture was lyophilized, and
the residue was purified by column chromatography (EtOAc/MeOH,
Adenine propenal (10 mM) was incubated with increasing
concentrations of N-α-acetyllysine (1, 2, 5, 10, 50, and 100 mM) in
reaction mixtures containing 10 mM NaCl and 10 mM MOPS (pH 6.5).
The mixtures were incubated for 2 h at room temperature and then
diluted 1:1000 in H2O. The absorbance at 300 nm of each dilution was
then obtained using a DU800 spectrophotometer (Beckman Coulter),
and the experimental extinction coefficient of 4 was used to estimate its
concentration in the reaction mixture. Note that, for reaction mixtures
containing >10 mM N-α-acetyllysine, complete consumption of adenine
propenal eliminates concerns of its contribution to the absorbance of the
final reaction mixture. The adenine product does not absorb significantly
at 300 nm (Figure S1). On the basis of its full UV spectrum (Figure S1),
the extinction coefficient of 3 at 300 nm is estimated to be 7.8 × 103 M−1
cm−1. The presence of this product at a concentration equal to
approximately 10% of the concentration of 4, as found in the reaction
mixtures, indicates that its contribution to the absorbance at 300 nm will
be no more than 3%. Consequently, the absorbance at 300 nm can be
used to estimate the concentration of 4 with reasonable accuracy.
Reaction of N-α-Acetyllysine with Adenine Propenal under
Reducing Conditions. N-α-Acetyllysine (10 mM) was incubated with
adenine propenal (10 mM) in 10 mM MOPS (pH 6.5) containing 10
mM NaCl in a 200 μL reaction mixture for 30 min. After incubation, 50
mM NaCNBH3 (in H2O) (Sigma-Aldrich) was added for an additional
30 min, and samples were then analyzed using LC-MS as described
below.
1
5:1) on silica gel (60 Å porosity, 40−63 μM particle size). H NMR
(D2O): δ (ppm) 1.33 (m, 2H), 1.56−1.61 (m, 3H), 1.71 (m, 1H), 1.93
(s, 3H), 2.91 (t, J = 7.3 Hz, 2H), 4.05 (m, 1H), 5.31 (m, 1H), 7.44 (d, J =
13.0 Hz, 1H), 8.59 (d, J = 8.5 Hz, 1H). 13C NMR (D2O): δ (ppm) 22.0,
23.4, 27.2, 32.0, 39.3, 55.3, 86.6, 163.7, 173.7, 179.1, 191.2. ESI-MS:
[MH]+, m/z 243.4. (See Supporting Information Figures S5−S7.)
Preparation of the Lysine−Lysine Cross-Link (4). The sodium
salt of MDA (60 mg, 0.319 mmol) was added to a solution of N-α-
acetyllysine (15 mg, 0.159 mmol) in 2 mL of anhydrous methanol. The
mixture was heated at reflux, and the progress of the reaction was
monitored by UV−vis spectroscopy. The product was then purified by
column chromatography (EtOAc/MeOH, 5:1) on silica gel (60 Å
porosity, 40−63 μM particle size) and HPLC. HPLC chromatography
was carried out on a Luna C18 column (250 × 4.60 mm, 5 μm) at a flow
rate of 1.25 mL/min. The eluting solvents included solvent A (10 mM
ammonium acetate) and solvent B (acetonitrile). The following solvent
gradient was used: 0−10.5 min linear gradient from 5% B to 20% B,
followed by a 2 min linear gradient to 80% B, hold for 6.50 min at 80% B,
followed by a 1 min linear gradient to the initial conditions of 5% B. The
product was observed with a retention time of 6.1 min. 1H NMR (D2O):
C
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Analysis of Reduced Intermediate (6) by LC-MS. LC-MS
analysis was performed on the Thermo Quantum triple quadrupole
mass spectrometer. Here, the instrument was operated in Q1 full-scan
negative ion mode using electrospray ionization. Q1 was scanned over
the range 100−400 amu over 0.5 s. Chromatography was carried out on
a Synergi Max-RP column (Phenomenex, 7.5 × 0.2 cm, 5 μm particle
size) using the following gradient: 0−5 min linear gradient from 2% B to
50% B, 5−7 min at 50% B, 7−10 min 50% B to 2% B. The mobile phase
solvents were H2O + 0.05% formic acid (v/v, solvent A) and 3:1
CH3CN/MeOH + 0.05% formic acid (v/v, solvent B), and they were
delivered at a flow rate of 400 μL/min. All data were acquired and
analyzed using Thermo Xcalibur software. Using this chromatographic
system, the reduced intermediate eluted at approximately 2.9 min.
Analysis of the Reaction of N-Acetylcysteine with Adenine
Propenal. N-Acetylcysteine (200 μM) was combined with 50 μM
adenine propenal in 50 mM sodium phosphate buffer, pH 7.4, and
incubated for 6 h at 37 °C. Unreduced sample was immediately frozen.
NaBH4 (50 mM) (Sigma-Aldrich) was used to reduce samples as
indicated. Analytes were separated on a Supelco Ascentis C18 column
(50 × 2.1 mm, 3 μm) with buffers A and B comprising 0.1% formic acid
in water and 0.1% formic acid in acetonitrile, respectively. The gradient
was as follows: 1−3 min, 1% B; 3−7 min, 98% B; 7−11 min, 98% B; 11−
17 min, 1% B. Samples were analyzed on a ThermoFinnigan TSQ
Quantum mass spectrometer with ESI source interfaced to a
ThermoFinnigan MS pump plus and autosampler plus (Thermo, San
Jose, CA). N-Acetylcysteine-adducted species were analyzed with data-
dependent scanning enabled in negative ion mode, scanning from m/z
150−650 over 0.5 s. The top two m/z peaks from each MS scan were
fragmented with 10 eV collision energy for 0.5 s.
LC-MS/MS Analysis of HSA Modification by Adenine
Propenal. HSA (15 μM) (Abcam, Cambridge, MA) was incubated
with increasing amounts of adenine propenal (0.15, 0.38, 1.5, 3, and 7
mM), representing a molar excess of adenine propenal to HSA of 10, 25,
100, 250, and 500, respectively. Reactions were incubated for 6 h at
room temperature in a buffer containing 10 mM NaCl and 10 mM
MOPS (pH 6.5). Following incubation, samples were precipitated with
25% trichloroacetic acid (Sigma-Aldrich) on ice for 1 h, washed with
cold acetone, dried, and reconstituted in 50 mM Tris (pH 8.0)
containing 50% 2,2,2-trifluoroethanol (Acros Organics, Pittsburgh, PA).
Samples were reduced with DTT (Sigma-Aldrich), carbamidomethy-
lated with iodoacetamide (Sigma-Alrich), and diluted 5-fold with 100
mM Tris (to obtain a final solution containing 10% 2,2,2-
trifluoroethanol). Following dilution, samples were digested with
sequencing-grade trypsin (Promega, Madison, WI) overnight, acidified,
and diluted in 0.1% formic acid. The resulting solutions of trypsin-
generated peptides were loaded onto a capillary reverse-phase analytical
column (360 μm o.d. × 100 μm i.d.) using an Eksigent NanoLC Ultra
HPLC and autosampler. The 20 cm analytical column was directly
packed into a laser-pulled emitter tip using Jupiter C18 reverse-phase
medium (3 μm beads, 300 Å pore size, Phenomenex). Mobile phase
solvents consisting of 0.1% formic acid in water (solvent A) and 0.1%
formic acid in acetonitrile (solvent B) at a flow rate of 500 nL/min were
used to elute the peptides. The 90 min gradient consisted of the
following: 0−10 min, 2% B; 10−50 min, 2−35% B; 50−60 min, 35−
95% B; 60−65 min, 95% B; 65−70 min 95−2% B; 70−90 min, 2% B.
Mass analysis of eluting peptides was performed on an LTQ Orbitrap
XL mass spectrometer (Thermo Scientific), equipped with a nano-
electrospray ionization source with detection in positive ion mode. The
LTQ Orbitrap was operated using a data-dependent method, enabling
dynamic exclusion. Full scan (m/z 400−2000) spectra were acquired
with the Orbitrap (resolution 60 000), and the five most abundant ions
in each MS scan were selected for fragmentation in the LTQ. An
isolation width of 2 m/z, 30 ms activation time, and collision energy
normalized to 35% were used to generate MS2 spectra. Dynamic
exclusion settings allowed for a repeat count of 2 within a 10 s duration,
with exclusion duration time set to 15 s. For identification of modified
peptides, tandem mass spectra were searched with SEQUEST (Thermo
Fisher Scientific) against a human subset database created from the
UniProtKB protein database (www.uniprot.org). Variable modifications
of +57.0214 on Cys (carbamidomethylation), +15.9949 on Met
(oxidation), and +54.0105 on Lys or Cys (oxypropenylation) were
included for database searching. Search results were assembled with
Scaffold 3.0 (Proteome Software) using threshold filtering criteria
consisting of 95% peptide probability to achieve a peptide false discovery
rate estimate of ≤0.2%. Sites of modification were validated by manual
interrogation of tandem mass spectra using Xcalibur 2.1 Qual Browser
software (Thermo Scientific). Calculations of mass errors (in ppm) of
precursor ions mass analyzed in the Orbitrap were performed using the
following equation: ((theoretical mass − observed mass)/theoretical
mass) × 106. To obtain theoretical masses, the Protein Prospector MS-
Product tool was used (v.5.10.4. UCSF). Analysis of cross-links was
performed in a similar manner with inclusion of +36 on Lys for database
searching.
Proteolytic Digestion of HSA Treated with Adenine Propenal
for Lysine−Lysine Cross-Link (4) Identification. Purified HSA
(0.75 μM) was incubated with increasing concentrations of adenine
propenal in reaction mixtures containing 10 mM NaCl and 10 mM
MOPS (pH 6.5) for 6 h at room temperature. Following incubation, the
protein was heated at 95 °C for 10 min, cooled to room temperature,
and then treated with 1 mg of Pronase (Calbiochem, Gibbstown, NJ)
per mg of protein at 37 °C for 24 h. Pronase was inactivated by heating at
95 °C for 10 min, followed by cooling to room temperature, and treating
with 1 μL of aminopeptidase M (Calbiochem, Gibbstown, NJ) at 37 °C
for 24 h. A final incubation at 95 °C for 10 min inactivated the
aminopeptidase M, and the samples were then cooled to room
temperature in preparation for adduct purification.
Purification of Lysine−Lysine Cross-Links (4) and Analysis by
MS. For use as an internal standard, [3H]-[14C]-4 was prepared by
reaction of MDA with [3H]-lysine containing [14C]-lysine as a tracer and
quantified using a method similar to that described for the [3H]-[14C]-
lysine-lactam internal standard as described.29 An Oasis HLB cartridge
(1 cm3) (Waters, Milford, MA) was equilibrated with 2 mL of methanol
followed by 2 mL of H2O. Following addition of [3H]-[13C]-4, the
sample was loaded onto the cartridge using gravity flow. The cartridge
was washed with 1 mL of H2O, and 4 was eluted with 3 mL 1:1
methanol/ethyl acetate and dried to 100 μL under N2 gas. The sample
was then diluted in 0.1% formic acid solution to a final volume of 1 mL
and filtered using a 0.22 μm nylon Spin-x centrifuge tube spun at 6000
rpm for 5 min.
Samples were purified using a Thermo Scientific Aquasil C18 5 μm
reverse-phase column using liquid scintillation counting to monitor
elution of the radioactive [3H]-[13C]-lysine−lysine cross-link standard.
The internal standard [3H]-[14C]-lysine−lysine cross-link was prepared
by reaction of MDA with [3H]-lysine containing [14C]-lysine as a tracer
and quantified using a method similar to that described for the [3H]-
[14C]-lysine-lactam internal standard.28 For quantitation, the transition
between m/z 341 and m/z 306 was monitored; it corresponds to the loss
of one molecule of water and one molecule of ammonia. Portions of
samples containing the standard were then concentrated using 1 cc
Oasis HLB columns and dried under nitrogen. The purified samples
were analyzed using a Thermo Scientific TSQ Vantage quadrupole mass
spectrometer equipped with Xcalibur software for data collection and
manipulation. All measured MDA-adduct signal in each sample was
divided by the signal for the [3H]-[13C]-lysine−lysine cross-link
standard of known concentration to quantify experimental results.
These methods will be published in detail elsewhere.
Proteomics Analysis of XPA Modification by Adenine
Propenal. XPA was expressed and purified as described.30 To modify
XPA, 3.2 μM of purified protein was incubated with 500× (1.6 mM) of
adenine propenal for 6 h at room temperature in a buffer containing 10
mM NaCl and 10 mM MOPS (pH 6.5). Modifications were identified as
described above for HSA.
Fluorescence Anisotropy DNA Binding Assay. A high-
throughput fluorescence anisotropy assay was used to measure the
DNA-binding activity of XPA.31,32 The substrate for this assay was a
fluorescein-labeled Y-shaped ssDNA−dsDNA junction substrate
containing 8 base pairs of duplex and 12 nucleotide noncomplementary
ssDNA overhangs at both the 3′ and 5′ ends. XPA was modified with
NHS−biotin as described previously.33 Modified or mock-treated XPA
protein and the DNA substrate were diluted in binding buffer (20 mM
D
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Figure 2. HPLC analysis of an N-α-acetyllysine and adenine propenal reaction mixture. N-α-Acetyllysine (50 mM) was incubated with adenine propenal
(10 mM) in a 50 μL reaction mixture for 2 h at room temperature. Following incubation, 4 μL of the reaction mixture was analyzed by HPLC. The
column eluate was monitored at 260 nm (A) or 300 nm (B). Analytes present in each peak were subjected to MS, UV, and NMR spectrometry. Numbers
correspond to the identified products, represented in (C).
Figure 3. HPLC analysis of product formation in the reaction of adenine propenal and N-α-acetyllysine. The indicated amounts of N-α-acetyllysine
(NAL) were incubated with 10 mM adenine propenal, and reactions were monitored hourly for 9−10 h. Peaks corresponding to 3 and 4 were monitored
at 280 and 300 nm, respectively, by diode-array detection. Data show the mean SD of three determinations.
HEPES, pH 7.9, 75 M KCl, 5 mM MgCl2, 5% glycerol, 1 mM DTT).
DNA substrate was added to a final concentration of 50 nM.
Fluorescence anisotropy was measured using a Synergy H1 plate reader
(λEx = 485 nm, λEm = 528 nm).30 Binding measurements were acquired
in triplicate for each DNA substrate. Apparent dissociation constants
(Kd) were determined for each individual titration by plotting
fluorescence anisotropy against protein concentration and fitting to a
simple two-state binding model with KaleidaGraph (v4.03) software.
RESULTS
■
Adenine Propenal Modifies N-α-Acetyllysine, Forming
Nε-Oxopropenyl-N-α-acetyllysine (3) and Lysine−Lysine
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Figure 4. Proposed scheme for the reaction of adenine propenal with N-α-acetyllysine.
Figure 5. LC-MS analysis of the reduced reaction intermediate (6). (A) Representative LC-MS chromatogram of 6. In this example, 5 mM N-α-
acetyllysine was reacted with 10 mM adenine propenal for 30 min followed by reduction with 50 mM NaCNBH3. (B) Mass spectrum of the parent ion of
the reduced intermediate displaying m/z 360 with detection in negative ion mode.
Cross-Links (4). Adenine propenal was reacted with N-α-
acetyllysine, N-α-acetylhistidine, N-α-acetylarginine, or N-α-
acetylcysteine in 10 mM NaCl buffered with 10 mM MOPS (pH
6.5). Reaction progress was monitored hourly by HPLC using a
C18 reverse-phase column for product separation. Adenine
propenal eluted at ∼8 min and displayed a maximum absorbance
of 260 nm (Figures 2 and S1). Reaction with N-α-acetyllysine
resulted in the appearance of three new chromatographic peaks
at 1.0, 4.8, and 5.3 min, which were analyzed by UV/vis, NMR,
and MS (Figures 2 and S1). The peak eluting at 1 min displayed a
UV absorbance maximum at 260 nm and an m/z of 136,
corresponding to protonated adenine ([M + H]+) (1). The 4.8
min peak displayed a UV maximum at 280 nm. On the basis of
NMR analysis, this product was identified as 3, and LC-MS
analysis gave an m/z of 243, which corresponds to the
protonated species ([M + H]+). The 5.3 min peak, with a
maximum absorbance at 300 nm and an m/z of 413, which
corresponds to the protonated species ([M + H]+), was
identified as 4 by NMR analysis. The total amount of product
formed was unaffected by variations in pH over a range of 6.5 to
9.5.
cm−1 at 260 nm). HPLC analysis of reaction mixtures of N-α-
acetyllysine and adenine propenal using diode-array detection
enabled direct comparison of peak areas for each compound
measured at its optimal wavelength (Figure 3). The essentially
identical absorbance coefficients of 3 and 4 allowed peak areas to
be used to estimate relative product quantity. These comparisons
indicated that the major product formed from the reaction was 4,
with much lower quantities of 3.
Variation of the amount of N-α-acetyllysine (from 2 to 100
mM) in reaction mixtures while holding the amount of adenine
propenal (10 mM) fixed demonstrated that the amount of 3
formed increased with N-α-acetyllysine concentrations from 2 to
10 mM and then decreased somewhat at the higher
concentrations. In contrast, the amount of 4 produced increased
with N-α-acetyllysine concentrations up to 50 mM. On the basis
of the relative peak area, the ratio of 4 to 3 in most reaction
mixtures was approximately 10:1. Estimation of the concen-
tration of 4 using absorbance at 300 nm indicated a maximum of
∼9 mM formed in reaction mixtures containing 50−100 mM N-
α-acetyllysine (Figure S2). These results are consistent with
complete consumption of adenine propenal under those
conditions.
Chemical synthesis of 3 and 4 yielded standards that verified
the product structure assignments. The molar extinction
coefficients of the pure compounds (ε = 33.6 × 103 M−1 cm−1
at 280 nm for 3 and ε = 33.6 × 103 M−1 cm−1 at 300 nm for 4)
were similar to that of adenine propenal (ε = 32.5 × 103 M−1
Our initial hypothesis for the production of 4 was that it arose
by reaction of N-α-acetyllysine with 3, but this reaction did not
occur under conditions comparable to those used for the reaction
of adenine propenal with N-α-acetyllysine. An alternative
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Figure 6. Human serum albumin modification by adenine propenal. (A) Modified lysine residues identified in Table 1 were mapped to the crystal
structure of HSA (1AO6). Lysine residues with an additional mass of +54.0105 are indicated in red. (B) The most highly modified residues by individual
electrophiles are highlighted with the modifying electrophile noted.
a
Table 1. Modified Lysine Residues within HSA
molar ratio adenine propenal:HSA
residue
peptide
10:1
25:1
100:1
250:1
500:1
Lys-28 (N/A)
Lys-36 (12)
DAHK*SEVAHR
FK*DLGEENFK
K*YLYEIAR
HPYFYAPELLFFAK*R
LDELRDEGK*ASSAK
LDELRDEGKASSAK*QR
AFK*AWAVAR
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
Lys-161 (137)
Lys-183 (159)
Lys-214 (190)
Lys-219 (195)
Lys-236 (212)
Lys-249 (225)
Lys-257 (233)
Lys-347 (323)
Lys-375 (351)
Lys-402 (378)
Lys-413 (389)
Lys-438 (414)
Lys-549 (525)
Lys-569 (545)
Lys-598 (574)
●
●
●
●
●
●
●
●
●
●
●
●
●
FPK*AEFAEVSK
AEFAEVSK*LVTDLTK
NYAEAK*DVFLGMFLYEYAR
LAK*TYETTLEK
VFDEFK*PLVEEPQNLIK
VFDEFKPLVEEPQNLIK*
K*VPQVSTPTLVEVSR
K*QTALVELVK
●
●
●
●
●
●
●
●
●
●
●
●
●
EQLK*AVMDDFAAFVEK
K*LVAASQAALGL
●
a
HSA was treated at the indicated molar ratios of adenine propenal and analyzed for lysine residues modified with +54.0105. The presence of each
modified lysine in a sample is indicated with ●. The modified lysine in each peptide sequence is indicated with an asterisk. Equivalent lysine residues
within the crystal structure of HSA are provided in parentheses.
reaction scheme, in which an enaminoimine cross-link between
adenine propenal and N-α-acetyllysine is first formed and then
reacts with H2O to produce 3 or another equivalent of N-α-
acetyllysine to form 4, is shown in Figure 4. If this scheme is
correct, then our data indicate that, at the concentrations of N-α-
acetyllysine and adenine propenal used here, reaction of the
proposed intermediate with another molecule of N-α-acetylly-
sine is more favorable than its reaction with water.
HPLC analysis with UV or in-line NMR was unable to detect
the putative intermediate 5 (Figure 4). Therefore, we attempted
to trap it by reduction with NaCNBH3. Adenine propenal was
incubated with N-α-acetyllysine in 10 mM NaCl/10 mM MOPS
(pH 6.5) in the presence of NaCNBH3. We utilized LC-MS
analysis of the reaction mixture to detect ions with m/z 360,
which corresponds to the partially reduced intermediate (6). A
peak corresponding to this mass eluted at 2.9 min, consistent
with the transient formation of the adenine propenal adduct to
N-α-acetyllysine (Figure 5).
Reactions of adenine propenal with N-α-acetylhistidine and N-
α-acetylarginine did not result in the formation of new
chromatographic peaks. Reaction with N-α-acetylcysteine
resulted in the formation of an oxopropenylated product,
which displayed m/z 216, corresponding to the deprotonated
molecular ion, and its major fragmentation product, m/z 86.9.
Treatment of the reaction mixture with NaBH4 yielded the
partially reduced product (m/z 218) and its major fragmentation
product (m/z 89) (Figure S3).
Adenine Propenal Reacts Predominantly with Lys-214
and Lys-375 in HSA. To ascertain adenine propenal’s ability to
react with protein, we incubated increasing amounts of the
electrophile with a fixed amount of purified HSA for 6 h, and the
samples were prepared for LC-MS/MS by trichloroacetic acid
precipitation, resolubilization of the precipitated protein pellet,
reduction and carbamidomethylation of cysteine residues, and
proteolytic digestion with sequencing-grade trypsin. The
resultant proteolytic peptides were analyzed via LC-MS/MS
using an LTQ-Orbitrap Velos mass spectrometer with collision-
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induced dissociation. Sequence coverage for unmodified HSA
was found to be 90%, but exposure to increasing concentrations
of adenine propenal reduced coverage to a minimum value of
62% (Table S1). HSA peptides were interrogated for the
presence of a +54.0105 mass increase, as would be expected from
the addition of an oxopropenyl group. To verify positively
identified residues, data were manually examined using Xcalibur
software. Extracted ion chromatograms were generated from
precursor peptide masses, and theoretical masses calculated from
Protein Prospector were compared to observed masses to
calculate ppm mass error. For modified precursor peptides
displaying a ppm mass error less than 10, MS/MS spectra were
interrogated with Xcalibur software. At least 40% of each
theoretical b- and y-product ion series was present in the
observed product ion spectra of positively identified peptides,
and 50% sequence coverage was obtained for each peptide
validated. To validate the site of adduction, at least two ions
within a product ion series with a +54.0105 adduction mass shift
were required. Multiple lysine residues were found to be
modified in adenine propenal-exposed HSA, including Lys26,
Lys36, Lys161, Lys183, Lys214, Lys219, Lys236, Lys249,
Lys257, Lys347, Lys375, Lys402, Lys413, Lys438, Lys549,
Lys569, and Lys598. Residues verified to have a +54.0105
adduction mass were mapped to the crystal structure of HSA
(PDB ID: 1AO6) and were found to be largely confined to the
protein surface (Figure 6A). The 17 modified lysine residues
represented 28% of the total lysines within the protein (Table 1).
Although we determined that adenine propenal can also form
oxopropenylated cysteine, searching for adducts on cysteine
residues within HSA did not result in the identification of any
modified sites. S-Oxopropenal-N-acetylcysteine was stable to
treatment with TCA as well a DTT, which are reagents used for
proteomic analysis, so any oxopropenylated cysteine residues on
HSA-derived peptides should have survived the workup prior to
mass spectrometry.34 Therefore, adenine propenal is acting as a
lysine-specific modifier of HSA.
The use of HSA as a biomarker has primarily been described in
the context of electrophilic modifications at Cys34.23 Adenine
propenal did not modify Cys34, so we sought to identify the most
reactive lysine residue(s) that might be used as a biomarker for
modification by adenine propenal. We reduced the levels of
adenine propenal relative to protein to a ratio of 10:1, which was
the lowest ratio at which modification was observed. Using the
interrogation methods described above to verify adduction sites,
two lysine residues, K190 and K351, displayed the highest
reactivity (Table 1). Mapping of those residues to the crystal
structure of HSA revealed that both are located within domain II,
a recognized binding region for many compounds (Figure 6A).35
Investigation of Cross-Link Formation in the Reaction
of Adenine Propenal with HSA. Decreased HSA sequence
coverage observed with increasing concentrations of adenine
propenal suggested that adducts impede protein digestion and/
or fragmentation required for peptide identification. As lysine−
lysine cross-links are formed from the reaction of adenine
propenal with N-α-acetyllysine, we hypothesized that at high
concentrations of adenine propenal, lysine−lysine cross-links
may be induced in HSA. To investigate the presence of cross-
links, we interrogated previously acquired proteomics data for a
+36 adduct mass, corresponding to an unreduced 3-carbon
linkage between lysine residues.31 This analysis did not result in
the identification of any lysine−lysine cross-links, which was not
surprising considering the decreased sequence coverage and
potential hindrance of peptide fragmentation by cross-links.
Although cross-linked sites were not identified with these
algorithm-based data searching methods, we manually inter-
rogated the data to look for the presence of precursor peptides
containing cross-linked residues. To predict lysine residues likely
to participate in cross-link formation, we performed in silico
analysis of the HSA crystal structure (1AO6). As a typical
carbon−carbon bond is approximately 1.5 Å in length, we
hypothesized that the 3-carbon cross-link would be ∼5−7 Å
long. Flexible, dynamic protein movements in solution may
result in cross-link formation between lysines at a distance
greater than 7 Å. We therefore examined the crystal structure to
identify lysine residue pairs separated by a distance of less than or
equal to 20 Å as potential sites of cross-link formation. Following
identification of these residues, tryptic peptide sequences were
determined from the primary sequence, and theoretical peptide
masses containing the cross-link mass of +36 were calculated
using Protein Prospector. We then thoroughly interrogated the
data manually for the presence of ions of the theoretical
precursor peptide masses. No evidence for the presence of cross-
link-containing peptides was obtained.
As proteomics-based mass spectrometric analyses were
unsuccessful, we employed a different analytical approach to
search for lysine−lysine cross-links, in which modified HSA was
degraded to the level of amino acids, and the resultant
hydrolysate was analyzed by MS. This methodology employed
selected reaction monitoring to identify analytes exhibiting a
mass transition from m/z 329 to m/z 294, as exhibited by analysis
of the lysine−lysine cross-link standard. Addition of a [3H]-
[13C]-4 internal standard (m/z 341 to m/z 306 transition)
enabled identification of analyte-containing fractions during
purification by scintillation counting and quantification by stable
isotope dilution. Application of this approach to HSA that had
been incubated with adenine propenal resulted in the
identification of 30 12 ng cross-link/mg protein in vehicle-
treated HSA and 57 17 ng cross-link/mg protein in adenine
propenal-treated HSA. These differences were not statistically
significant, supporting the conclusion that adenine propenal did
not induce intramolecular cross-links in HSA.
Adenine Propenal Oxopropenylates XPA. Having
confirmed adenine propenal’s capacity to modify lysine residues
in HSA, we investigated its reaction with XPA. Adenine propenal
was incubated with XPA for 6 h at room temperature, and the
samples were prepared for and analyzed by LC-MS/MS using the
same methods as described for HSA. As for HSA, peptides were
interrogated for the presence of a +54.0105 mass addition, and
rigorous criteria were applied to peptide and site of adduction
identification. The data revealed six modified residues, listed in
Table 2. Of note, three of these, K145, K183, and K204, are
Table 2. XPA Lysine Modifications Induced by Adenine
a
Propenal
residue
peptide
Lys-63
Lys-67
Lys-86
Lys-145
Lys-183
Lys-204
PYSATAAAATGGMANVK*AAPK
AAPK*IIDTGGGFILEEEEEEEQK
IIDTGGGFILEEEEEEEQK*IGK
TEAK*QEYLLK
NPHHSQWGDMK*LYLK
SLEVWGSQEALEEAK*EVR
a
Purified XPA was incubated with a 500-fold molar excess of adenine
propenal and analyzed for lysine residues modified with +54.0105. The
modified lysine in each peptide sequence is indicated with an asterisk.
H
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Figure 7. Adenine propenal modifies XPA and reduces its DNA-binding activity. (A) Lysine residues modified with an adduct mass of +54.0105 were
mapped to the XPA NMR solution structure (1D4U). (B) Plots of the change in fluorescence anisotropy for a Y-shaped 8/12 ssDNA−dsDNA junction
substrate (50 nM) versus added protein treated with DMSO, adenine propenal, or NHS-biotin. Each data point represents the mean SD of three
titrations.
located in the proposed DNA-binding domain of XPA (Figure
7A),12,13 and two, K63 and K67, are known sites of acetylation
that reduces XPA’s interaction with replication protein A.25
Modification of residues within the XPA DNA-binding
domain has the potential to affect DNA-binding activity. Using
a plate-based fluoresence anisotropy assay, the effect of adenine
propenal-mediated oxopropenylation on XPA’s affinity for a Y-
shaped ssDNA−dsDNA junction substrate was evaluated.32
Fitting of the data for binding of unmodified XPA to the
fluorescein-labeled 20-nucleotide substrate provided a dissocia-
tion constant (Kd) of 300 30 nM (Figure S4). Binding affinities
for the 20-nucleotide substrate were measured for purified XPA
incubated with vehicle (DMSO), adenine propenal, or NHS−
biotin (biotin N-hydroxysuccinimide ester) (Table 3).33 As
cellular function, including mutagenesis, stress responses, cellular
dysfunction, and ultimately cell death. All of these may
contribute to pathological processes such as carcinogenesis,
neurodegeneration, atherosclerosis, and chronic inflammation.
However, elucidation of the exact mechanisms by which specific
macromolecular damage leads to pathology requires a better
understanding of the sites and nature of that damage.
The most frequently used biomarker of electrophile damage to
protein during oxidative stress is the change in global protein
carbonylation.38 Although useful as a general indicator that stress
has occurred, this approach does not identify the exact type of
modification or the modifying electrophile. Recent develop-
ments in highly sensitive MS-based proteomics techniques have
allowed for expansion of this approach to the investigation of the
specific types of modifications induced and the impact of these
modifications on protein activity. The resultant increased ability
to identify specific protein modifications induced by a single
electrophile enables the search for biomarkers that distinguish
between different mechanisms of oxidant damage.
Table 3. Dissociation Constants for XPA Binding to Y-Shaped
a
ssDNA−dsDNA Junction Substrate
treatment
Kd (μM)
unmodified
DMSO
0.30 0.03
0.31 0.03
The generation of adenine propenal occurs predominantly in
intact DNA, suggesting that most of its targets will likely be
DNA-associated proteins in the nucleus or mitochondria.
Nevertheless, we initiated our investigation of adenine propenal’s
protein-modifying profile, using HSA as the model protein.
HSA’s reactivity with electrophilic agents has been studied
extensively in the past,7,39−45 allowing us to place adenine
propenal-dependent modifications in the context of those
existing data. For example, teucrin A was found to preferentially
modify Lys351 and Lys545, while 4-hydroxynonenal modifies
Lys195, Lys199, Lys233, and Lys525, and polycyclic aromatic
hydrocarbon epoxides modify Lys137.44−46 MDA has been
shown to modify multiple lysine residues including Lys136,
Lys174, Lys240, Lys281, Lys525, and Lys541.7 As MDA and
adenine propenal both produce oxopropenyl adducts on lysine
residues, it is interesting to note that the major sites of adduction
for the two electrophiles are different: Lys190 and Lys351 for
adenine propenal versus Lys525 for MDA. These findings
suggest that distinct HSA modification patterns may be used as
markers for different electrophiles (Figure 6B). Clearly, the use
of HSA as a biomarker of electrophile damage is attractive, given
its high concentration in the blood and ease of sampling.
However, as noted above, adenine propenal generation is largely
confined to intact DNA, so it remains to be seen if enough of the
adenine propenal
NHS−biotin
1
0.1
n.d.
a
Dissociation constants (Kd) were determined by fitting fluorescence
anisotropy data to a simple two-state binding model. Kd values
represent the mean SD of at least three titrations. The biotin-
modification of XPA causes such a large loss of DNA-binding affinity
that there is no significant change in fluorescence anisotropy; hence, a
Kd value could not be determined.
expected, incubation of XPA with DMSO had no effect on its
DNA-binding activity. In contrast, exposure to NHS−biotin,
which efficiently modifies lysine residues and disrupts the DNA-
binding activity of many proteins,33,36,37 resulted in a nearly
complete loss of XPA’s DNA-binding affinity. XPA modified with
adenine propenal bound to the substrate, but with a greater than
3-fold reduction in affinity as compared to that of the unmodified
protein (Figure 7B). In fact, oxopropenylation of the protein
reduced the affinity to such an extent that it could not be
accurately measured; thus, the Kd value reported in Table 3 (1
0.1 μM) is an estimate.
DISCUSSION
■
Covalent modifications of DNA and proteins by electrophiles
produced during oxidative stress have multiple consequences on
I
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electrophile reaches the circulation to allow the modification of
HSA to serve as a biomarker of exposure.
mentation group A; HSA, human serum albumin; MOPS, 4-
morpholinepropanesulfonic acid
The impact of electrophiles has long been studied in the
context of covalent modification of DNA and its potential to lead
directly to mutations. However, adenine propenal produced in
the nucleus also has the potential to modify DNA-associated
proteins, including those involved in DNA repair. We report here
that adenine propenal covalently modifies the NER protein XPA
in vitro and that this modification leads to a reduction in XPA’s
DNA-binding activity. Oxopropenylation of residues known to
be acetylated further suggests that adenine propenal-dependent
modification could result in failure of regulation of XPA’s activity
through SIRT-1-dependent deacetylation.25 XPA is a critical
component of the NER machinery, without which repair activity
ceases.47 Mutations in XPA, particularly in the DNA-binding
domain, are associated with severe XP clinical phenotypes.48
Thus, it is possible that decreased XPA-binding activity resulting
from adenine propenal-dependent modification could result in
reduced NER activity in cells and increased potential for
mutagenesis and carcinogenesis. It should be noted that, in this
model, the modification of repair proteins may have a synergistic
effect on mutagenesis by preventing the repair of the original
DNA lesion. In this case, even moderate reductions in the activity
of the repair proteins have the potential to result in significant
increases in genomic instability. Further analysis will be required
to determine whether reactive base propenal species modify
DNA repair proteins in cells under oxidative stress and to
characterize the effects of modification on DNA repair activity in
vivo.
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ASSOCIATED CONTENT
■
S
* Supporting Information
UV/vis spectra of adenine propenal reactant and products with
N-α-acetyllysine; quantification of lysine cross-link formation;
MS spectrum of adenine propenal−cysteine adduct; XPA-
junction substrate-binding data; COSY spectrum, HMBC, and
HSQC of 3 and 4; and sequence coverage of HSA. This material
is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 615-343-7329. Fax: 615-343-7534. E-mail: larry.
marnett@vanderbilt.edu.
Funding
This work was supported by research and training grants from
the National Institutes of Health [R37 CA087819 (L.J.M), R01
ES1065561 (W.J.C.), DK020593 (J.A.O.), GM15431 (J.A.O.),
and F32 CA159701-01 (S.C.S.)]. S.M.S is supported by
postdoctoral fellowship 119569-PF-11-271-01-DMC from the
American Cancer Society. Partial support for the NMR and MS
was provided by P30 ES000267. Funds for the 600 MHz
spectrometer was provided from the National Institutes of
Health (S10 RR019022), and we thank Dr. Don Stec for
providing this service.
Notes
The authors declare no competing financial interest.
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ABBREVIATIONS
■
MDA, malondialdehyde; M1dG, 3-(2′-deoxy-β-D-erythro-pento-
furanosyl)-pyrimido[1,2-α]-purin-10(3H)-one; NER, nucleo-
tide excision repair; XPA, xeroderma pigmentosum comple-
J
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Chemical Research in Toxicology
Article
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