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					                                                 Free Radical Biology & Medicine 39 (2005) 453 – 462
                                                                                                              www.elsevier.com/locate/freeradbiomed

                                                             Original Contribution

         Redox proteomics analysis of oxidatively modified proteins in
        G93A-SOD1 transgenic mice—A model of familial amyotrophic
                               lateral sclerosis
      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,*
                                        a
                                         Department of Chemistry, University of Kentucky, Lexington KY 40506, USA
  b
   Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA
                 c
                  Oklahoma Center for Neuroscience, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA
    d
     Kidney Disease Program and Core Proteomics Laboratory, University of Louisville School of Medicine and VAMC, Louisville, KY 40292, USA
                                      e
                                       Mass Spectrometry Facility, University of Kentucky, Lexington, KY 40506, USA
                   f
                     Department of Pharmacology, University of Louisville School of Medicine and VAMC, Louisville, KY 40292, USA
                                g
                                 Section of Biochemistry, Department of Chemistry, University of Catania, Catania, Italy
                                    h
                                     Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506, USA
                                  i
                                    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



Abstract

   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: dabcns@uky.edu (D.A. Butterfield).                       radical to hydrogen peroxide and oxygen. Over 100
0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2005.03.030
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 [5]. 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) [1].                                   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) [6]. 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
                                                   À
superoxide reductases [14] 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            [37], 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 [6].           mating transgenic males with B6/SJLF1 females as pre-
The proteinaceous inclusions found in tissues from ALS                 viously described [46].
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
[27], 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.
[18,28,29]).
    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 [36]. 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 [32] and eventually develop motor                      Samples of spinal cord proteins were prepared according
neuron disease and die within 5 –6 months [37]. G93A-                  to the procedure previously described [47]. 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 [11] and has a higher capacity to generate            temperature (25-C) for 20 min. Proteins were then
free radicals [9] 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 [38]. 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.
[39], 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 [32], 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 [47], 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
ANOVA.
                                                                           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 [48]. In this assay, 39 Al of buffer
previously [47]. 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 spectrometry
                                                                       Immunoprecipitation
   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 [49]. 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 [14]. 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 [32]. 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 [46]. 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 [32]. 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 [54]. 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 [32], 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
  controlled tumor
                                                                               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
  carboxyl-terminal
  hydrolase
                                                                               processes calcium-binding activity (reviewed in [55]) and
  isozyme L1                                                                   has a tubulin binding region [56]. Overexpression of TCTP
aB crystallin         Not detected    20.1 T 8.4     39.7        <0.0001a      stabilizes microtubules and alters cell morphology [57].
 a
   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 [58] and the interaction with myeloid cell leu-
                                         H.F. Poon et al. / Free Radical Biology & Medicine 39 (2005) 453 – 462                                          457


Table 2
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
  carboxyl-terminal
  hydrolase isozyme L1
aB crystallin                        gi|6753530            5                     26                   7.05, 20.1            60              Not significant



kemia 1 protein [59]. TCTP levels are highly regulated in                         ization as an antiapoptotic protein [67], these observations
response to various stress conditions and extracellular sig-                      suggest that TCTP may exert a cytoprotective function for
nals, including growth signal [55], 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
[63], and proapoptotic/cytotoxic signals [64,65]. Along with                      that the putative cytoprotective function and the calcium
the structural similarity to chaperones [66] 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                           [72]. Loss of UCH-L1 function causes neuroaxonal dys-
function of proteins [49 – 51,68,69]. Consistent with this                        trophy [74 –76], significant protein oxidization [45], and
notion, free cytosolic calcium was increased in lymphocytes                       accumulation of synuclein protein in gracile axonal dys-
from ALS patients [70], suggesting that oxidative modifica-                       trophy mice [77]. 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 [78]. 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 [71]. 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 [72]. 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
[73]. 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 [36].
                                                                             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 [92]. 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
                                                                             Acknowledgments
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
                                                                             Association.
[85], and overexpression of aB-crystallin protects cells
against apoptosis and necrosis during myocardial ischemia
and reperfusion [86] by acting as a chaperone [87].                          References
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