Free Radical Biology & Medicine 39 (2005) 453 – 462
Redox proteomics analysis of oxidatively modified proteins in
G93A-SOD1 transgenic mice—A model of familial amyotrophic
H. Fai Poona, Kenneth Hensleyb,c, Visith Thongboonkerdd, Michael L. Merchantd,
Bert C. Lynna,e, William M. Piercef, Jon B. Kleind, Vittorio Calabreseg, D. Allan Butterfielda,h,i,*
Department of Chemistry, University of Kentucky, Lexington KY 40506, USA
Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA
Oklahoma Center for Neuroscience, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA
Kidney Disease Program and Core Proteomics Laboratory, University of Louisville School of Medicine and VAMC, Louisville, KY 40292, USA
Mass Spectrometry Facility, University of Kentucky, Lexington, KY 40506, USA
Department of Pharmacology, University of Louisville School of Medicine and VAMC, Louisville, KY 40292, USA
Section of Biochemistry, Department of Chemistry, University of Catania, Catania, Italy
Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506, USA
Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536, USA
Received 22 December 2004; revised 22 March 2005; accepted 28 March 2005
Available online 14 April 2005
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease characterized by the loss of neuronal function in the motor
cortex, brain stem, and spinal cord. Familial ALS cases, accounting for 10 – 15% of all ALS disease, are caused by a gain-of-function mutation
in Cu,Zn-superoxide dismutase (SOD1). Two hypotheses have been proposed to explain the toxic gain of function of mutant SOD (mSOD).
One is that mSOD can directly promote reactive oxygen species and reactive nitrogen species generation, whereas the other hypothesis
suggests that mSODs are prone to aggregation due to instability or association with other proteins. However, the hypotheses of oxidative stress
and protein aggregation are not mutually exclusive. G93A-SOD1 transgenic mice show significantly increased protein carbonyl levels in their
spinal cord from 2 to 4 months and eventually develop ALS-like motor neuron disease and die within 5 – 6 months. Here, we used a parallel
proteomics approach to investigate the effect of the G93A-SOD1 mutation on protein oxidation in the spinal cord of G93A-SOD1 transgenic
mice. Four proteins in the spinal cord of G93A-SOD1 transgenic mice have higher specific carbonyl levels compared to those of nontransgenic
mice. These proteins are SOD1, translationally controlled tumor protein (TCTP), ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1), and,
possibly, aB-crystallin. Because oxidative modification can lead to structural alteration and activity decline, our current study suggests that
oxidative modification of UCH-L1, TCTP, SOD1, and possibly aB-crystallin may play an important role in the neurodegeneration of ALS.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Redox proteomics; ALS; Mechanisms of neurodegeneration; Oxidatively modified proteins; Enzyme activity decline; Free radicals
Amyotrophic lateral sclerosis (ALS) is a fatal motor cord. ALS typically presents in middle age and progresses
neuron degenerative disease characterized by the loss of rapidly. Life expectancy of victims of ALS usually is 3 – 5
neuronal function in the motor cortex, brain stem, and spinal years after diagnosis [1,2]. Inherited ALS accounts for 10–
15% of cases, and among all of the familial ALS (FALS)
patients, 20 –30% of them are caused by a gain-of-function
* Corresponding author. Department of Chemistry, University of Ken-
mutation in Cu,Zn-superoxide dismutase (SOD1) [3,4].
tucky, Lexington, KY 40506, USA. Fax: (859) 257 5876. SOD1 catalyzes the disproportionation of superoxide anion
E-mail address: email@example.com (D.A. Butterfield). radical to hydrogen peroxide and oxygen. Over 100
0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
454 H.F. Poon et al. / Free Radical Biology & Medicine 39 (2005) 453 – 462
different missense substitutions in the 153-amino-acid Alzheimer disease (AD) patients and models thereof [40 –
SOD1 have been described in individuals and kindreds 45] and provided important insights into the role of protein
affected by SOD1-linked FALS . One of the most oxidation in AD. In order to better understand the role of
common mutations of SOD1 is the substitution of glycine oxidative modification of proteins in ALS, we employed
by alanine at residue 93 (G93A) . quantitative redox proteomic analysis to identify the specific
Two principal hypotheses have been proposed to explain oxidized proteins in the spinal cord of G93A-SOD1 mice.
the toxic gain of function of mutant SOD (mSOD) . One
is that mSOD can directly promote reactive oxygen species
and reactive nitrogen species generation [7 –12]. Evidence Methods
supporting this hypothesis showed that mSODs enhance
oxidative activity by acting as peroxidases [9,11,13] or Animals
superoxide reductases  or by producing O2 to form
peroxynitrite [15,16]. The second hypothesis is that mSODs Transgenic mice expressing the human SOD1 gene with
are prone to aggregation due to their instability or a G93A mutation, strain B6SJL/TgN (SOD1-G93A)-2Gur)
association with other proteins [17 – 20]. These aggregates , were purchased from The Jackson Laboratory (Bar
facilitate toxicity [21,22] and deplete the mSOD-associated Harbor, ME, USA) and maintained as hemizygotes by
proteins and thus perturb the normal functions of cells . mating transgenic males with B6/SJLF1 females as pre-
The proteinaceous inclusions found in tissues from ALS viously described .
patients [23 – 25] and mSOD transgenic mice [22,26] All studies of live animals were authorized and overseen
reportedly are rich in mSOD, ubiquitin, and neurofilament by the Institutional Animal Care and Use Committee of the
proteins. It is noteworthy that the hypotheses of oxidative Oklahoma Medical Research Foundation and conducted by
stress and protein aggregation are not mutually exclusive trained and certified technical and veterinary staff. Every
, although the roles of oxidative stress and aggregation effort was made to avoid unnecessary discomfort to the
in ALS are highly controversial (recently reviewed in experimental animals.
Increased oxidative modification of macromolecules was Sample preparation
demonstrated in neuronal tissues of SOD1-related FALS
patients and transgenic mice [30 – 32]. Enhanced suscepti- Whole frozen mouse spinal cords (n = 6) were ho-
bility of exogenous oxidative stress in mSOD1 cell cultures mogenized in 10 mM sodium acetate buffer, pH 7.2,
was also observed in in vitro studies [33 – 35]. Exogenous containing 0.1% Triton X-100 and mammalian protease
oxidative stress can even inhibit the rapid degradation of inhibitor cocktail (Sigma Chemical Co., St. Louis, MO,
mSOD . These studies are consistent with the notion USA).
that oxidative stress plays an important role in ALS
development. G93A-SOD1 transgenic mice show signifi- Two-dimensional (2D) gel electrophoresis
cantly increased protein carbonyl levels in their spinal cord
from 2 to 4 months  and eventually develop motor Samples of spinal cord proteins were prepared according
neuron disease and die within 5 –6 months . G93A- to the procedure previously described . Briefly, 300 Ag
SOD1 catalyzes the oxidation of a model substrate by H2O2 of protein was incubated with 4 vol of 2 N HCl at room
at a higher rate  and has a higher capacity to generate temperature (25-C) for 20 min. Proteins were then
free radicals  compared to wild-type SOD1. Also, precipitated by addition of ice-cold 100% trichloroacetic
elevation of inflammation-related genes (e.g., induced nitric acid (TCA) to obtain a final concentration of 15% TCA. The
oxide synthase, proinflammatory cytokines) occurs at 11 samples were then mixed with 185 Al of rehydration buffer
weeks of age in the presymptomatic stage before motor (8 M urea, 20 mM dithiothreitol, 2.0% (w/v) Chaps, 0.2%
neuron death in G93A-SOD1 transgenic mice, suggesting Biolytes, 2 M thiourea, and bromophenol blue).
that neuroinflammation-mediated oxidative stress is also In the first-dimension electrophoresis, 200 Al of sample
present in G93A-SOD1 mice . Therefore, the expression solution was applied to a ReadyStrip IPG strip (Bio-Rad).
of G93A-SOD1 is believed to elevate the generation of The strip was then actively rehydrated in a protean
oxygen radicals in vulnerable tissue, such as spinal cord isoelectric focusing (IEF) cell (Bio-Rad) for 16 h at 50 V.
, creating oxidative stress that may be responsible for The focused IEF strip was stored at À80-C until second-
the ALS-like syndrome observed in the G93A-SOD1 mice. dimension electrophoresis was performed. For the second-
One of the oxidatively modified proteins in G93A-SOD1 dimension electrophoresis, thawed IPG strips, pH 3– 10,
transgenic mice is SOD1 , indicating that oxidation of were equilibrated for 10 min in 50 mM Tris – HCl (pH 6.8)
SOD1 is likely important to the development of this model containing 6 M urea, 1% (w/v) sodium dodecyl sulfate, 30%
of ALS. However, other oxidatively modified proteins were (v/v) glycerol, and 0.5% dithiothreitol and then reequilib-
not identified. Recent quantitative proteomic studies rated for 15 min in the same buffer containing 4.5%
enabled the identification of oxidized brain proteins in iodoacetamide in place of dithiothreitol. Linear gradient (8–
H.F. Poon et al. / Free Radical Biology & Medicine 39 (2005) 453 – 462 455
16%) precast Criterion Tris –HCl gels (Bio-Rad) were used Peptides resulting from in-gel digestion with trypsin were
to perform second-dimension electrophoresis. Precision analyzed on a 384-position, 600-Am AnchorChip Target
protein standards (Bio-Rad) were run along with the sample (Bruker Daltonics) and prepared according to AnchorChip
at 200 V for 65 min. The protein levels on the 2D gels were recommendations (AnchorChip Technology, rev. 2; Bruker
detected by Bio-Safe Coomassie blue (Bio-Rad). Daltonics). Briefly, 1 Al of digestate was mixed with 1 Al of
a-cyano-4-hydoxycinnamic acid (0.3 mg/ml in ethanol:ace-
Western blotting tone, 2:1 ratio) directly on the target and allowed to dry at
room temperature. The sample spot was washed with 1 Al of
As previously described , 300 Ag of protein was a 1% TFA solution for approximately 60 s. The TFA droplet
incubated with 2,4-dinitrophenyl hydrazine. The 2D gels was gently blown off the sample spot with compressed air.
were prepared in the same manner as for 2D electro- The resulting diffuse sample spot was recrystallized (refo-
phoresis. The proteins from the 2D gels were then cused) using 1 Al of a solution of ethanol:acetone:0.1% TFA
transferred to nitrocellulose paper (Bio-Rad) and detected (6:3:1 ratio). Reported spectra are a summation of 100 laser
immunochemically. shots. External calibration of the mass axis was used for
acquisition and internal calibration using either trypsin
Image analysis autolysis ions or matrix clusters was applied postacquisition
for accurate mass determination.
The gels and nitrocellulose blots were scanned and saved The MALDI spectra used for protein identification from
in TIFF format using a Scanjet 3300C (Hewlett –Packard). tryptic fragments were searched against the NCBI protein
An Investigator HT analyzer (Genomic Solutions, Inc., Ann databases using the MASCOT search engine (http://www.
Arbor, MI, USA) was used for matching and analysis of matrixscience.com). Peptide mass fingerprinting used the
visualized protein spots among differential gels and oxy- assumption that peptides are monoisotopic, oxidized at
blots. The average mode of background subtraction was methionine residues, and carbamidomethylated at cysteine
used to normalize intensity values, which represents the residues [41 – 45]. Up to one missed trypsin cleavage was
amount of protein (total protein on gel and oxidized protein allowed. Mass tolerance of 150 ppm was the window of
on oxyblot) per spot. After completion of spot matching, the error allowed for matching the peptide mass values.
normalized intensity of each protein spot from individual
gels (or oxyblots) was compared between groups using Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) assay
The activities of UCH-L1 in the spinal cord were measured
Trypsin digestion by determining the rate of conversion of ubiquitin-C-terminal
7-amido-4-methylcoumarin (Ub-AMC) (Calbiochem) to
Samples were prepared using the techniques described ubiquitin and free AMC . In this assay, 39 Al of buffer
previously . The selected protein spots were excised with (50 mM Hepes, pH 7.0, 10 mM DTT, and 0.1 mg/ml
a clean blade and transferred into clean microcentrifuge ovalbumin) was mixed with 10 Al of 2 mg/ml spinal cord
tubes. After being washed with ammonium bicarbonate homogenate of six individual transgenic animals and six
(NH4HCO3) and acetonitrile, the protein spots were treated individual control animals for 1 h. Then 10 Al of 200 nM Ub-
with dithiothreitol and iodoacetamide separately. Then the AMC was added to the enzyme solution, and cleavage of
gel pieces were washed with NH4HCO3 and acetonitrile AMC from Ub-AMC was monitored at Eex 355 nm and Eem
again before rehydration with modified trypsin (Promega, 460 nm using a SpectrMAX Gemini XS fluorescence micro-
Madison, WI, USA). The gel pieces were chopped into titer plate reader. UCH-L1 activities of each individual were
smaller pieces and incubated at 37-C overnight in a shaking assayed by the change of Eem 460 nm over time. The average
incubator. UCH-L1 activities of six transgenic animals were compared
to those of six control animals using Student’s t test.
Mass spectra reported in this study were acquired from
both the University of Kentucky Mass Spectrometry Facility Immunoprecipitation was performed essentially as
(UKMSF) and the Department of Pharmacology at the described previously . A rabbit anti-aB-crystallin anti-
University of Louisville School of Medicine and VAMC. A body (5 Al) (Chemicon) was added directly to the spinal
Bruker Autoflex MALDI-TOF (matrix-assisted laser desorp- cord homogenate. Antibody/lysate solutions were incubated
tion ionization-time of flight) mass spectrometer (Bruker on a rotary mixer overnight at 4-C. The aB-crystallin/
Daltonics, Billerica, MA, USA) at the UKMSF or a TOF antibody complexes were precipitated with protein A-
Spec 2E (Micromass, UK) MALDI-TOF mass spectrometer conjugated agarose beads. Protein A beads were added in
at the University of Louisville operated in the reflectron 50-Al aliquots from a stock of 300 mg/ml in PBS and mixed
mode was used to generate peptide mass fingerprints. on a rotary mixer for1 h at room temperature. Beads were
456 H.F. Poon et al. / Free Radical Biology & Medicine 39 (2005) 453 – 462
then centrifuged and 2D electrophoresis was performed on of aB-crystallin, we identified the protein spot immuno-
the supernatant. chemically. The spot was absent in the 2D gels upon
immunoprecipitation (Fig. 3). Therefore, the protein iden-
tification of aB-crystallin is ensured.
Results In order to confirm that oxidative modification inactivated
protein activity, we compared the activity of UCH-L1 in the
We used a parallel approach to investigate the effect of G93A-SOD1 transgenic mice to that in the control mice.
G93A-SOD1 on protein oxidation [40 – 45]. The specific Consistent with our prior studies that demonstrate loss of
carbonyl levels were obtained by dividing the carbonyl level activity of oxidatively modified proteins [49 – 51], UCH-L1
of a protein spot on the nitrocellulose membrane by the activity was significantly decreased (29%) in the G93A-
protein level of its corresponding protein spot on the gel. SOD1 transgenic mice compared to that of nontransgenic
Such numbers give the carbonyl level per unit of protein. We control (Fig. 4).
found that in comparison to nontransgenic mice, four proteins
in the spinal cords of G93A-SOD1 transgenic mice have
significantly higher specific carbonyl levels than those in Discussion
nontransgenic littermate controls. These proteins were
identified as SOD1, translationally controlled tumor protein The G93A-SOD1 transgenic mouse is frequently used as
(TCTP), UCH-L1, and aB-crystallin. The specific carbonyl an animal model of human FALS due to the neuropatho-
levels of the proteins that are significantly different are logical similarity of this mouse model to the human disease
summarized in Table 1. The summary of the mass spectro- [52,53]. Moreover, this mouse model can provide insight
metry results for the proteins is presented in Table 2. into the mechanisms of the neurotoxicity of mutant SOD in
Fig. 1 shows representative 2D-electrophoresis gels of vivo. It is well established that mutant SOD1 enhances
G93A-SOD1 transgenic mice (Fig. 1A) and nontransgenic oxidative activity by acting as a peroxidase [9,11,13] or a
mice (1B) after Coomassie blue staining. Fig. 2 shows the superoxide reductase . Protein carbonyl levels in the
representative 2D Western blots of the spinal cord of a spinal cord of 3- to 4-month-old G93A-SOD1 transgenic
G93A-SOD1 transgenic mouse (Fig. 2A) and a non- mice show a 557% increase compared to nontransgenic
transgenic mouse (2B). We report here that the specific animals at the same age . Such protein oxidation in the
carbonyl levels of human SOD1, TCTP, UCH-L1, and, spinal cord of G93A-SOD1 transgenic mice followed an
possibly, aB-crystallin are significantly increased in the alteration in cytokine expression . In the current study,
spinal cord of G93A-SOD1 transgenic mice compared to we identified the proteins that demonstrate increased
that of nontransgenic mice. Spots close to human SOD1 carbonyl levels compared to those of the nontransgenic
are modified SOD1, possibly phosphorylated SOD1. mice as SOD1, TCTP, UCH-L1, and aB-crystallin.
However, these modifications cannot be resolved by SOD1 previously was identified immunochemically as
MALDI mass spectrometry. one of the oxidatively modified proteins in G93A-SOD1
Although our mass spectra do not significantly match the transgenic mouse spinal cord . Here, we used a parallel
mass spectra of aB-crystallin, the molecular weight and pI proteomics approach to confirm that the specific carbonyl
value of aB-crystallin agree with the location of the gel level of SOD1 is increased in the spinal cords of G93A-
spots on the 2D gel map. In order to ensure the identification SOD1 transgenic mice. Although G93A-SOD1 shows
dismutation activity identical to that of wild-type SOD1,
Table 1 the activity of SOD1 in FALS patients with mutations is
Increased specific carbonyl level of oxidized proteins in G93A-SOD1 mice decreased 50% in motor cortex, parietal cortex, and
(n = 6) cerebellum . Moreover, free radical production in the
Proteomics Nontransgenic G93A-SOD1 F p value G93A-SOD1 transgenic animals is induced by SOD1
identified mice transgenic F crit = 4.7 mutation , alteration of tumor necrosis factor a (TNF-
protein (AU T SD) mice
(AU T SD)
a), and TNF-a-modulating cytokines [38,46]. Although the
issue of whether oxidative stress plays an early role in ALS
Transcriptionally 15.1 T 10.1 30.6 T 14.1 5.6 <0.05
remains unclear, our current study is consistent with the
protein 1 notion that oxidative modification of SOD1 plays a role in
Cu,Zn-superoxide Not detected 2.88 T 1.78 18.2 <0.005a the neurotoxicity of mutant SOD1 in the disease.
dismutase Another oxidatively modified protein in G93A-SOD1
Ubiquitin 0.74 T 0.23 2.31 T 1.27 10.3 <0.0001 transgenic mice identified by proteomics was TCTP. TCTP
processes calcium-binding activity (reviewed in ) and
isozyme L1 has a tubulin binding region . Overexpression of TCTP
aB crystallin Not detected 20.1 T 8.4 39.7 <0.0001a stabilizes microtubules and alters cell morphology .
Because specific carbonyl levels are not detected in nontransgenic mice, Other molecular interactions of TCTP include self-
a value of 0 was used to calculate the p value. interaction  and the interaction with myeloid cell leu-
H.F. Poon et al. / Free Radical Biology & Medicine 39 (2005) 453 – 462 457
Summary of proteins identified by mass spectrometry
Identified protein gi accession No. peptides % coverage pI, M r (kDa) Mowse Probability of
number matched matched score a random
peptides identification hit
Transcriptionally controlled gi|6678437 5 28 4.76, 19.5 79 1.25 Â 10À8
tumor protein 1
Cu,Zn-superoxide dismutase gi|2982081 8 42 5.73, 16.1 85 3.16 Â 10 – 9
Ubiquitin gi|18203410 8 39 5.29, 24.6 86 2.5 Â 10 – 9
hydrolase isozyme L1
aB crystallin gi|6753530 5 26 7.05, 20.1 60 Not significant
kemia 1 protein . TCTP levels are highly regulated in ization as an antiapoptotic protein , these observations
response to various stress conditions and extracellular sig- suggest that TCTP may exert a cytoprotective function for
nals, including growth signal , cytokines [60,61], cells. The current study showed that TCTP was oxidatively
starvation [55,62], heat shock, heavy metals, calcium stress modified in the spinal cord of G93A-SOD1 mice, suggesting
, and proapoptotic/cytotoxic signals [64,65]. Along with that the putative cytoprotective function and the calcium
the structural similarity to chaperones  and character- binding affinity of TCTP are impaired in G93A-SOD1 mice
Fig. 1. (A) Representative gel of proteins from the spinal cords of G93A-SOD1 transgenic mice after 2D electrophoresis. (B) Proteins from the spinal cords of
nontransgenic mice after 2D electrophoresis. Insets: (Left) Expansion of the region of the 2D gel of G93A-SOD1 transgenic mouse spinal cord outlined by the
box. (Right) Expansion of the region of the 2D gel of nontransgenic mouse spinal cord outlined by the box.
458 H.F. Poon et al. / Free Radical Biology & Medicine 39 (2005) 453 – 462
Fig. 2. (A) Carbonyl Western blot from the spinal cords of G93A-SOD1 transgenic mice (left) and expansion of the blot outlined by the box (right). (B)
Carbonyl Western blot from the spinal cords of nontransgenic mice (left) and expansion of the blot outlined by the box (right).
because oxidative modification alters the structure and . Loss of UCH-L1 function causes neuroaxonal dys-
function of proteins [49 – 51,68,69]. Consistent with this trophy [74 –76], significant protein oxidization , and
notion, free cytosolic calcium was increased in lymphocytes accumulation of synuclein protein in gracile axonal dys-
from ALS patients , suggesting that oxidative modifica- trophy mice . Similarly, decreased UCH-L1 activity by
tion of TCTP may also play an important role in neuro- mutation also enhances protein aggregation in Escherichia
toxicity of G93A-SOD1 and thus neurodegeneration in the coli . Therefore, based on the prior literature, oxidative
spinal cords of G93A-SOD1 transgenic mice. inactivation of UCH-L1 presented in the current study
UCH-L1 belongs to a family of ubiquitin carboxyl- possibly contributes to both the protein aggregation and the
terminal hydrolases that play important roles in the oxidative stress observed in G93A-SOD1 transgenic mice
ubiquitin –proteolytic pathway . The ubiquitin –protea- and ALS patients. Consistent with this notion and consistent
some system is a major pathway for selective protein with our finding (Fig. 4) that UCH-L1 activity is decreased
degradation . Ubiquitinated proteins form polyubiquitin in G93A-SOD1 mouse spinal cord, the inclusions of human
chains that are eventually degraded by the 26S proteasome ALS and mSOD1 (including G93A) mice are excessively
. UCH-Ls then recycle ubiquitin from ubiquitinated ubiquitinated [79 – 82].
protein complexes or polyubiquitin chains by cleaving the aB-Crystallin belongs to the small heat shock protein
amide linkage next to the C-terminal glycine of ubiquitin (sHSP) class of the heat shock protein family. HSPs are
Fig. 3. (A) 2D electrophoresis gel from supernatant of the spinal cords of mice described in this study after immunoprecipitation. (B) 2D electrophoresis gel
from spinal cords of the mice described in this study. Arrow indicates that aB-crystallin disappears after immunoprecipitation, confirming its identity.
H.F. Poon et al. / Free Radical Biology & Medicine 39 (2005) 453 – 462 459
accumulation of mutant SOD1 is demonstrated in Caeno-
rhabditis elegans expressing human mutant SOD1 .
Based on our current observations, the increased oxidative
modification of SOD1, UCH-L1, and aB-crystallin plays a
significant role in the protein aggregation in the spinal cords
of G93A-SOD1 transgenic mice. Our current study provides
insight into the mechanism of G93A-SOD1 neurotoxicity in
vivo, which involves oxidative modification of a Ca2+
regulating protein (TCTP) and proteins involved in inclu-
sion formation (SOD1, UCH-L1, and aB-crystallin), sug-
gesting a potential relationship between protein oxidation,
protein aggregation, and Ca2+ regulation in ALS. Moreover,
one can speculate that the oxidative modification of these
proteins impairs protein stability (aB-crystallin), Ca2+
binding (TCTP), protein degradation (UCH-L1), and anti-
oxidant capacity (SOD1). It should be noted that other
oxidative modifications could also play a role in the
pathogenesis of ALS involving other proteins . More-
over, because only symptomatic mice were compared to the
Fig. 4. Activity of UCH-L1 in G93A-SOD1 transgenic mice as a percentage nontransgenic mice in this study, it is possible that the
of the nontransgenic control. The activity of UCH-L1 is significantly oxidative modification of the proteins described is related to
decreased in the spinal cord of G93A-SOD1 transgenic mice compared to the consequences of the degenerative process. Therefore,
nontransgenic control. Columns represent mean T SEM. *p < 0.05, n = 6 future studies will address whether similar changes are
for each group.
observed in presymptomatic G93A-SOD1 mutant mice.
cellular constituents synthesized by living organisms
under stress conditions as well as normal conditions.
The major function of sHSP is to stabilize other proteins
This work was supported in part by grants from the NIH
under stress conditions, whereas the high-molecular-
to D.A.B. (AG-05119, AG-10836) and to K.H. from the
weight HSPs normally play roles in protein folding during
Oklahoma Center for Advancement of Science and Tech-
biosynthesis [83,84]. Incorporation of aB-crystallin into
nology (HR02-149R), the NIH (NS044154), and the ALS
red cell ghosts protects ATPase against oxidative stress
, and overexpression of aB-crystallin protects cells
against apoptosis and necrosis during myocardial ischemia
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