Proteomic identification of nitrated brain proteins in traumatic ...
Proteomic Identiﬁcation of Nitrated
Brain Proteins in Traumatic Brain-Injured
Rats Treated Postinjury With
Gamma-Glutamylcysteine Ethyl Ester:
Insights Into the Role of Elevation of
Glutathione as a Potential Therapeutic
Strategy for Traumatic Brain Injury
Tanea T. Reed,1
William M. Pierce,2
Patrick G. Sullivan,3
and D. Allan Butterﬁeld1,4*
Department of Chemistry, University of Kentucky, Lexington, Kentucky
Department of Pharmacology, University of Louisville, Louisville, Kentucky
Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky
Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky
Traumatic brain injury (TBI) occurs suddenly and has
damaging effects to the brain that are dependent on
the severity of insult. Symptoms can be mild, moder-
ate, or severe. Oxidative damage is associated with
traumatic brain injury through reactive oxygen/nitrogen
species production. One such species, peroxynitrite, is
elevated in TBI brain tissue (Orihara et al.  Foren-
sic Sci. Int. 123:142–149; Deng et al.  Exp. Neu-
rol. 205:154–165). Peroxynitrite can react with carbon
dioxide and decompose to produce NO2 and carbonate
radicals, which in turn can lead to 3-nitrotyrosine, an
index of protein nitration. Gamma-glutamylcysteine
ethyl ester (GCEE) is an ethyl ester moiety of gamma-
glutamylcysteine, an agent that up-regulates glutathi-
one (GSH) production in brain (Drake et al. 
J. Neurosci. Res. 68:776–784). Many preclinical studies
of TBI have employed pretreatment of animals with
proposed beneﬁcial agents prior to the injury itself.
However, in the real world of TBI, treatment begins
postinjury. Hence, insights into agents that improve out-
come following injury are desperately needed. This
study is one of the ﬁrst to investigate a potential GSH-
based therapy for TBI postinjury. Protein carbonyls, an
index of protein oxidation, were signiﬁcantly elevated in
brain of animals subjected to TBI. However, if, after
TBI, GCEE was administered i.p., protein carbonyl lev-
els were signiﬁcantly reduced. Similarly, 3-nitrotyrosine
levels were elevated in brain following TBI but signiﬁ-
cantly decreased following TBI if GCEE was adminis-
tered i.p. Redox proteomics analysis showed that sev-
eral brain proteins were nitrated after TBI. However, if
GCEE was given i.p. following TBI, many of these pro-
teins were protected from nitration. The results are
encouraging and are discussed with reference to
potential therapeutic strategies for TBI involving ele-
vated GSH. VVC 2008 Wiley-Liss, Inc.
Key words: oxidative stress; brain; antioxidant
Traumatic brain injury (TBI) is deﬁned as an event
in which sudden trauma and secondary injury cause
brain damage. Symptoms of TBI can be mild, moderate,
or severe, depending on extent of brain damage. Mild
symptoms can include but are not limited to confusion,
lightheadedness, double vision, personality changes, and
memory impairment. Moderate and severe TBI cases
show the same symptoms but worsened, slurred speech,
seizures, nausea, unresponsiveness, and possible death.
TBI can be focused on one particular area (focal) or
involve more than one area of the brain (diffuse).
Worldwide, TBI occurs in 10 million people annually,
with 1.4 million cases in the United States alone (Lan-
glois et al., 2006). Males between the ages of 15 and 24
Tanea T. Reed’s current address is Department of Chemistry, Eastern
Kentucky University, Richmond, Kentucky.
Contract grant sponsor: NIH; Contract grant number: AG-10836 (to
*Correspondence to: Prof. D. Allan Butterﬁeld, Department of Chemis-
try, Center of Membrane Sciences, University of Kentucky, Lexington,
KY 40506. E-mail: firstname.lastname@example.org
Received 9 May 2008; Revised 22 June 2008; Accepted 5 July 2008
Published online 19 September 2008 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.21872
Journal of Neuroscience Research 87:408–417 (2009)
' 2008 Wiley-Liss, Inc.
years have the highest incidence of TBI (Rutland-
Brown et al., 2006). The outcome can range from com-
plete patient recovery to permanent neurological dys-
function. There is no known cure for TBI, although
ﬂuoxetine, neurosteroids, bromocriptine, and dexanabi-
nol show therapeutic promise (Gorska, 2000; Bedell and
Prough, 2002; Djebaili et al., 2005; Mostert et al.,
2008); however, immediate medical care after injury is
most advantageous for patient recovery.
Oxidative damage has been associated with TBI
through formation of reactive oxygen species (ROS),
(Kontos and Povlishock, 1986; Kontos
and Wei, 1986; Opii et al., 2007), peroxynitrite (Hall
et al., 2004), and OHÁ
(Hall et al., 1994). Peroxynitrite
is a potent nitrating agent involved in neurodegenerative
diseases (Butterﬁeld, 2006; Sultana et al., 2006b). Perox-
ynitrite is formed by the combination of the two radi-
cals, nitric oxide and superoxide. In the presence of
CO2, peroxynitrite forms NO2
radicals can react with DNA, lipids, and proteins, includ-
ing MnSOD (Jackson et al., 1998; Tangpong et al.,
2007), Cu/ZnSOD (Alvarez et al., 2004), creatine kinase
(Wendt et al., 2003), and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH; Szabo, 2003). Inactivation of
these proteins can result in inefﬁcient energy metabo-
lism, reduced ATP production, and an excess hydrogen
peroxide, leading to overall increase in ROS production
and oxidative stress (Sultana et al., 2006a; Reed et al.,
2008). Peroxynitrite is elevated in brain tissue following
TBI (Orihara et al., 2001; Deng et al., 2007). This ele-
vation likely is due to superoxide leakage from the mito-
chondria reacting with NOÁ
and is supported by research
using nitric oxide synthase (NOS) inhibitors to decrease
3-NT levels in TBI-treated mice (Gahm et al., 2005).
Peroxynitrite- derived radicals can lead to cellular dam-
age in proteins, lipids, RNA, and DNA (Szabo et al.,
1996; Aoyama et al., 2000; Masuda et al., 2002; Botti
et al., 2005; Sultana et al., 2006b, 2007). Increased oxi-
dative damage in the form of elevated levels of protein
carbonyls, protein nitration, and lipid peroxidation was
observed in a diffuse, closed-head-injury mouse model
(Hall et al., 2004).
Gamma-glutamylcysteine ethyl ester (GCEE) is a
modiﬁed molecule of gamma-glutamylcysteine, the ﬁrst
two amino acids of the tripeptide glutathione (GSH).
Gamma-glutamylcysteine synthetase catalyzes the forma-
tion of gamma-glutamylcysteine. The ethyl ester moiety
increases its efﬁcacy to cross both the plasma membrane
of cells and the blood–brain barrier. GCEE leads to up-
regulated GSH in the brain and protects the brain against
peroxynitrite-mediated oxidative stress (Drake et al.,
2002). Glutathione (g-Glu-Cys-Gly) is a powerful anti-
oxidant found in brain. Cysteine (Cys) is the limiting
amino acid in GSH biosynthesis. Cys is in micromolar
levels in the brain, whereas glutamate and glycine are in
millimolar concentrations (Cooper and Kristal, 1997). It
has well documented that several free radical scavengers
are neuroprotective in various TBI animal models
(Ozdemir et al., 2005; Dohi et al., 2006; Singh et al.,
2007; Sonmez et al., 2007). Importantly, these and other
agents were administered prior to TBI. In contrast, we
investigated nitrated brain proteins following TBI and
their protection against oxidative stress induced by post-
treatment with GCEE. Only a few drugs have shown
cognitive beneﬁt when administered post-TBI, making
this study imperative in the investigation of TBI thera-
MATERIALS AND METHODS
All chemicals were of the highest quality and were pur-
chased from Sigma-Aldrich (St. Louis, MO) unless otherwise
mentioned. The OxyBlot kit used for protein carbonyl deter-
mination was purchased from Intergen (Purchase, NY).
GCEE was purchased from Bachem (Torrance, CA).
Surgical Procedures and Brain Processing
All the surgical, injury, and animal care protocols
described below have been approved by the University of
Kentucky Institutional Animal Care and Use Committee and
are consistent with the animal care procedures set forth in the
guidelines of the U.S. Public Health Service Policy on
Humane Care and Use of Laboratory Animals.
Eighteen Wistar adult male rats (Harlan Laboratories, In-
dianapolis, IN) wieghing 300–350 g were used in this study.
The rats were anesthetized with isoﬂurane (3.0%), shaved, and
then placed in a stereotaxic frame (David Kopf Instruments,
Tujunga, CA). Surgery was completed in the same fashion as
previously described (Sullivan et al., 2002). A 1.5-mm drop of
a metal ball was used to produce TBI in each rat as described
elsewhere (Sullivan et al., 2002), except for sham animals.
Each group (sham, TBI, and GCEE-treated) consisted of six
animals. After surgery and injury, a 4-mm disk made from
dental cement was placed over the craniotomy site and
adhered to the skull with cyanoacrylate. To prevent immedi-
ate hypothermia following the skin suturing, rats were placed
on a warm mat until they regained consciousness (increased
attention and mobility). Six rats were given GCEE (150 mg/
kg) approximately 10 min postinjury. This concentration was
chosen based on prior studies (Joshi et al., 2007). Six injured
rats were given saline 10 min after injury. The remaining six
rats were treated as sham controls, in which they received a
craniotomy but not cortical contusion. All rats were kept alive
24 hr postinjury and then sacriﬁced. This time point was cho-
sen based on prior research in which improvement with post-
neurosteroidal treatment was observed (Djebaili et al., 2005).
Also, maximal glutathione deletion from TBI is observed at
this time point (Ansari et al., 2008b). Upon sacriﬁce, rats
were decapitated, and the whole brain was rapidly removed
and placed in a –808C freezer until use. Brain samples were
minced and suspended in 10 mM HEPES buffer (pH 7.4)
containing 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4,
0.1 mM EDTA, and 0.6 mM MgSO4 as well as proteinase
inhibitors: leupeptin (0.5 mg/ml), pepstatin (0.7 lg/ml), type
II S soybean trypsin inhibitor (0.5 lg/ml), and phenylmethyl-
sulfonyl ﬂuoride (PMSF; 40 lg/ml). Homogenates were cen-
trifuged at 14,000g for 10 min to remove debris. Protein con-
Elevated Glutathione as a Potential Therapy for TBI 409
Journal of Neuroscience Research
centration in the supernatant was determined by the BCA
Measurement of Protein Carbonyls
Five microliters of sample were derivatized with 10 mM
2,4-dinitrophenylhydrazine (DNPH) in the presence of 5 ll
12% sodium dodecyl sulfate for 20 min at room temperature.
The samples were then neutralized with 7.5 ll neutralization
solution (2 M Tris in 30% glycerol). Derivatized samples (250
ng) were loaded onto a nitrocellulose membrane with a slot
blot apparatus. The blot was then blocked with 3% bovine
serum albumin (BSA) and incubated with rabbit polyclonal
anti-DNPH antibody (1:100 dilution) for 2 hr. The blot was
washed in wash blot buffer (10 mM Tris-HCl, pH 7.5, 150
mM NaCl, 0.05% Tween 20) three times before incubation
with a secondary antibody (goat anti-rabbit antibody conju-
gated with alkaline phosphatase). The blot was washed again
and then developed using SigmaFast tablets (Sigma Aldrich),
and densiometric levels were quantiﬁed in Scion Image
Measurement of HNE and 3-NT Levels
Measurement of HNE-bound proteins is one way of
indexing lipid peroxidation, whereas measuring 3-NT levels is
an effective method for quantifying the level of nitrated pro-
teins. For both measurements, 5 ll brain homogenate was
incubated with 5 ll Laemmli buffer [0.125 M Tris base, pH
6.8, 4% (v/v) sodium dodecyl sulfate (SDS), and 20% (v/v)
glycerol] for 20 min at room temperature. The resulting sam-
ples (250 ng) were loaded per well in the slot blot apparatus
containing a nitrocellulose membrane under pressure. Each
sample was loaded in duplicate. After the sample was loaded,
the membrane was blocked with 3% (w/v) BSA in phos-
phate-buffered saline containing 0.01% (w/v) sodium azide
and 0.2% (v/v) Tween 20 (PBST) for 1 hr and then incubated
with a 1:5,000 dilution of anti-HNE (or a 1:2,000 dilution
of anti-3NT) polyclonal antibody in PBST for 2 hr. After
primary antibody incubation, the membrane was carefully
washed three times for 5 min each. A secondary antibody of
anti-rabbit IgG alkaline phosphatase in PBST (1:3,000 dilu-
tion) was added to the membrane for 1 hr. The membrane
was again washed three times in PBST and developed using
SigmaFast BCIP/NBT (5-bromo-4-chloro-3-indolyl phos-
phate/nitro blue tetrazolium) tablets (Sigma). Blots were dried,
scanned in Adobe Photoshop (San Jose, CA), and quantitated
in Scion Image software.
Two-Dimensional Gel Electrophoresis
Two-dimensional (2D) gel electrophoresis was per-
formed to separate proteins into single detectable protein spots
based on size and isoelectric point. Proteins (200 lg) were
precipitated by addition of cold 100% trichloroacetic acid
(TCA) to obtain a ﬁnal concentration of 15% TCA and
placed on ice for 10 min. Precipitates were centrifuged at
14,000g for 2 min at 48C. The pellets were washed three
times with 1 ml of 1:1 (v/v) ethanol:ethyl acetate solution.
The samples were then dissolved with 200 ll of rehydration
buffer [8 M urea, 2 M thiourea, 20 mM dithiothreitol, 2.0%
(w/v) CHAPS, 0.2% Biolytes, and bromophenol blue]. Iso-
electric focusing separates proteins according to their isoelec-
tric point (pI). For ﬁrst-dimension electrophoresis, 200 ll of
sample solution was applied to a 110-mm ReadyStrip IPG
strip, pH 3–10 (Bio-Rad, Hercules, CA). The strips were
allowed to soak in the sample solution for 1 hr to allow
uptake of the proteins. Two milliliters of mineral oil were
added on top of each strip to prevent evaporation throughout
this entire process. The strip was then actively rehydrated in a
Protean IEF cell (Bio-Rad) for 16 hr at 50 V. The isoelectric
focusing was performed at 300 V for 2 hr linearly, 500 V for
2 hr linearly, 1,000 V for 2 hr linearly, 8,000 V for 8 hr line-
arly, and 8,000 V for 10 hr rapidly. All the processes described
above were carried out at 208C. The focused IEF strip was
stored at –808C until 2D gel electrophoresis was performed.
SDS-PAGE separates proteins based on size and shape. For
2D electrophoresis, thawed strips were equilibrated for 10 min
in equilibration buffer A, which consisted of 50 mM Tris-
HCl (pH 6.8) containing 6 M urea, 1% (w/v) SDS, 30% (v/
v) glycerol, and 0.5% dithiothreitol, then reequilibrated for 10
min in the same buffer containing 4.5% iodacetamide instead
of dithiothreitol. Linear Gradient (8–16%) Precast Criterion
Tris-HCl gels (Bio-Rad) were used to perform 2D electro-
phoresis. Precision Protein Standards (Bio-Rad) were run
along with the sample at 200 V for 65 min.
SYPRO Ruby Staining
After electrophoresis, the gels were incubated in ﬁxing
solution [7% (v/v) acetic acid, 10% (v/v) methanol] for 20
min. Approximately 40 ml of SYPRO Ruby Gel Stain (Bio-
Rad) was used to stain the gels for 2 hr, on a gently continu-
ous rocker. The gels were placed in deionized water overnight
for destaining. SYPRO Ruby ﬂuorescent stain achieves a lin-
ear and sensitive staining of gel slabs.
Western Blotting and Immunochemical Detection
Western immunoblotting was performed to detect pro-
teins immunochemically on a nitrocellulose membrane. Pro-
tein samples (200 lg) were loaded onto an 8–16% Criterion
gel (Bio-Rad), and electrophoresis was carried out as described
above. The proteins from the 2D gel were transferred to a
nitrocellulose membrane (Bio-Rad) using a TransBlot SD
Semi-Dry Transfer Cell (Bio-Rad) at 15 V for 2 hr. Nitrated
proteins were detected on nitrocellulose paper using anti-3-ty-
rosine antibody (1:2,000) for 2 hr at room temperature while
rocking, followed by a secondary goat anti-rabbit IgG (Sigma)
antibody conjugated to alkaline phosphatase (1:3,000) diluted
in wash blot buffer for 1 hr at RT. The resultant membrane
was developed using 5-bromo-4-chloro-3-indolyl phosphate/
nitroblue tetrazolium (BCIP/NBT) solution (SigmaFast tablets;
In-gel digestion of selected gel spots was performed
according to techniques established by Thongboonkerd et al.
(2002). The signiﬁcant protein spots were excised from
SYPRO Ruby-stained 2D gels with a clean blade and trans-
410 Reed et al.
Journal of Neuroscience Research
ferred into clean microcentrifuge tubes. The protein spots
were then washed with 0.1 M ammonium bicarbonate
(NH4HCO3; Sigma) at room temperature for 15 min. Aceto-
nitrile (Sigma) was added to the gel pieces and incubated at
room temperature for 15 min. The solvent was removed, and
the gel pieces were dried in a ﬂow hood. The protein spots
were incubated with 20 ll of 20 mM dithiothreitol (DTT;
Bio-Rad) in 0.1 M NH4HCO3 at 568C for 45 min. The
DTT solution was then removed and replaced with 20 ll of
55 mM iodoacetamide (Bio-Rad) in 0.1 M NH4HCO3. The
solution was incubated at room temperature in the dark for
30 min. The excess iodoacetamide was removed and replaced
with 0.2 ml of 50 mM NH4HCO3 and incubated at room
temperature for 15 min. Two hundred microliters of acetoni-
trile was added. After a 15-min incubation, the solvent was
removed, and the gel spots were dried for 30 min in a ﬂow
hood. The gel pieces were rehydrated with 20 ng/ll modiﬁed
trypsin (Promega, Madison, WI) in 50 mM NH4HCO3 with
the minimal volume to cover the gel pieces. The gel pieces
were chopped into smaller pieces and incubated with shaking
overnight at 378C.
The immunoreactivity of the Western blot was normal-
ized to the actual protein content as measured by the intensity
of a SYPRO Ruby protein stain (Bio-Rad). Images from
SYPRO Ruby-stained gels, used to measure protein content,
were obtained with a UV transilluminator (kex 5 470 nm,
kem 5 618 nm; Molecular Dynamics, Sunnyvale, CA). Gels
were then stored in deionized water until spot excision for in-
gel trypsin digestion. Western blots were scanned in Adobe
Photoshop on a Microtek Scanmaker 4900. PDQuest 2-D
Analysis software (Bio-Rad) was used to match and analyze
visualized protein spots among differential gels and membranes
to compare protein and immunoreactivity content between
injured animal samples and age-matched controls. Powerful
automatching algorithms quickly and accurately match gels or
blots, and sophisticated statistical analysis tools allow spots of
interest to be identiﬁed. The principles of measuring intensity
values by 2D analysis software were similar to those of densi-
tometric measurement. The average mode of background sub-
traction was used to normalize intensity values, which repre-
sent the amount of protein (total protein on gel and protein
bound on membrane) per spot. After completion of spot
matching, the normalized intensity of each protein spot from
individual gels (or membranes) was compared between groups
using statistical analysis (Student’s t-test).
All mass spectrometry was performed at the University
of Louisville’s Mass Spectrometry Facility (ULMSF) and Vet-
eran’s Affairs Medical Center (VAMC). A Bruker Autoﬂex
matrix-assisted laser desorption ionization-time of ﬂight
(MALDI-TOF) mass spectrometer (Bruker Daltonics, Biller-
ica, MA) operated in the reﬂectron mode was used to gener-
ate peptide mass ﬁngerprints. Peptides resulting from in-gel
digestion with trypsin were analyzed on a 384-position, 600-
lm AnchorChip Target (Bruker Daltonics, Bremen, Germany)
and prepared according to AnchorChip recommendations
(AnchorChip Technology, Rev. 2; Bruker Daltonics). Brieﬂy,
1 ll digestate was mixed with 1 ll a-cyano-4-hydroxycin-
namic acid (0.3 mg/ml in ethanol:acetone, 2:1 ratio) directly
on the target and allowed to dry at room temperature. The
sample spot was washed with 1 ll of a 1% TFA solution
for approximately 60 sec. The TFA droplet was gently blown
off the sample spot with compressed air. The resulting dif-
fuse sample spot was recrystallized (refocused) using 1 ll of a
solution of ethanol:acetone:0.1% TFA (6:3:1 ratio). Reported
spectra are a summation of 100 laser shots. External calibra-
tion of the mass axis was used for acquisition and internal
calibration using either trypsin autolysis ions or matrix clus-
ters and was applied postacquisition for accurate mass
Analysis of Peptide Mass Fingerprints
Results from MALDI spectra used for protein identiﬁca-
tion from tryptic fragments were searched against the NCBI
database using the MASCOT search engine (http://www.ma-
trixscience.com). The search parameters used were previously
described by Castegna et al. (2002a). Peptide mass ﬁngerprint-
ing used the assumption that peptides are monoisotopic, oxi-
dized at methionine residues, and carbamidomethylated at
cysteine residues (Castegna et al., 2002a,b; Butterﬁeld et al.,
2003). Carbamylation occurs when urea is broken down in a
sample solution. Urea can degrade to cyanate, which can then
react with protein amino groups and remove the positive
charge, making them more acidic. Database searches allowed
up to one missed trypsin cleavage. Mass tolerance of 100 ppm
was the window of error allowed for matching the peptide
Probability-based MOWSE scores were estimated by
comparison of search results against estimated random match
population and were reported as –10 Á log10 (P), where P is
the probability that the protein identiﬁcation is not correct.
Proteins with a P value less than 0.05 were considered signiﬁ-
cant. All protein identiﬁcations were in the expected size and
pI ranges based on their gel position.
There was a signiﬁcant increase (19%) in protein
carbonyls observed in TBI rats compared with sham ani-
mals (Fig. 1). GCEE treatment 10 min postinjury pro-
vided protection against TBI (70% of control). There
was a signiﬁcant increase (19%) in protein nitration in
brain from TBI rats. Protein nitration was reduced to
below control levels by administration of GCEE as
shown in Figure 2. Protein-bound HNE levels were
found to be elevated in TBI rats compared with sham
(27%), but there was no signiﬁcant decrease in HNE
levels post-GCEE treatment (data not shown).
Elevated Glutathione as a Potential Therapy for TBI 411
Journal of Neuroscience Research
Identiﬁcation of Nitrated Proteins in
GCEE-Treated TBI Rats
Two-dimensional gels were performed for TBI,
sham, and TBI rats treated with GCEE 10 min follow-
ing TBI (Fig. 3a–c, respectively). The 2D Western blots
probed with anti-3NT antibody to detect 3-NT immu-
noreactivity are seen in Figure 4a–c, respectively. Pro-
teomics analysis for excessively nitrated proteins in TBI
was performed as described in Materials and Methods.
Seven proteins were found to be signiﬁcantly nitrated in
TBI rats compared with sham rats. These proteins
include synapsin 1, gamma enolase, guanosine diphos-
phate dissociation inhibitor 1 (GDP), phosphoglycerate
mutase 1, heat shock protein 70, ATP synthase, and a-
spectrin. A list of these proteins can be found in Table I.
If GCEE is given to animals that suffered a TBI, these
proteins are no longer excessively nitrated compared
with sham controls.
Sudden brain trauma is described as TBI. There is
no known cure, but immediate medical care after injury
is most advantageous for patient recovery. ROS produc-
tion occurs in TBI, as can be observed by increased pro-
tein nitration and protein carbonyls (Hall et al., 2004;
Opii et al., 2007). Ansari et al. (2008b) demonstrated
that oxidative stress occurs within 3 hr postinjury. Cur-
rently, several drugs show promise in treating TBI,
including ﬂuoxetine (Mostert et al., 2008), progesterone
(Djebaili et al., 2005), bromocriptine (Gorska, 2000),
and dexanabinol (Bedell and Prough, 2002). Nicotina-
mide, atomoxetine, erythropoietin, and amandatine have
shown cognitive improvement when administered post-
injury (Grasso et al., 2007; Hoane et al., 2008; Reid and
Hamm, 2008; Sawyer et al., 2008). The most investi-
gated therapeutic strategy for TBI patients over the past
10 years has been the use of mild hypotheremia expo-
sure. This lowers core body temperature in an effort to
prevent the likelihood of further complications. How-
ever, there is little efﬁcacy in this method of treatment
(Tisherman et al., 1999; Ginsberg, 2002; Polderman,
2004; Qiu et al., 2007).
GCEE, an ester moiety of the dipeptide gamma-
glutamylcysteine, is a vital antioxidant that can easily
cross the plasma membrane and up-regulate GSH in the
brain (Drake et al., 2002). GCEE prevents oxidative
stress induced by amyloid-b peptide and other moieties
by scavenging and free radicals (Boyd-Kimball et al.,
2005b; Joshi et al., 2007). In the current study, GCEE
reduced levels of 3NT and protein carbonyls to those of
control. In this study, posttreatment of GCEE appears to
lower protein oxidation signiﬁcantly in TBI rats, leading
to possible promise as a potential postinjury therapeutic.
In this work, seven proteins were found by redox
proteomics analysis to be nitrated in TBI. The functions
of these proteins include energy metabolism, neurotrans-
mission, and chaperone function. Synapsin 1 is a synaptic
protein that binds to synaptic vesicles and regulates neu-
rotransmission. Synapsin aids in maintaining an ample
supply of ‘‘reserve’’ vesicles at the synapse for release.
This phosphoprotein binds to the SH3 (high-afﬁnity
phosphotyrosine binding site) domain of the c-Src kinase
in synaptic vesicles, causing stimulation of c-Src activity
(Onofri et al., 2000). Src kinase phosphorylates Tyr
residues on proteins rather than Ser or Thr residues.
Synapsin-deﬁcient vesicles show signiﬁcantly reduced
Src activity and Tyr phosphorylation (Onofri et al.,
2007). This synaptic protein has also been found to be
modiﬁed oxidatively in recent work involving traumati-
cally brain-injured animals (Ansari et al., 2008a). Nitra-
tion of synapsin 1 is consistent with reported reduced
neurotransmission and synaptic plasticity exhibited by
TBI patients (Schwarzbach et al., 2006).
Several proteins (g-enolase, phosphoglycerate mu-
tase, and ATP synthase) nitrated in TBI are associated
with energy metabolism. Enolase catalyzes the conver-
sion of 2-phosphoglycerate to phosphoenolpyruvate,
ﬁnally leading to ATP production. Gamma-enolase is
one of the three isoforms (a, b, and g) of enolase, a
protein frequently modiﬁed in several in vivo and in
vitro systems of neurodegeneration (Schonberger et al.,
2001; Poon et al., 2004, 2005a,b; Boyd-Kimball et al.,
2005a; Perluigi et al., 2005; Butterﬁeld, 2006; Ekegren
Fig. 2. 3-Nitrotyrosine levels in traumatically brain-injured rats and
remedied by GCEE treatment. Percentage 6 SEM; N 5 6; *P <
0.01,**P < 0.05, ***P < 0.009.
Fig. 1. Protein carbonyl content in traumatically brain-injured rats
and remedied by GCEE treatment. Percentage 6 SEM; N 5 6;
*P < 0.04, **P < 1.45 3 1025
, ***P < 7.87 3 10–6
412 Reed et al.
Journal of Neuroscience Research
et al., 2006). Phosphoglycerate mutase 1 (PGM1) cata-
lyzes the interconversion of 3-phosphoglycerate to 2-
phosphoglycerate, leading to a second equivalent of ATP
produced in glycolysis. Protein dysfunction and increased
glycolytic intermediates lead to decreased pyruvate pro-
duction and decreased ATP production. PGM1 is found
to be modiﬁed oxidatively in several models of oxidative
stress and genetic mutation in neurodegenerative diseases
(Bowling and Beal, 1995; Castegna et al., 2004). ATP
synthase a-chain is a mitochondrial regulating subunit of
complex V that plays a key role in energy production.
ATP synthase goes through a sequence of coordinated
conformational changes of its major subunits (a, b) to
produce ATP. ATP synthase a-subunit has been previ-
ously shown to be modiﬁed oxidatively in various mod-
els of aging and in neurodegeneration (Boyd-Kimball
et al., 2005c; Butterﬁeld, 2006). The oxidation of ATP
synthase leads to the inactivation of this mitochondrial
complex, resulting in decreased electron transport chain
activity and impaired ATP production. ATP, the energy
source of the cell, is extremely important at nerve termi-
nals for normal neural communication. Decreased levels
of cellular ATP at nerve terminals may lead to loss of
synapses and synaptic function, decreased function of ion
pumps, loss of ion gradients, and ATPase dysfunction
and may ultimately contribute to cell death and memory
loss observed in TBI patients. Energy metabolism is sig-
niﬁcantly reduced in TBI patients (Marklund et al.,
2006), and nitration of this protein can provide a reason
for the lethargy associated with TBI.
GTP-coupled proteins are lipid anchored to the
plasma membrane and bind guanine nucleotides (GDP
or GTP). G-proteins require binding to GTP for activa-
tion. Normally, a guanosine exchange factor binds to the
inactive G protein–GDP complex, alters the conforma-
tion, and exchanges GDP for GTP, thereby activating
the G protein. Once active, G proteins can bind to a
target protein (effector) and stimulate signal transduction.
Guanosine diphosphate dissociation inhibitors (GDI) in-
hibit the exchange of GDP for GTP; therefore, the G
protein will remain inactive. This inactivity prevents
effector binding downstream signaling (i.e., glycogen ca-
Fig. 3. Two-dimensional gel of TBI-treated rats (a), sham rats (b), and TBI rats treated with
GCEE 10 min postinjury (c).
Elevated Glutathione as a Potential Therapy for TBI 413
Journal of Neuroscience Research
tabolism, etc.). In TBI, glycogen levels are increased
compared with control, supporting the downstream
effects impaired by GDI (Otori et al., 2004).
Heat shock protein 70 (Hsp70) is a member of the
heat shock protein family. Heat shock proteins are cyto-
protective to injury and various metabolic disturbances
while also assisting in the stress response (Kiang and Tso-
kos, 1998; Poon et al., 2004). The main function of heat
shock proteins/cognates is to act as chaperone proteins by
refolding misfolded proteins or shepherding these proteins
for recycling through proteasomal degradation. Hsp70 is
altered in TBI, bolstering the importance of functioning
heat shock proteins in the cell (Lai et al., 2004; Poon
et al., 2004; da Rocha et al., 2005). Hsp70 dysfunction
may exacerbate protein misfolding, protein aggregation,
and reduced effective proteasomal activity.
Fig. 4. a: Two-dimensional Western blot of TBI rat probed with anti-3-nitrotyrosine antibody.
b: Nitrated proteins in sham rat. c: Nitrated proteins in TBI rat treated with GCEE 10 min
TABLE I. Nitrated Proteins in TBI-Treated Rats
Apparent MW based
on migration rate (Mr)
(% control) Probability
Synapsin 1 169 9.81 74,114 39 122.5 6 54.2 <0.05
Gamma-enolase 185 5.91 61,098 34 54.03 6 8.9 <0.02
Guanosine diphosphate dissociation
inhibitor 1 (GDP)
140 5.00 51,074 50 161.4 6 13.4 <0.05
Phosphoglycerate mutase (PGM) 84 6.75 28,797 31 728.6 6 27.4 <0.04
Heat shock protein 70 (Hsp70) 186 5.07 72,402 36 280.8 6 28.6 <0.05
ATP synthase 72 5.54 50,548 37 164.2 6 49.5 <0.03
Alpha-spectrin 217 5.22 285,689 18 365.2 6 23.1 <0.05
414 Reed et al.
Journal of Neuroscience Research
Alpha-spectrin plays a critical role in maintaining
cytoskeletal structure and membrane integrity. This pro-
tein interacts with actin and tropomyosin, two proteins
found in thin ﬁlaments and associated with muscle con-
traction, in a hexagonal arrangement. Spectrin also binds
to synapsin 1 (also known as band 4.1 in RBC mem-
branes), which enhances spectrin-actin binding. In dif-
fuse TBI, spectrin is cleaved by calpain, a Ca21
ent cysteine protease causing membrane degradation and
cell death (Pike et al., 1998; Buki et al., 1999; Kupina
et al., 2003; Yu and Geddes, 2007). Calpain activity
plays a dominant role in posttraumatic brain injury and
neurodegeneration (Saatman et al., 1996). Oxidative
modiﬁcation of spectrin has also been observed in the
senescence-accelerated (SAMP8) mouse model (Poon
et al., 2004), which has diminished learning and mem-
ory. Spectrin oxidative modiﬁcation in TBI is consistent
with the memory and learning deﬁcits associated with
TBI (Schwarzbach et al., 2006).
This study suggests that elevation of GSH by
GCEE 10 min post-TBI is neuroprotective against oxi-
dative stress associated with TBI. This is one of the ﬁrst
studies to demonstrate a potential postinjury therapeutic
strategy for the treatment of TBI, a condition that affects
millions of persons annually. Speciﬁc proteins are pro-
tected against nitrosative stress associated with TBI,
which conceivably modulates loss of function of the pro-
teins in TBI. GCEE offers promise as a potential postin-
jury therapeutic for TBI.
We thank TBI nurse Marcia Butterﬁeld, RN, for
useful discussions. T.T.R. was supported by a T32 train-
ing grant administered by the University of Kentucky
Spinal Cord and Brain Injury Research Center and
funded by NIH grant AG-1012806160.
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