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Targets of protein nitration during aging

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Targets of protein nitration during aging

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Content TARGETS OF PROTEIN NITRATION IN MITOCHONDRIA DURING AGING

by

Catherine Brégère
________________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

May 2008

Copyright 2008 Catherine Brégère

ii
Acknowledgments
I would like to acknowledge the excellent mentorship by Professor Rajindar S.
Sohal. I sincerely appreciated his patience throughout my graduate studies, and his
precious advice on scientific writing. I am also extremely grateful to Dr. Igor Rebrin
for his guidance in my research, his invaluable scientific advice and friendship.
I would like to thank my committee members, Professors Jean Shih, Wei-Chiang
Shen, and Neil Kaplowitz, for being part of my thesis committee over the years.
I would also like to express my gratitude to the members of the laboratory, Barbara
Sohal, Dr. Dikran Toroser, Dr. Sergey Kamzalov, Dr. Robin Mockett, Dr. Melissa
Ferguson, Kathleen Rice and Marta Orona, for their support and kindness at all
times. I owe special thanks to Dr. Timothy K. Gallaher, who provided skillful help in
mass spectrometry.
Lastly, I would like to thank my family and friends for the constant support they
provided me. I dedicate this work to the memory of my mother.
iii
Table of Contents
Acknowledgments ii
List of Tables vi
List of Figures vii
Abstract x
Chapter One: Introduction 1
I. The oxidative stress hypothesis of aging 2
A. Principal tenet of the oxidative stress hypothesis of aging 2
B. Main oxidizing species relevant to the aging process: reactive oxygen and
nitrogen species 3
C. Mitochondria are key sites for the production of pro-oxidizing species 5
D. Proteins: key target of oxidative damage 6
II. The contribution of oxidatively modified proteins to the aging process 7
A. Protein oxidation as a marker of aging 7
B. Current knowledge on protein nitration during aging-Rationale of the
study 10
III. Hypothesis and specific goals 12
A. Hypothesis 12
B. Specific aims 13
1. Identify in vivo nitrated proteins and quantify nitration content with age 13
2. Determine the functional consequences of protein nitration 14
3. Assess the impact of calorie restriction (CR) on the content of nitration
and the catalytic activity of the proteins of interest 14
Chapter Two: Materials and Methods 16
I. Reagents and Materials 16
II. Animals and tissues 17
III. Methods 17
IV. Statistical analysis of data 29

iv
Chapter Three: Results 30
I. Identification of proteins targets of nitration among the mitochondrial soluble
proteins in different rat organs 30
A. Western blot analysis of the soluble mitochondrial proteins using an anti-3-
nitrotyrosine antibody 30
B. Specificity of the reaction with the anti-3NT antibody 32
C. Purification and identification of the 58 kDa immunopositive protein 35
1. Purification procedures 35
2. Identification of the two immunopositive proteins 40
D. Background information on SCOT and ECH 41
E. Summary of the findings 45
II. Identification of the nitrated residue(s) in SCOT 45
A. Autolytic fragmentation of SCOT 45
B. Amino acid analysis by HPLC/electrochemical detection 49
C. Mass spectrometric characterization of the amino acid X 52
1. MALDI mass spectrometry 52
2. In vitro synthesis of nitro-hydroxytryptophan and characterization by
HPLC and MALDI analysis 54
3. SI-MS/MS analysis 57
D. Localization of tryptophan 372 in the SCOT three dimensional structure 62
E. Summary of the findings 63
III. Effects of age and calorie restriction on SCOT nitrohydroxytryptophan
content, protein amount and catalytic activity 66
A. Age-associated variations in SCOT nitrohydroxytryptophan content in
mitochondria of various rat organs 66
B. Impact of age on SCOT parameters in heart and kidney mitochondria 67
C. Impact of calorie restriction on SCOT nitrohydroxytryptophan content,
protein amount and specific activity 72
D. Summary of the effects of aging and calorie restriction on SCOT
parameters 74
E. Specific activity of nitrated SCOT 76
F. Summary of the findings 77
IV. Assessment of functional consequences of SCOT nitration via in vitro
experiments 77
A. Impact of peroxynitrite-induced nitration on SCOT catalytic activity 77
B. Susceptibility of SCOT to proteolysis 81
1. Susceptibility to trypsin induced-degradation 82
2. Susceptibility to chymotrypsin-mediated proteolysis 87
3. Susceptibility to other proteases induced-degradation 89
C. Summary 91
V. Impact of age and calorie restriction on SCOT parameters in different mice
strains 91
A. SCOT nitration in three different mice strains 92
B. Impact of age on SCOT parameters in C57 and DBA mice 93
v
C. Impact of calorie restriction 96
D. Summary 99
VI. Effect of age and calorie restriction on enoyl-CoA hydratase protein amount
and catalytic activity in rat kidney and liver mitochondria 99
A. Effect of age on ECH activity and protein amounts in kidney and liver 100
B. Impact of calorie restriction 101
VII. Succinyl-CoA:3-oxo-acid CoA-transferase and enoyl-Coenzyme A hydratase
nitration in the kidney from species exhibiting life spans that differ from the rat 103
A. SCOT nitration in kidney mitochondria of pig, dog, cow and rabbit 103
B. Purification of the SCOT protein from kidney of different species 104
C. Identification of the site of nitration in the SCOT protein from various
species 111
Chapter Four: Discussion 113
Chapter Five: Conclusions 136
References 139

vi
List of Tables
Table 1: Results of mass spectrometric analysis of proteins identified in
enriched SCOT isolates from heart and kidney mitochondria.
59
Table 2: Summary of SCOT parameters measured in the heart and kidney of
young (4 months), old (24 months), ad-libitum fed and 40% calorie
restricted (19 months) rats
75
Table 3: SCOT tryptic peptide containing the +61 Da mass increment in C57
mice, cow and pig
112
vii
List of Figures
Figure 1: Localization of nitrated protein band among the mitochondrial
soluble proteins of various organs
31
Figure 2: Controls for establishing the specificity of the immunoreaction
with the anti-3-nitrotyrosine antibody and for validation of the
experimental procedures
34
Figure 3: Purification of the immunopositive protein in rat heart
mitochondria by chromatofocusing and gel filtration
38
Figure 4: Purification of the immunopositive protein in rat kidney
mitochondria by chromatofocusing and gel filtration
39
Figure 5: N-terminal sequencing of the purified immunopositive proteins
and their expression in various rat tissues
41
Figure 6: Ketolysis and β-oxidation of fatty acids 44
Figure 7: Autolytic fragmentation of SCOT revealed the presence of nitrated
amino acid(s) in the 21 kDa carboxyl terminal fragment of the
protein
48
Figure 8: Detection of an unknown amino acid, X, in the SCOT protein 51
Figure 9: MALDI mass spectra of the amino acid X, and various tryptophan
derivatives
56
Figure 10: Structure of 6-nitro-5-hydroxy-tryptophan and its derivatives
under MALDI conditions and after reducing treatment
57
Figure 11: Identification of tryptophan 372 as the site of nitration in SCOT 61
Figure 12: Ribbon diagram of SCOT monomer (A) showing amino acids
involved in catalysis and the proximity of Trp 372 (B) and
structure of the substrate, succinyl-CoA (C)
65
Figure 13: Age-associated changes in SCOT nitration content in different
tissues of the rat
67
viii
Figure 14: Impact of age on SCOT nitrohydroxytryptophan content, protein
amounts and specific activity in rat heart mitochondria
70
Figure 15: Impact of age on SCOT nitrohydroxytryptophan content, protein
amounts and specific activity in rat kidney mitochondria
71
Figure 16: Effect of calorie restriction on SCOT parameters in rat heart and
kidney of rat
73
Figure 17: Peroxynitrite-induced nitration of SCOT in kidney and heart and
its impact on nitration and activity
80
Figure 18: Degradation of SCOT during proteolysis in vitro 84
Figure 19: Degradation of carboxy and amino terminal fragments of SCOT
upon partial trypsin induced-proteolysis in vitro
86
Figure 20: Lack of degradation of the SCOT protein in response to
chymotrypsin treatment
88
Figure 21: SCOT susceptibility to degradation by proteases with different
cleavage specificities
90
Figure 22: Nitration of the protein SCOT in kidney mitochondria from three
different strains of mice
93
Figure 23: Impact of age on SCOT nitration, protein amount and catalytic
activity in C57 and DBA mice
95
Figure 24: Impact of calorie restriction on SCOT catalytic activity in the
kidney of the young (6 months) and old (23 months) C57BL/6
and DBA/2 mice
97
Figure 25: Impact of calorie restriction on SCOT protein amount and relative
nitration content, estimated by the ratio OD
nitrated SCOT
to OD
total
SCOT
, in C57BL/6 and DBA/2 mice at 23 months of age
98
Figure 26: Age-associated variations in ECH protein content and catalytic
activity in kidney and liver mitochondria
101
Figure 27: Impact of calorie restriction on ECH catalytic activity and protein
amounts
102
ix

Figure 28: SCOT nitration in kidney mitochondria from pig, dog, cow and
rabbit
104
Figure 29: Purification of SCOT from kidney mitochondria of C57 mice,
pig, cow, dog and rabbit
107
Figure 30: SCOT fragmentation in pig, C57 mice, cow and dog 112

x
Abstract
The main goal of this study was to test the hypothesis that specific proteins
undergo an age-related increase in nitration, which results in their functional
alteration, and that calorie restriction (CR), a regimen which prolongs the life span of
many rodents, can attenuate or postpone such age-associated changes. Succinyl-CoA
transferase (SCOT) and enoyl-CoA hydratase (ECH), two enzymes involved in
energy production, were detected immunochemically with an anti-3-nitrotyrosine
antibody to be targets of nitration in mitochondria from Fischer rat tissues. Mass
spectrometric studies revealed that, rather than tyrosine, tryptophan 372, located in
the vicinity of key catalytic residues, was the site of a novel posttranslational
modification, namely nitrohydroxytryptophan in the SCOT protein. This amino acid
alteration was also detected in mice, pig and cow, but not rabbit, suggesting that
oxidative/nitrative mechanisms targeting this particular tryptophan residue are
relatively well conserved, but not universal, among mammals. In rat heart
mitochondria, an increase in nitrohydroxytryptophan content was associated with an
elevation in SCOT specific catalytic activity during aging, while SCOT protein
amounts remained unchanged. In contrast to heart, the amounts of SCOT protein
declined significantly with age in kidney, whereas the nitration and specific activity
remained unchanged. CR selectively attenuated the age-related changes in the
amount of SCOT protein in kidney mitochondria, but had no effect in the heart. Both
in vivo and in vitro data indicated that nitration/oxidation of tryptophan may activate
xi
the SCOT protein in rat heart during aging. Nitrated SCOT was relatively more
susceptible to undergo in vitro proteolysis than the unmodified enzyme. In C57BL/6
and DBA/2 mice, SCOT protein amount, degree of its nitration and catalytic activity
remained unaffected with age. CR induced a similar quantitative decrease in the
content of SCOT protein in both strains. Overall, SCOT catalytic activity seemed to
be modulated by its degree of nitration and the amounts of protein. Age and CR-
related changes in ECH catalytic activity appeared to be mostly correlated with
levels of ECH protein. In general, the results imply that there is a shift in the
utilization of ketone bodies during aging in Fischer rats, but not in C57BL/6 and
DBA/2 mice. The degree of SCOT nitration remained unaffected by CR, suggesting
that the CR effect does not involve SCOT. The impact of age and CR were tissue and
species specific.
1
To grow old is to pass from passion to compassion
Albert Camus
Chapter One: Introduction
Aging corresponds to the terminal phase of life of organisms, during which
there is a gradual decline in the efficiency of various physiological processes, leading
ineluctably to death. Typically, the most prominent manifestations of the progressive
functional impairments in mammals during aging include: (i) an exponential increase
in the likelihood of death and vulnerability to many diseases, (ii) a decline in fertility
and mobility, and (iii) a compromised ability to maintain homeostasis, i.e.
equilibrium among various physiological functions, and to endure environmental
stresses.
Presently, the sole experimental regimen that has been shown to delay the
onset of these age-associated decrements, and to significantly prolong median life
span (i.e. the average life expectancy of any individual within a population), and
maximum life span (i.e. the mean longevity of the 10% longest lived individuals
within a population) of a spectrum of rodents and other species is calorie restriction
(CR) (Sohal and Weindruch 1996). CR entails reduction of food intake ranging from
25-50% of the intake of ad libitum-fed animals, without causing malnutrition. For
instance, it has been shown that a 40% long-term CR regimen in Fischer 344 rats,
initiated at 3-4 months of age, induces a 40% increase in maximum life span. Thus
studies aiming at understanding the biological basis of aging have often included the
study of CR, since any causal factor in the aging process should be retarded by CR.
2
The ultimate causes of the deleterious changes occurring during the aging
process, and the mechanisms responsible for the retardation of aging induced by CR
are presently not well understood. Although numerous theories of aging have been
proposed, most lack credence due to limited empirical support. The main purpose of
this study was to test one of the presently most prominent theories of aging, namely
the oxidative stress hypothesis of aging.
I. The oxidative stress hypothesis of aging
A. Principal tenet of the oxidative stress hypothesis of aging
The oxidative stress hypothesis of aging proposes that accumulation of
oxidative damage to macromolecules (lipids, nucleic acids, carbohydrates and
proteins) causes age-associated deteriorations in function (Harman 1956). Such
accrual of molecular oxidative damage is explained by an age-associated increase in
oxidative stress, defined as an imbalance between the generation of oxidizing species
and their removal by antioxidants in the cells. An enhancement in oxidative stress
during aging is suggested to be the result of (i) an age-related increase in the
production of pro-oxidizing species and/or (ii) an age-related decline in antioxidant
defenses and/or (iii) an age-associated decrease in the removal of oxidative damage
or (iv) a combination of all these factors.

3
B. Main oxidizing species relevant to the aging process: reactive oxygen
and nitrogen species
The basis of the oxidative stress hypothesis is that oxygen, although
indispensable for the survival of aerobic organisms, can be converted into an array of
oxidizing species, the reactive oxygen species (ROS) that can be toxic to
biomolecules. This dual nature of oxygen, i.e. vital but potentially noxious, is
commonly referred to as the “oxygen paradox”. The discovery that nitric oxide
(NO
.
), a free radical, was produced endogenously (Palmer, Ferrige et al. 1987)
broadened the scope of the oxidative stress theory of aging. Thus oxidizing species
of relevance to the aging process implicate both ROS and nitric oxide derived
species, or reactive nitrogen species (RNS). It is widely proposed that the molecular
damage inflicted by these oxidizing species causes or contributes to aging.
ROS and RNS comprise free radicals and non-radical species. Free radicals
are reactive molecules due to the presence of one or more unpaired electrons on their
atomic orbital. Their potential toxicity stems from the fact that, to stabilize
themselves, they tend to abstract hydrogens from, or add onto stable molecules, thus
forming new radicals and setting off chain reactions, or altering the chemical
properties of the structure that they target. Oxygen centered species of interest in
aging include the superoxide anion radical (O
2
.-
), hydroxyl radical (
.
OH) and
hydrogen peroxide (H
2
O
2
,

a non radical species)
.
Their generation in cells involves
both spontaneous and catalytic reactions. Superoxide,

which results from the one-
electron reduction of oxygen, can be produced in mitochondria, when electrons leak
out from the electron transport chain (ETC), and into the cytosol, or via the activity
4
of enzymes such as xanthine oxidase, NADPH oxidase and nitric oxide synthase (if
L-arginine concentrations are low). The half-life of O
2
.-
is relatively short, and
cytosolic or mitochondrial superoxide dismutases (SODs) catalyze its dismutation to
hydrogen peroxide. Thus intracellular sources of hydrogen peroxide are, to a certain
extent, O
2
.-
dependant, but also derive from enzymatic activities. For instance,
monoamine oxidases A and B (MAO-A/B), enzymes located on the outer membrane
of mitochondria, catalyze the deamination of polyamines, and generate H
2
O
2
as a
byproduct. Hydrogen peroxide is not very reactive per se, but in the presence of
metals, it can yield the highly cytotoxic hydroxyl radical through Fenton reaction. As
soon as the hydroxyl radical is formed, it reacts indiscriminately with virtually every
kind of molecule in its vicinity. Examples of potentially deleterious damage, induced
by these ROS, include: (i) lipid peroxidation, which impairs the fluidity and
elasticity of cellular membranes, (ii) various type of DNA lesions, e.g. strand
breakage, mutations (iii) and protein carbonylation (Halliwell and Gutteridge 1984).
Reactive nitrogen species relevant to the aging process include mainly nitric
oxide, a free radical, and peroxynitrite, (ONOO
-
), a non radical species. Nitric oxide
effects are subtle as it can act both as a pro-oxidizing species as well as an
antioxidant (Pacher, Beckman et al. 2007). Three isoenzymes are responsible for its
generation, namely neuronal, inducible and endothelial nitric oxide synthase (nNOS,
iNOS, and eNOS). They convert L-arginine to L-citrulline and NO
.
, using molecular
oxygen and NADPH as substrates, and tetrahydrobiopterin (BH
4
), FAD, FMN and
heme as cofactors.
5
If many of the physiological effects of NO
.
are linked to its capacity to bind
to iron containing enzymes, such as guanylyl cyclase, it is thought that detrimental
effects of NO
.
are mediated through the formation of peroxynitrite (Beckman 1990),
which arises from the recombination of superoxide anion and NO
.
, as follows:
O
2
-.
+ NO
.
→ ONOO
-

Similar to the hydroxyl radical, peroxynitrite is a very potent oxidant: it can
initiate lipid peroxidation (Radi, Beckman et al. 1991), induce damage to nucleic
acids, through the formation of 8-nitroguanine (Yermilov, Rubio et al. 1995) and
modify specific amino acids in proteins (Ischiropoulos and al-Mehdi 1995). A
plausible marker of peroxynitrite-mediated functional alterations in proteins is the
addition of a nitro (NO
2
-) group onto aromatic amino acids, namely 3-nitrotyrosine
(Ischiropoulos 1998).
C. Mitochondria are key sites for the production of pro-oxidizing
species
In eukaryotic cells 90% of inhaled oxygen is metabolized by mitochondria to
produce energy (adenosine triphosphate, ATP), via oxidative phosphorylation. It is
currently estimated that between 0.1-2% of the oxygen consumed by mitochondria is
univalently and bivalently reduced to O
2
-.
and H
2
O
2
, respectively, because of

electron leakage in the electron transport chain (Chance, Sies et al. 1979; Hansford,
Hogue et al. 1997). Despite this low percentage, other cellular compartments might
not exceed ROS generation by mitochondria, because most oxygen utilizing enzymes
exhibit lower binding affinities to oxygen than cytochrome c oxidase. Therefore, in
6
vivo, mitochondria might be the main or at least key intracellular producers of pro-
oxidizing species. Being the most relevant intracellular sites for the production of
ROS, it was hypothesized that mitochondria and its components might also be the
most susceptible targets of ROS-mediated damage, and that, as a result,
mitochondrial dysfunction might play a key role in the aging process (Harman 1972;
Miquel, Economos et al. 1980).
Currently, the main evidence supporting the oxidative stress hypothesis of
aging is: (i) the rate of mitochondrial ROS production as well as the amount of
oxidatively damaged macromolecules increase with age (Agarwal and Sohal 1994),
(ii) the rates of mitochondrial ROS generation and accumulation of oxidative damage
are inversely correlated to the maximum lifespan of different species (Ku, Brunk et
al. 1993), (iii) a decrease in mitochondrial rates of ROS generation, accompanied by
a reduction in molecular oxidative damage levels, is observed in calorically restricted
animals, the sole experimental regimen known to prolong life span of rodents (Sohal,
Ku et al. 1994). Nevertheless, such observations are correlative, and a causal link to
the aging process remains to be established.
D. Proteins: key target of oxidative damage
Among molecules susceptible to oxidative damage, proteins are thought to be
the key targets, because oxidatively modified proteins often lose catalytic activity,
and are preferentially degraded or elicit immune responses (Stadtman 1992;
Stadtman 2001; Thomson, Christie et al. 2007). In this context, this investigation has
addressed the role of ROS and RNS in mediating potentially deleterious
7
modifications to proteins, which may contribute to age-related alterations. The
particular focus of this study was to investigate the relevance of protein nitration to
the aging process.
II. The contribution of oxidatively modified proteins to the aging process
It has been observed that proteins purified from organs of aged animals
showed biochemical alterations, such as conformational changes, partial loss or toxic
gain in enzymatic activity, and higher sensitivity to heat denaturation, in comparison
to the ones isolated from young animals, and that such impaired proteins
accumulated in aging cells (Rothstein 1977). In the early 1980s, the pioneering work
of Stadtman and coworkers indicated that in vitro oxidation of proteins often elicited
catalytic inactivation of enzymes, and their eventual proteolytic degradation (Levine,
Oliver et al. 1981; Fucci, Oliver et al. 1983), thereby suggesting that oxidative events
in protein might be a mechanism by which senescent alterations occur. Stadtman’s
establishment of protein carbonylation as marker of protein oxidation and its role
during aging will be reviewed below. The necessity to examine other markers of
protein oxidation, such as protein nitration, to extend the understanding of the
contribution of oxidative stress to the aging process, is also discussed.
A. Protein oxidation as a marker of aging
Protein oxidative modifications induced by ROS or their by-products are
quite diverse. These include carbonylation, oxidation of aromatic and sulfur amino
acids. Carbonyl groups (aldehyde or ketone) can be introduced directly on the side
8
chains of some specific residues (threonine, arginine, lysine and proline) through
metal catalyzed oxidation, or indirectly through the addition of carbonyl containing
groups, such as lipid peroxidation (e.g. malondialdehyde (MDA) and 4-
hydroxynonenal (HNE)) and glycation products.
Biological consequences from oxidative modifications depend on several
factors, such as the nature of the modification, the site of the modified amino acid in
the three-dimensional structure, for instance its distance to key catalytic residues.
Thus, physiological repercussions might range from changes in surface
hydrophobicity to protein misfolding, peptide bond cleavage, and protein
aggregation, all of which might ultimately not only modulate catalytic activity, but
also either accelerate or limit their turnover.
Central questions pertaining to the involvement of oxidatively modified
proteins in senescence-related impairments are: (i) whether they accumulate during
aging, (ii) which proteins are oxidatively modified (iii) what are the functional
consequences of such modifications in vivo. In this context, the role of carbonylated
proteins is by far the most documented, and their measurement is commonly used as
a mean to assess cellular oxidative stress state. Thus, results from Stadtman’s group,
our laboratory as well as others have indicated that (i) the content in carbonylated
proteins increase exponentially with age in various animal models and tissues
(Stadtman 2001) and that (ii) calorie restriction could attenuate this age-related
accrual in carbonylated proteins (Dubey, Forster et al. 1996; Lass, Sohal et al. 1998).
In addition, removal and degradation of oxidized proteins was reported to be
compromised with age, due to a decline in proteasome activity (Petropoulos,
9
Conconi et al. 2000). Nevertheless, it should be noted that not all oxidatively
modified proteins accumulate with age, and that the presence of an oxidative
modification is not always associated with an obvious functional impact. Thus, it
was recently observed that two mitochondrial proteins isolated from mouse heart,
namely very long chain acyl coenzyme A dehydrogenase (VLCAD) and α-
ketoglutarate dehydrogenase, despite being putative MDA-containing proteins, did
not exhibit an age-associated alteration in their MDA content, nor did their catalytic
activity vary with age (Yarian, Rebrin et al. 2005). These examples underlie the
necessity to study individual proteins that are modified and to characterize any
potential functional effect.
Sohal and coworkers further reported that protein oxidation during aging was
a selective, rather than a stochastic process. Indeed, results of their studies
demonstrated that, in fly mitochondria, two proteins, namely aconitase (Yan, Levine
et al. 1997; Das, Levine et al. 2001) and adenine nucleotide translocase (Yan and
Sohal 1998) exhibited an age-associated increase in carbonyls, which correlated with
a corresponding loss of their activity. Their results accord with the fact that only few
enzymes lose catalytic activity with age, but challenge the commonly held view that
ubiquitous and non-discriminatory oxidative damage underlies age-associated
cellular dysfunction. Nonetheless, difficulties arose in the interpretation of such data,
because carbonyls can sometimes be derived from non oxidative reactions.
Therefore, the present study has used another marker of in vivo oxygen and nitrogen
derived species, namely 3-nitrotyrosine.
10
B. Current knowledge on protein nitration during aging-Rationale of
the study
As noted earlier, the discovery of nitric oxide as an endogenously synthesized
gas prompted the exploration of NO-mediated chemistry in vivo, notably reactions of
NO
.
with proteins. Protein nitration, i.e. the addition of a nitro group (-NO
2
) to
aromatic amino acids, such as 3-nitrotyrosine, represents one potential marker of in
vivo protein oxidative modification.
Accumulation of nitrated proteins occurs during normal aging (Kanski,
Behring et al. 2005; Kanski, Hong et al. 2005; Gokulrangan, Zaidi et al. 2007; Hong,
Gokulrangan et al. 2007). Notwithstanding, the functional consequences of such
modification remain largely ambiguous. In vitro (Han, Canali et al. 2005) and in vivo
nitration have been shown to decrease catalytic activity of target proteins
(MacMillan-Crow, Crow et al. 1996), but also in one instance to enhance (Ji,
Neverova et al. 2006) or have no impact on enzyme function (Soulere, Claparols et
al. 2001). Three major limitations of these studies could explain such a discrepant
picture. (i) The susceptibility of proteins to in vitro nitration does not imply that they
are in fact nitrated in vivo. (ii) Because nitrating agents also induce oxidation and
nitration of different amino acids, besides tyrosine, it is often difficult to relate
directly the presence of a nitro group on a tyrosine residue to the observed impact on
catalytic activity. (iii) The specific nitrated residue(s) in the in vivo protein have to be
identified, and their relative amount to the total the protein quantified. So far, only
two proteins, namely sarcoplasmic reticulum (SR) Ca
2+
-ATPase (SERCA2a isoform)
and glycogen phosphorylase b from rat skeletal muscle were shown to accumulate a
11
significant amount of 3-nitrotyrosine with age (Viner, Ferrington et al. 1999; Sharov,
Galeva et al. 2006). For both proteins, nitrated residues were identified, and the
accumulation in nitrated enzymes with time correlated with an age-related decrease
in catalytic activity.
It should also be noted that the studies on protein nitration have been mainly
focused on the study of tyrosine-nitrated proteins, mostly because of the commercial
availability of the anti-3-nitrotyrosine antibody. Nevertheless, mass spectrometric
studies clearly show that tryptophan residues in proteins are also sensitive targets to
in vitro nitrating agents (Yamakura, Matsumoto et al. 2005; Salavej, Spalteholz et al.
2006). To date, in vivo tryptophan nitration has been demonstrated in a bacterial
dipeptide (Kers, Wach et al. 2004). An anti-6-nitrotryptophan has been recently
developed, but its use was limited to the detection of nitrated proteins after
peroxynitrite treatment of rat cultured cells (Ikeda, Yukihiro Hiraoka et al. 2007).
Thus, the in vivo nitration of tryptophan residues in mammalian proteins has never
been reported.
In this context, additional nitrated proteins in vivo need to be identified in
order to gain further insight into the functional impact of protein nitration during
aging.
Thus, the overall goal of this study was to examine the potential contribution
of ROS/RNS-mediated protein nitration to age-associated dysfunctions. The major
questions raised were: (i) what are the specific proteins targets of nitration? (ii) Is
there an age-related change in the content of nitrated proteins? (iii) Which residue(s)
12
are nitrated, and (iv) can we directly relate the modification of target residue(s) to
specific functional consequences?
III. Hypothesis and specific goals
A. Hypothesis
The hypothesis tested in this study was that nitrated protein(s) accumulate
with age in vivo, and that this process can modulate the catalytic activity of the
target protein(s). It was also postulated that calorie restriction, an experimental
regimen known to delay the aging process, can attenuate the age-related
changes in protein nitration.
To test this hypothesis, the research plan proceeded through these steps. In
vivo nitrated proteins were identified as follows: (1) the mitochondrial proteins from
Fischer rats of different ages, ad libitum (AL)-fed and calorie restricted (CR)
animals, were subjected to Western blot analysis using an anti-3-nitrotyrosine
antibody, and (2) the nitrated proteins were purified and sequenced. Content in
nitration with age was quantified by densitometric analysis and HPLC. The
assessment of the biological impact of nitration during aging was performed by
enzymatic activity measurements, as well as additional in vitro experiments detailed
below. Three additional animal models were used in this study: C57BL/6, DBA/2
and monoamine oxidase A/B double knockout mice. Monoamine oxidase A/B
(MAO-A/B) knockout mice were used to investigate the potential importance of
mitochondrial hydrogen peroxide on protein nitration since MAO-A/B, located on
13
the outer mitochondrial membrane, generate H
2
O
2
as a byproduct of their activity.
The rationale for the use of C57BL/6 and DBA/2 will be explained below.
B. Specific aims
As mentioned above, this study intended to examine whether protein nitration
contributes to the modulation of protein catalytic activity during the aging process.
Accordingly, the specific aims were to:
1. Identify in vivo nitrated proteins and quantify nitration
content with age
Mitochondrial proteins isolated from various organs, namely brain, heart,
skeletal muscle, kidney, liver, lung and testis, of 4 and 24 months-old Fischer rats,
were used to identify nitrated proteins by Western blot analysis with an anti-3-
nitrotyrosine antibody for immunodetection, followed by purification and
sequencing. Age-associated variations in the content of nitrated proteins were
quantified by both densitometry analysis and HPLC measurements.
Even though the nature of nitrating species in vivo remains controversial,
peroxynitrite represents a likely candidate in mediating protein nitration. As
mentioned above, peroxynitrite arises from the recombination of superoxide and
nitric oxide. Mitochondria are a key site of superoxide generation, and it has been
shown that the rate of O
2
-.
production increases with age (Sohal, Ku et al. 1994).
These organelles are also an important site for the production of reactive nitrogen
species, due to the putative, albeit very controversial (Csordas, Pankotai et al. 2007),
presence of a mitochondrial nitric oxide synthase (NOS) isoenzyme (Elfering,
14
Sarkela et al. 2002). Therefore, mitochondria might be a relevant site for nitrating
compounds generation in vivo, such as peroxynitrite.
2. Determine the functional consequences of protein nitration
An oxidative/nitrative modification is relevant only if it significantly affects
the function of the target protein. Three complementary approaches are necessary to
relate the presence of a nitro group on aromatic residue to an effect on activity. First,
the modified residue(s) needs to be identified by mass spectrometric analysis in order
to (i) ascertain their presence within the protein, and to (ii) determine its localization
in the three dimensional structure of the protein. Second, the activity of the identified
nitrated enzyme(s) as a function of age has to be measured in the intact cellular
fraction of interest. Third, additional in vitro experiments have been carried out to
gain further insight in the relationship between the modification and its biological
effect: the impact of in vitro nitrating agents on catalytic activity, as well as the
propensity of nitrated proteins to proteolysis was tested.
3. Assess the impact of calorie restriction (CR) on the content of
nitration and the catalytic activity of the proteins of interest
Since CR is believed to mediate extension of life span through, in part, a
reduction in oxidative stress, it may attenuate any age-related changes in the nitration
content of protein and catalytic activity. The impact of CR was assessed by Western
blot analysis and enzymatic assays, as described above. For this particular goal, the
comparison between two strains of mice which differ in their response to the CR
regimen, namely C57BL/6 and DBA/2, should help distinguish between effects
15
induced by calorie restriction that play a role or not in the prolongation of longevity.
Indeed, the C57BL/6 strain life span is prolonged by CR regimen, whereas DBA’s
life span is not. It should be noted that the impact of such a CR regimen on
individual nitrated proteins has not been determined before.
16
Chapter Two: Materials and Methods
I. Reagents and Materials
Unless stated otherwise, all reagents were purchased from Sigma-Aldrich Co
(St. Louis, MO). Suppliers of other materials were: Acrylamide/Bis solution 40% T,
3.3% C and broad range of prestained molecular weight markers (myosin, β-
galactosidase, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor,
lysozyme and aprotinin, with molecular masses of 209, 124, 80, 49.1, 34.8, 28.9,
20.6 and 7.1 kDa, respectively), Bio-Rad (Hercules, CA); Polyvinylidene fluoride
(PVDF) transfer membranes (0.45 µm), Pall Corporation (Pensacola, FL); BioLight
films, Kodak (Eastman Kodak, Rochester, NY); Mouse monoclonal anti-
nitrotyrosine, clone 1A6, and peroxynitrite, Upstate (Lake Placid, NY); Anti-
lipoamide dehydrogenase and horseradish peroxidase conjugated goat anti-mouse
IgG F(ab’)
2
, Rockland (Gilbertsville, PA); Horseradish peroxidase conjugated goat
anti-rabbit and anti-mouse IgG (H+L), Pierce (Rockford, IL); ECL Plus, Amersham
Biosciences (UK); Percoll and chromatofocussing reagents, Amersham Corp.
(Arlington Heights, IL); sequencing grade modified trypsin, Promega (Madison,
WI); pronase from Streptomyces griseus and complete protease inhibitor cocktail,
Boehringer Mannheim (Indianapolis, IN); 5-nitrotryptophan, WAKO Pure Chemical
Industries (Richmond, VA). Rabbit polyclonal anti-SCOT and anti-enoyl-Coenzyme
A hydratase antibodies were produced against their most hydrophilic peptides,
KGPRFEKRIERLTTRDSP and REGMSAFVEKRKANFKDH, respectively,
17
conjugated to keyhole limpet hemocyanin, KLH, BioSource International
(Camarillo, CA). The IgG fraction from rabbit immune serum was purified by
ammonium sulfate precipitation and ion-exchange chromatography (Dunbar and
Schwoebel 1990).
II. Animals and tissues
Male Fischer 344 rats, C57BL/6 and DBA/2 mice were obtained from the
National Institute on Aging-National Institute of Health and housed at the USC
animal facility. For purification of SCOT, 200 rat kidneys and 100 hearts were
shipped overnight in ice-cold antioxidant buffer. Kidneys from dog, pig and cow
were obtained from Pel-Freez Biologicals (Rogers, AR). Kidneys from rabbit, and
monoamine oxidase A/B double knock out mice were kindly provided by Prof. Sarah
F. Hamm-Alvarez and Prof. Jean C. Shih, respectively, from University of Southern
California.
III. Methods
Isolation of mitochondria
Rats were killed by decapitation, and mice by cervical dislocation. Organs,
such as heart, kidney, brain, hind limb skeletal muscle, testis, lung, spleen and liver
were pooled from two animals, and placed in ice-cold antioxidant buffer, containing
150 mM potassium phosphate, 2 mM EDTA, and 0.1 mM butylated hydroxytoluene,
pH 7.4. Isolation buffers for each tissue consisted of: heart-0.3 M sucrose, 0.03 M
nicotinamide, 0.02 M EDTA, pH 7.4; kidney-220 mM D-mannitol, 70 mM sucrose,
18
2 mM HEPES, 10 mM EGTA, 0.5 mg/ml bovine serum albumin, pH 7.4; brain,
testis, lung and spleen-0.32 M sucrose, 1 mM EDTA, 10 mM Tris, pH 7.4; hind limb
skeletal muscle- buffer 1, 0.12 M KCl, 2 mM MgCl
2
, 1 mM EGTA, 0.5 mg/ml BSA
and buffer 2, 0.3 M sucrose, 0.1 mM EGTA, 2 mM HEPES, pH 7.4. Mitochondria
from each of the aforementioned tissues were prepared by differential centrifugation,
i.e. a low-speed followed by a high-speed centrifugation, as follows: heart- 700 g for
10 min and 10,000 g for 5 min; kidney-600 g for 10 min and 8500 g for 10 min;
liver- 1000 g for 10 min and 17,500 g for 10 min; brain, testis, lung and spleen- 1300
g for 3 min and 21,200 g for 10 min; skeletal muscle- 600 g for 12 min and 17,000 g
for 12 min in buffer 1, and 1200 g for 12 min and 12,000 g for 12 min in buffer 2.
Isolation of mitochondria from various tissues was completed within 1 to 2 h after
tissue dissection, except from the brain, which required a longer isolation time (up to
3 h) due to Percoll gradient centrifugation (Sims 1993). Mitochondrial pellets were
resuspended in appropriate volumes of the respective tissue homogenization buffers,
to achieve a concentration of 5-10 mg/ml protein, and stored in small aliquots at –
80
o
C.
Preparation of soluble proteins from mitochondria
Mitochondrial preparations were placed on ice, sonicated for 30 sec (duty
cycle 30, output control 5), put on ice for 2 min, and sonicated again using similar
conditions. Such disrupted mitochondria were then centrifuged at 100,000 g for 1 h
at 4ºC, to separate the soluble and the membrane proteins. Supernatants were
collected, and, to extract more soluble proteins, the pellets were resuspended in a
buffer consisting of 50 mM imidazole (pH 7), 50 mM sodium chloride and 5 mM 6-
19
aminohexanoic acid, followed by sonication and ultracentrifugation as described
above. Supernatants from both ultracentrifugations were combined, and the protein
concentration was measured immediately. Samples were stored at -80ºC until use.
Preparation of nitrated and non nitrated bovine serum albumin
Fatty acid-free bovine serum albumin (BSA) was nitrated with peroxynitrite,
and used as a positive control for the Western blot analysis. Due to instability of
BSA in the presence of degraded peroxynitrite and the occurrence of crosslinking
reactions, the nitrated protein was further purified by high performance gel
permeation chromatography. Briefly, a 25 mg/ml solution of BSA was incubated
with 1 mM peroxynitrite for 15 min on ice in 25 mM sodium phosphate buffer, pH
7.4, containing 75 mM NaCl. Nitrated BSA (50 µl, 1.25 mg protein) was injected
onto the gel filtration column, and eluted with buffer (25 mM sodium phosphate, pH
7.4, 75 mM NaCl) at a flow rate of 0.5 ml/min. The bulk of a peak with a retention
time of 17 min, containing the nitrated BSA monomer, was collected, stored in small
aliquots at –80
o
C, and used in SDS-PAGE and Western blot analysis. A negative
control of BSA (non-nitrated) was prepared by a similar procedure in the absence of
the peroxynitrite. The molar content of 3NT in the nitrated BSA control was
determined to be 100 ± 12 mmol/mol BSA (molecular weight 67 kDa) after analysis
of amino acid composition, as described below.
Western Blot analysis
One dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed according to the method developed by Schägger and Jagow (Schagger and
von Jagow 1987). Proteins (5-20 µg) were denatured by boiling for 5 min in 4X
20
sample buffer (200 mM Tris-HCl, pH 6.8, 40% glycerol, 0.07 % (w/v) bromphenol
Blue, 8% (w/v) SDS and 400 mM dithiothreitol) and subjected to electrophoresis on
1.0-mm gels, consisting of 4% stacking and 10% separating gels, using a Bio-Rad
Miniprotean III gel apparatus. The electrophoretic separation was carried out at a
constant current of 30 V during 40 min for the stacking, and 100 V during 90 min for
separating gels. For each experiment, one gel was stained with Coomassie Blue, and
the other one was electrotransferred to a PVDF membrane to perform
immunodetection steps. Coomassie staining was carried out as follows: gels were
agitated for 15 min in fixing buffer, consisting of 50% methanol, 10% acetic acid
and 40% water, stained for 45 min with Coomassie staining solution containing 50%
methanol, 10% acetic acid, 40% water and 0.1% (w/v) Coomassie brilliant blue
R250, fixed twice for 15 min and destained overnight in a buffer containing 17.5 %
methanol, 7.5 % acetic acid and 75 % water. Protein electrotransfer onto PVDF
membranes was performed at 4
o
C in a buffer containing 16 mM Tris base, 120 mM
glycine and 10% (v/v) methanol at a constant current of 120 V for 80 min. For
immunodetection, PVDF membranes were incubated with blocking solution
containing 5% dry milk and 0.1% Tween 20 in Tris Buffered Saline (TBS, 10 mM
Tris/HCl buffer, pH 7.5, and 150 mM NaCl) for 45 min at 37
o
C, or overnight at 4
o
C.
Membranes were then quickly rinsed in distilled water and incubated with the
primary antibody. Specific conditions used for each primary antibody were: anti-3-
nitrotyrosine-1:2,000 dilution, and incubation at 37
o
C for 1 h, or overnight at 4
o
C;
anti-SCOT serum-1:1,000 dilution, and incubation at 37
o
C for 1 h; purified IgG from
anti-enoyl-CoA hydratase serum-1:1,000,000 dilution, and incubation for 1 h at
21
37ºC; anti-lipoamide dehydrogenase-1:25,000 dilution and incubation for 1 h at
37ºC. After quickly rinsing in distilled water, and washed four-five times, 5 min
each, with Tween-TBS, PVDF membranes were incubated with the appropriate
secondary antibody. Conditions for each secondary antibody were: HRP-conjugated
goat anti-mouse IgG (H+L)- dilution of 1:20,000 and incubation for 1 h at 37ºC;
HRP-conjugated goat anti-rabbit-dilution of 1:100,000 or 200,000 and incubation for
1 h at 37ºC; HRP-conjugated goat anti-mouse IgG F(ab’)
2
- 1:20,000 dilution and
incubation for 2 h at room temperature. After vigorous washing with Tween-TBS for
5 min 4-5 times, the membranes were developed using the chemiluminescence
detection kit, ECL-Plus. Images of the immunoblots were digitized by using a
flatbed scanner (Epson 2450). Analysis of densitometric data was performed using
the software LabWorks 4.0.0.8. Contents of SCOT protein and nitration in different
samples of mitochondrial matrix were calculated by comparisons with standard
curves fitting increasing amounts of purified SCOT (10 to 40 ng) or nitrated BSA
(30 to 70 ng) and mitochondrial matrix extracts from 4-month-old rat (2.5 to 15 µg).
The relation between amounts of protein and band densities was found to be
exponential, rather than linear, within a limited dynamic range of amounts of protein
(indicated above) and was also dependent on the duration of film exposure during the
chemiluminescence reaction.
Purification of the 58 kDa nitrated protein
Mitochondrial soluble proteins (~100 mg) were dialyzed against 25 mM
imidazol buffer containing 0.2 mM phenylmethanesulphonylfluoride (PMSF), pH
7.85, for 2 h, and applied onto a chromatofocusing column (6×100 mm), equilibrated
22
with the same buffer, and eluted with 100 ml of Polybuffer 74 (dilution 1:10), pH
3.9. The flow rate was 6 ml/h and fractions of 2 ml each were collected.
Measurements of pH were made in every fifth fraction. Each consecutive fraction
was subjected to one dimensional SDS-PAGE electrophoresis. Gels were
electrotransferred onto a PVDF membrane for immunodetection with the anti-3NT
antibody. The fractions containing the immunopositive band were then pooled, and
concentrated in a volume of 200 µl, using Centricon 30 concentrators. Gel filtration,
i.e. separation of proteins according to their size, was performed at room temperature
using a Shimadzu Class VP HPLC system and BioSep-SEC-S 3000 gel permeation
column (5μm, 7.5×300 mm) obtained from Phenomenex (Torrance, CA). The
column was equilibrated with 25 mM Tris buffer, pH 7.4, containing 75 mM NaCl,
at a flow rate of 0.5 ml/min. Fifty µl of concentrated sample containing the
immunopositive protein band were injected onto the column, and the absorbance
(200-600 nm) was monitored with a diode-array UV detector. Fractions (0.5 ml) of
the eluate were collected from the gel filtration column in between 12 and 20 min.
Three consecutive injections were made. The purity of the immunopositive protein
was assessed by SDS-PAGE. This purification procedure yielded ~200 µg of
electrophoretically pure (>85%) 58 kDa nitrated protein.
N-terminal sequencing
Fractions from heart and kidney mitochondria containing the purified 58 kDa
nitrated protein and the co-purifying immunopositive 29 kDa protein were separated
by SDS-PAGE and electroblotted onto PVDF membranes. The membranes were
then stained with Coomassie Blue, and the bands of interest were subjected to N-
23
terminal Edman degradation at the Microchemical Core Facility Laboratory of the
University of Southern California. Sequences were ascertained by using the BLAST
network service at the National Center for Biotechnology Information (NCBI).
Numbering of amino acid residues in the text corresponds to the amino acid
sequence of mature rat SCOT (NCBI accession number NP_001012221), or of
mature rat ECH (NP_511178).
SCOT autolytic fragmentation
The purified SCOT protein was incubated in the absence or presence of 1
mM acetoacetyl-Coenzyme A for 5 min at room temperature, and then for 1 h at
70ºC, as described by Howard et al., (1986). Buffer consisted of 50 mM sodium
phosphate, pH 7.4. The protein mixtures were then separated by one dimensional
SDS-PAGE electrophoresis, following which the gels were either stained with
Coomassie Blue or electrotransferred to PVDF membranes for further analysis.
HPLC amino acid analysis
The PVDF membranes prepared after fragmentation of the purified SCOT
protein were stained with Coomassie Blue. The bands (10-100 µg) corresponding to
the full length SCOT protein (58 kDa), the amino (37 kDa) and carboxy terminal (21
kDa) fragments were cut and placed in 0.5 ml plastic tubes containing 100 µl of 0.1
M sodium acetate buffer, pH 7.4, mixed with 5% (w/w) pronase to hydrolyze the
proteins into amino acids. The hydrolysis was carried out overnight at 50
o
C, and was
stopped by the addition of 100 µl 10% (w/v) meta-phosphoric acid. The samples
were then centrifuged at 18,000 g for 20 min, and the supernatants transferred to
autosample micro vials for injection on HPLC column. Amino acids (tyrosine, 3-
24
nitrotyrosine, tryptophan, 4-nitrotryptophan, 5-nitrotryptophan, 5-
hydroxytryptophan, and kynurenine) were separated by HPLC, fitted with a
Shimadzu Class VP solvent delivery system using a reverse phase C18 Gemini
column (4.6×150 mm, 5 µm, Phenomenex, Torrance, CA). The mobile phase for
isocratic elution consisted of 25 mM monobasic sodium phosphate, 12.5% methanol,
pH 2.7, adjusted with 85% phosphoric acid. The flow rate was 1 ml/min. Under these
conditions, the separation was completed in 30 min, 5-nitrotryptophan being the last
eluted peak, with a retention time of approximately 27 min. For the analysis of
tyrosine and 3NT, the methanol was omitted from the solvent. The elution of 3NT
occurred towards the end, with a retention time of approximately 25 min. The amino
acids used as calibration standard were prepared in 5% meta-phosphoric acid. Amino
acids were detected with a model 5600 CoulArray electrochemical detector (ESA,
Chelmsford, MA), equipped with a four-channel analytical cell, using potentials of
+600, +700, +800 and +900 mV. With the signal to noise ratio of 4:1, the lower limit
for electrochemical detection was 300 fmol for 3-nitrotyrosine, 4- and 5-
nitrotryptophan, and 200 fmol for tyrosine, tryptophan, 5-hydroxytryptophan and
kynurenine. For quantification, each sample was injected twice, and the peak areas
were averaged.
Mass spectrometric analysis
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)
MALDI-TOF mass spectra were acquired on AXIMA CFR (Shimadzu,
Columbia, MD) operated in the positive ion linear mode, using α-cyano-4-
hydroxycinnamic acid (10 mg/ml in 70% acetonitrile) as a matrix compound. Spectra
25
represented the average of at least 100 laser shots. To optimize the fragmentation
spectra, various settings of laser power (60, 90, 120 and 150) were examined.
External mass calibration was achieved using methionine, tryptophan, 5-
hydroxytryptophan and 5-nitrotryptophan, whose protonated ions [M+H]
+
masses are
150.1, 205.1, 221.09 and 250.08, respectively. One mg of dried sample of amino
acids was dissolved in 100 µl of 0.1% trifluoroacetic acid (TFA). One µl of the
sample solution was then mixed with 1µl of matrix solution, and the resulting
mixture deposited on the stainless-steel sample holder and let dry for several minutes
on air. The HPLC fraction containing the putative peak corresponding to
nitrohydroxytryptophan was collected between 20 and 22 min during HPLC
separation. The eluate was then concentrated using SpeedVac (Thermo Savant,
Boston, MA) and dissolved in 10 µl of 0.1% TFA. Three independent MALDI
measurements were made for each sample to evaluate the reproducibility of the ion
peaks.
Electrospray Ionization tandem mass spectrometry (ESI-MS/MS)
In-solution and in-gel tryptic digestion of purified full length, carboxy-
terminal and amino-terminal fragments of SCOT, analysis of tryptic peptide
sequence tags by tandem mass spectrometry, and protein identification were
performed as described in detail previously (Yarian, Rebrin et al. 2005). Briefly,
proteins from solution and/or from Coomassie stained gel pieces were reductively
alkylated and digested with sequencing grade trypsin overnight at 37
o
C. Tryptic
digest products were extracted, dried and resuspended in 10 µl of 60% (w/v) acetic
acid. Chromatographic separation of the tryptic peptides was achieved using a
26
ThermoFinnigan Surveyor MS-pump in conjunction with a BioBasic-18 reverse
phase capillary column (100×0.18 mm, ThermoFinnigan). Mass analysis was
performed using a ThermoFinnigan LCQ Deca XP Plus ion trap mass spectrometer
equipped with a nanospray ion source employing a 4.5 cm needle, using data-
dependent acquisition mode.
Protein identification was carried out with the MS/MS search software
Mascot (Matrix Science), with confirmatory or complementary analyses by
TurboSequest (Bioworks Browser 3.2, build 41, from ThermoFinnigan and Sonar
MS/MS from Genomics Solutions). Alkylated modification (carbamidomethyl) was
designated as fixed; in contrast, oxidation of methionine and tryptophan as well as
nitration of tyrosine and tryptophan were considered as variable modifications in the
TurboSequest search. Rat, mouse, bovine and pig protein databases, complemented
with the non-redundant protein database were downloaded from the NCBI server.
The theoretical m/z values for the peptides and their fragmentation ions were
assessed using “MS/MS Fragment Ion Calculator” from The Institute for Systems
Biology at http://db.systemsbiology.net:8080/proteomicsToolkit/. Acceptable cross-
correlation scores (X
corr
) for the positive identification of a modification were set at
>1 and > 2 for a singly and doubly charged ion, respectively. In addition, MS/MS
spectra of particular interest were inspected manually.
Synthesis of nitrated 5-hydroxy-tryptophan
5-Hydroxynitrotryptophan was synthesized by the reaction between 5-
hydroxytryptophan and tetranitromethane. The procedure was carried out on ice in
the dark (amber glass vial). 10 mg of solid 5-hydroxytryptophan were dissolved in
27
50 µl of tetranitromethane, and the solution was overlaid with 450 µl of 70%
acetonitrile, and stirred gently. It should be noted that, due to the insolubility of
tetranitromethane (bottom) in acetonitrile (top), the solution in the vial separated into
two phases. In due course, the dark red tetranitromethane phase migrates into the
upper acetonitrile-containing phase. Progress of the reaction was monitored by
examining 50 µl aliquot (100-fold dilution of upper phase of the reaction mixture in
5% meta-phosphoric acid) by HPLC, as described above. The highest yield (~70%)
of 5-hydroxynitrotryptophan was obtained after a prolonged period of reaction (up to
6 h). Further incubation resulted in the appearance of additional minor peaks with
retention times of more than 30 min, probably due to the occurrence of multiple
nitration reactions on the indole ring of tryptophan. In contrast, if the reaction was
carried out at pH 8.0, using 50 mM phosphate buffer, the formation of 5-
hydroxynitrotryptophan was accompanied by relatively high amounts of multiple
nitration by-products, and a rapid decay of 5-hydroxynitrotryptophan was detected
within several minutes in phosphate buffer in the pH range of 7.0 to 8.0. Therefore,
to obtain high yield and purity as well as prolonged stability, the buffer was
excluded. No significant decomposition of 5-hydroxynitrotryptophan was observed
after storage at 4
o
C for up to 5 days if the reaction was acidified with 0.1%
trifluoroacetic and 5% meta-phosphoric acid.
Succinyl Coenzyme A transferase (SCOT) catalytic activity assay
SCOT enzyme activity was performed by following the appearance of the
complex magnesium-acetoacetyl-CoA at 313 nm at 30ºC, according to the procedure
developed by Williamson (Williamson, Bates et al. 1971). Briefly, the final
28
concentration of reactants in the assay mixture (500 µl) was: 100 mM Tris-HCl pH
8.5, 10 mM magnesium chloride, 4 mM iodoacetamide, 0.2 mM succinyl Coenzyme
A, and 50 mM acetoacetate. Various protein concentrations were tested to obtain
linear reaction for 2 min; 40 µg or 5 µg of soluble mitochondrial proteins from
kidney or heart, respectively, were determined to be optimal amounts to reach
linearity of the reaction. After initiation of the reaction by acetoacetate and succinyl-
CoA, the change in absorbance was followed at 313 nm for 2 min using a Beckman
DU-640 spectrophotometer. Enzyme assays were carried out in duplicate and the
average absorbance was used for calculations. Specific activity of SCOT was
expressed as mol acetoacetyl-CoA formed/min/mol SCOT enzyme. The molar
content of SCOT in samples of the soluble fractions of rat heart and kidney
mitochondria was estimated in Western blots using the anti-SCOT antibody, and
purified SCOT as the standard, and expressed as pmol SCOT per mg mitochondrial
soluble proteins.
Enoyl Coenzyme A hydratase (ECH) catalytic activity assay
ECH activity was determined by following the decrease in absorbance of
crotonyl-CoA at 280 nm at 25ºC, according to the method developed by Steinman et
al. (1975). A standard assay mixture contained 16 mM dibasic potassium phosphate
pH 7.4, 4 mM EDTA, 0.8 mg/ml BSA, 0.1 mg/ml ADP and 5 µg of soluble
mitochondrial proteins from kidney or liver in a total volume of 0.5 ml. The reaction
was initiated with 100 µM of crotonyl-CoA, and the decrease in absorbance was
linear for 2 min. Enzyme assays were carried out in duplicate and the average
absorbance was used for calculations.
29
IV. Statistical analysis of data
Statistical analysis of data was performed using analysis of variance
(ANOVA), including Bonferroni correction. The results are means ± SD. P values of
<0.05 were considered statistically significant.
30
Chapter Three: Results
I. Identification of proteins targets of nitration among the mitochondrial
soluble proteins in different rat organs
A. Western blot analysis of the soluble mitochondrial proteins using an
anti-3-nitrotyrosine antibody
The main purpose of this preliminary experiment was to localize proteins
with nitrative additions in the mitochondrial soluble fraction from different tissues of
the rat. Accordingly, mitochondria from heart, kidney, brain, skeletal muscle, lung,
spleen, testis and liver were isolated of young animals (5 months-old) by differential
centrifugation as described in Material and Methods. To separate mitochondrial
membrane proteins from soluble proteins, present in the intermembrane space and
the matrix, mitochondria were sonicated, and the submitochondrial particles, which
are composed of membranes, were separated from the soluble proteins by 100,000g
high speed centrifugation. Proteins in the supernatant were then resolved, according
to their size, by one dimensional SDS-PAGE electrophoresis, and the gels were
either stained with Coomassie blue, or transferred to a membrane for
immunoblotting with an anti-3-nitrotyrosine antibody (anti-3NT).
The Coomassie stained gel allowed the visualization of the electrophoretic
profile of the soluble proteins, which was found to vary in different organs (Fig. 1A).
Western blot analysis using anti-3NT antibody, showed a single discernable
immunopositive band in the heart, kidney, brain cortex, hind limb skeletal muscle,
lung, spleen, but not in testis or liver. By comparison with proteins of known
31
molecular weight, the band was inferred to be ~58 kDa. The intensity of the
immunopositive band was highest in the heart and kidney, followed by skeletal
muscle, brain, lung and spleen (Fig. 1B). Thus, an ubiquitous immunopositive
protein band was localized among mitochondrial soluble proteins in various tissues.

Figure 1: Localization of nitrated protein band among the mitochondrial
soluble proteins of various organs. Proteins in the soluble mitochondrial
subfraction from various organs of young rats (5 months-old) were separated by one-
dimensional SDS-PAGE. Gels were either stained with Coomassie blue (panel A) or
transferred to PVDF membranes for Western blot analysis with an anti-3-
nitrotyrosine antibody (anti-3NT) (panel B). M refers to the protein standards,
myosin (210 kDa), β-galactosidase (120 kDa), bovine serum albumin, (80 kDa),
ovalbumin (49 kDa), carbonic anhydrase (35 kDa), soybean trypsin inhibitor (29
kDa), lysosyme (20 kDa), aprotinin (7 kDa) and 0.5 µg of control non-treated bovine
serum albumin (BSA) (67 kDa). Lane C contains 0.5 µg of peroxynitrite-treated
BSA. Lanes 2-9 contain 10 µg of soluble mitochondrial proteins from heart, kidney,
brain cortex, hind limb skeletal muscle, lung, spleen, testis and liver. The arrow on
the right indicates the position (58 kDa) of the nitrated band.

A
210
120
80
49
35
29
20
7
M C 1 2 3 4 5 6 7 8
Coomassie
B
Anti-3NT
210
120
80
49
35
29
20
7
32
B. Specificity of the reaction with the anti-3NT antibody
To assess the specificity and reliability of the immunoreaction with the anti-
3NT antibody, and the reliability of the experimental procedures, several control
experiments were conducted. Internal negative and positive controls, consisting of
respectively untreated or peroxynitrite-nitrated bovine serum albumin (BSA),
displaying presence or absence of cross reaction with the anti-3NT antibody, were
loaded in gels designated for immunoblotting. The immunoreaction was found not to
occur: (i) in the absence of the anti-3NT antibody, (ii) after pre-incubation of the
anti-3NT antibody with 1.5 mM 3-nitrotyrosine, and (iii) after treatment of the
membrane with dithionite, an agent which reduces 3-nitrotyrosine to amino-tyrosine.
In contrast, the pre-incubation of the primary antibody with 5-nitrotryptophan, 5-
hydroxytryptophan, or kynurenine did not prevent the immunoreaction (Fig. 2A). In
two additional control experiments, the potential capability of substances used during
the experimentations, to reduce the nitro (-NO
2
) groups present in proteins to amino
(-NH
2
) derivatives, was tested. The concentration of dithiothreitol (DTT), a strong
reducing agent, in the sample buffer was decreased from 100 mM to 10, 1 and 0 mM.
Butylated hydroxy-toluene (BHT) was omitted from the antioxidant buffer used
during mitochondria isolation. Subsequent Western Blot analysis with an anti-3NT
showed that (ii) the intensity of the immunopositive band remained unchanged in
comparison to the control, and (ii) no additional immunopositive bands were
detected (Fig. 2B & C). Altogether, these controls confirmed that, among the soluble
mitochondrial proteins, the 58 kDa protein band was a specific target of nitration.

33
Figure 2: Controls for establishing the specificity of the immunoreaction with
the anti-3-nitrotyrosine antibody and for validation of the experimental
procedures. Heart mitochondrial soluble proteins (each lane contained 10 μg
proteins) were separated by SDS-PAGE electrophoresis, and various immunoblot
analyses were performed to ascertain both specificity and validity of
experimentations. Panel A: the immunoreaction was abolished in the absence of the
anti-3NT antibody, after pre-incubation of the primary antibody with 1.5 mM free 3-
nitrotyrosine (3NO
2
-Tyr), and after reduction of the PVDF membrane with 100 mM
dithionite. Pre-incubation of the primary antibody with 1.5 mM free 5-
nitrotryptophan (5NO
2
-Trp), 5-hydroxytryptophan (5-OH-Trp) or kynurenine, did
not prevent the immunoreaction. Panel B: Compared to the usual 100 mM (lane 1),
lower concentrations of dithiothreitol in the sample buffer, 10, 1 and 0 mM (lanes 2-
4), did not affect the pattern of the immunoblot. Panel C: The immunoblot was
identical in the presence (lane 1) or absence (lane 2) of butylated hydroxy-toluene in
the antioxidant buffer. M and C refer to the protein standards mixed with control
BSA and peroxynitrite-treated BSA, respectively, as described in the legend of Fig.
1.

34

Figure 2

Anti-3NT + - + + + + +
1.5 mM 3NO
2
-Tyr - - + - - - -
Dithionite - - - + - - -
1.5 mM 5NO
2
-Trp - - - - + - -
1.5 mM OH-Trp - - - - - + -
1.5 mM Kynurenine - - - - - - +
M C 1 M C 1 M C 1 M C 1
M C 1 M C 1 M C 1
A
B
M C 1 2 3 4
210
120
80
49
35
29
20
7
C
M C 1 2
210
120
80
49
35
29
20
7

35
C. Purification and identification of the 58 kDa immunopositive protein
1. Purification procedures
To obtain sufficient amounts (around 10 µg) of protein, for the purpose of
identification, the 58 kDa nitrated protein was purified. Because it displayed the
most intense immunoreaction with the anti-3NT antibody in heart and kidney, these
two tissues were selected for protein purification.
Mitochondria were isolated from 100 hearts and 200 kidneys of rats, and
soluble proteins were prepared by sonication, followed by a 100,000g centrifugation.
Proteins were fractionated according to their isoelectric point (pI) on a
chromatofocusing column in the pH range from 4 to 8 (Fig. 3A). Fractions were
resolved by one dimensional SDS-PAGE electrophoresis, and the gels were either
stained with Coomassie, or electrotransferred to a membrane for Western blot
analysis with an anti-3NT antibody. Several protein bands can be visualized on the
Coomassie stained gels, each fraction containing proteins of close pI. In both heart
and kidney, the 58 kDa protein exhibited an identical isoelectric point, i.e. 6.9-7.1.
The immunoblots showed that fractions #12-31 and 11-25 of heart and kidney,
respectively, contained the immunopositive protein. Furthermore, an additional faint
immunopositive ~ 29 kDa band, present in fractions #14-20, was observed in the
kidney.
Eluates containing the 58 kDa immunopositive protein were pooled,
concentrated using Centricon 30 concentrators, and further fractionated by size,
employing HPLC gel filtration. The proteins from resulting fractions were

36
electrophoresed, and the gels were either visualized with Coomassie, or transferred
to a membrane for Western Blot analysis with anti-3NT antibody. Immunoblots
indicated that the 58 kDa protein band was present in the fractions #17-19 of heart
and #16.5-22 of kidney (Fig. 3C and 4B). The enriched protein was also clearly
visible in the Coomassie gels of both tissues. It was however quite obvious that the
degree of purity achieved in heart was higher than that in the kidney. In the latter,
several other co-purifying proteins were observed on the Coomassie gel,
furthermore, the immunointensity of the 29 kDa protein, present in fractions #17-18,
was increased by comparison to the faint band observed during the previous
purification stage (Fig. 4B). Nevertheless, the 29 kDa protein could not be rationally
assigned to a particular band on the Coomassie stained gel.

37
Figure 3: Purification of the immunopositive protein in rat heart mitochondria
by chromatofocusing and gel filtration. Panel A shows a chromatogram obtained
from the fractionation of soluble heart mitochondrial proteins according to their
isoelectric point (pI) in the range of pH 4-8. The fractions collected were submitted
to SDS-PAGE and gels were either stained with Coomassie blue or transferred to
PVDF for Western blot analysis with anti-3NT antibody (panel B).
Chromatofocusing fractions containing the nitrated protein (12-31) were pooled and
further separated by HPLC gel filtration (panel C). For panel B and C, M refers to a
mixture of prestained standards and control BSA, as described in legend of Fig. 1.
Lane C contains the positive control, i.e. 0.5 µg of peroxynitrite-treated BSA. S
represents the initial sample loaded onto the HPLC column, i.e. concentrated soluble
proteins from heart mitochondria and from the pooled chromatofocusing fractions
containing the immunopositive protein, for panel B and C, respectively. Lanes 5-61
and lanes 14-22 correspond to the chromatofocusing and gel filtration fraction
numbers, respectively. The arrows on the right indicate the position of the 58 kDa
immunopositive protein.

38

Figure 3
0 10 20 30 40 50 60
Fraction number
0.00
0.25
0.50
Absobance at 280 nm
pH
8.0
4.0
A
B
M C S 5 7 9 11 13 15 17 19 21 23 25 29 31 33 35 37 39 41 43 45 47 27 49 51 53 55 57 59 61
Coomassie
210
120
80
49
35
29
20
7
210
120
80
49
35
29
20
7
Anti-3NT
C
Coomassie
S C M 14 15 16 17 18 19 20 21 22
210
120
80
49
35
29
20
7 Anti-3NT
S C M 14 15 16 17 18 19 20 21 22

39

Figure 4: Purification of the immunopositive protein in rat kidney mitochondria
by chromatofocusing and gel filtration. The same purification procedure as the one
described for the heart was used for kidney mitochondria. Panel A shows the
Coomassie gel and the immunodetection of nitrated proteins in the chromatofocusing
fractions. The arrows indicate the positions of the 58 kDa immunopositive protein
and another nitrated protein of 29 kDa which co-elutes with the first nitrated protein.
The HPLC fractions 10-28 were pooled and further separated by gel filtration (panel
B). The gel filtration fractions were analyzed by SDS-PAGE followed by
immunodetection with an anti-3NT antibody. The second 29 kDa nitrated protein
was further enriched in the fractions 17-18. M refers to the mixture of prestained
standards and control BSA, and C to the positive control, i.e. nitrated BSA, as
described in the legend of Fig. 1.
A
Coomassie
M C 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 S 53 55
210
120
80
49
35
29
20
7
Anti-3NT
210
120
80
49
35
29
20
7
58
29
B
Coomassie
M C S 15 16 16.5 17 17.5 18 18.5 19 20 21 22
210
120
80
49
35
29
20
7
Anti-3NT
M C S 15 16 16.5 17 17.5 18 18.5 19 20 21 22
58
29

40
2. Identification of the two immunopositive proteins
The two purified proteins were microsequenced by Edman degradation, and
the two N-terminal sequences thus obtained, assigned by NCBI-BLAST search
system to succinyl-CoA: 3-ketoacid-CoA transferase (SCOT, EC 2.8.3.5) for the 58
kDa protein, and short chain enoyl-Coenzyme A hydratase (ECH, EC 4.2.1.17) for
the 29 kDa protein (Fig. 5A).
The identity of these proteins was further confirmed by Western blot analysis
of mitochondrial soluble proteins from various tissues of young rats using two
polyclonal antibodies directed against the most hydrophilic regions of the two
proteins (Fig. 5B). The intensity of the two protein bands varied in different organs.
The rank order was: heart> kidney> skeletal muscle> lung> brain> spleen for SCOT,
and heart> brain> skeletal muscle> kidney> liver> lung and spleen for ECH.
Immunoreaction was not observed (i) in testis and liver or (ii) in testis with the anti-
SCOT and anti-ECH polyclonal antibodies, respectively.
Densitometric analysis was carried out to determine whether SCOT was
nitrated to the same extent in organs where it is expressed. The ratio of OD
nitrated
SCOT
/OD
SCOT protein
averaged 0.74±0.26, thereby indicating that SCOT nitration
content relative to the total amount of SCOT protein, was quite similar in various rat
organs.
ECH was nitrated in kidney, but did not appear to be nitrated in the heart,
where it is more highly expressed. Purification of ECH was attempted in liver, where
ECH also appeared to be nitrated (results not shown). However, a reasonable degree

41
of purity could not be achieved, and further enrichment attempts in other organs
were not pursued.

Figure 5: N-terminal sequencing of the purified immunopositive proteins and
their expression in various rat tissues. Panel A shows the peptides generated after
Edman degradation. Using Blast search, the 58 kDa in heart and kidney was
identified as succinyl-CoA:3-oxo-acid CoA-transferase (SCOT). The 29 kDa nitrated
protein in the kidney was identified as short chain enoyl-CoA hydratase (ECH).
Polyclonal antibodies against the most hydrophilic peptides were made and western
blot analysis using either the anti-SCOT or the anti-ECH serum confirmed the
identity of the two mitochondrial proteins (Panel B). The expression of these two
enzymes in various tissues showed that SCOT is mainly expressed in heart and
kidney, whereas ECH is abundant in heart, brain, skeletal muscle and liver. Lanes 1-
8 contains 10 µg soluble mitochondrial proteins isolated from heart, kidney, brain
cortex, hind limb skeletal muscle, lung, spleen, testis and liver. M refers to the
mixture of prestained standards and control BSA, and C to the positive control, as
described in legend of Fig. 1.
D. Background information on SCOT and ECH
Both nitrated proteins are located in the mitochondrial matrix, and are
involved in energy production. SCOT catalyzes the first step of ketone bodies
degradation, or ketolysis. Ketolysis is a process by which the main ketone body,
58 kDa immunopositive protein: VKFYTDPVKAVEGI
SCOT: VKFYTDPVKAVEGI

29 kDa immunopositive protein: ANFQYITTEKGGNNS
ECH: ANFQYITTEKGGNNS
A
B
M 1 2 C 3 4 5 6 7 8
Anti-SCOT
Anti-ECH
58 kDa
29 kDa

42
acetoacetate, is converted to acetyl-CoA, the principal intermediate substrate, which
is ultimately oxidized for ATP generation. Ketolysis occurs only in the extra hepatic
organs.
Short chain enoyl-CoA hydratase catalyzes the second step of the β-oxidation
of short chain unsaturated fatty acids, converting enoyl-CoAs to hydroxyacyl-CoA
derivatives. Each cycle of fatty acid degradation generates acetyl-CoA, used for
energy production (Fig. 6).

43
Figure 6: Ketolysis and β-oxidation of fatty acid. Succinyl-CoA transferase
(SCOT) and enoyl-coenzyme A hydratase (ECH) are involved in two crucial energy
pathways, namely ketolysis and the oxidation of fatty acids. Both pathways lead to
the production of acetyl-CoA, which is then oxidized in the Krebs cycle, and
reducing equivalents NADH,H
+
and FADH
2
, are generated. Electrons from
NADH,H
+
and FADH
2
enter the electron transport chain, leading eventually to ATP
synthesis.

44

Figure 6

CoA-SH
NAD
NADH, H
+

Acetoacetate Acetoacetyl-CoA Acetyl-CoA
Succinyl-CoA Succinate
SCOT
ATP
Krebs cycle
Reducing
Compounds
Electron
Transport
Chain
H
2
O
Fatty acyl-CoA
FAD
FADH
2

Enoyl-CoA
3-Ketoacyl-CoA
3-Hydroxyacyl-CoA
Fatty acyl-CoA
shortened
by two carbons
ECH
3-Hydroxyacyl-CoA
dehydrogenase
Thiolase
Acyl-CoA
dehydrogenase
Ketolysis
Fatty acid oxidation

45
E. Summary of the findings
Using one-dimensional SDS-PAGE electrophoresis, followed by
immunoblotting with an anti-3NT antibody, SCOT, an enzyme involved in ketolysis,
was identified as a specific target of nitration among the mitochondrial soluble
proteins, isolated from different organs of young rats. SCOT nitration content,
relative to the protein amount present in each tissue, was similar in all organs.
ECH nitration was clearly detectable only after the second stage of
purification, in kidney and liver mitochondria. Despite a high level of expression in
the heart, ECH did not seem to be nitrated in this organ.
Because SCOT was highly abundant in heart and kidney mitochondria,
subsequent studies on the functional impact of nitration were mostly focused on
SCOT, and performed in these two tissues.
II. Identification of the nitrated residue(s) in SCOT
The aims of the following experiments were: (i) to confirm the presence of
the nitrated amino acid(s) within the SCOT protein, (ii) to identify the nature and the
specific site(s) of the modification, and (iii) to localize the nitrated residue(s) within
the three dimensional structure of SCOT.
A. Autolytic fragmentation of SCOT
Known information about the catalytic properties of the SCOT protein was
used to identify the nitrated amino acid(s). Thus, during catalysis, SCOT can

46
undergo a cleavage at its active site by a process referred to as autolytic
fragmentation. The covalent binding of the substrate, succinyl-CoA or acetoacetyl-
CoA, to the active site, glutamate 303 generates a high energy thioester, which can
form an internal oxyproline. The latter is unstable and can trigger SCOT cleavage
(Howard, Zieske et al. 1986) (Fig. 7A). Such SCOT autolysis generates two
fragments, a 37 kDa amino-, and a 21 kDa carboxy-terminal fragment. The mature
rat SCOT contains 11 tyrosine and 3 tryptophan residues (NP_001012221) (Fig. 7B).
This autolytic fragmentation can be amplified under specific conditions, as
described below. Purified SCOT from rat heart was incubated in the absence or
presence of acetoacetyl-CoA for 5 min at room temperature, and then heated at 70ºC
for one h. The different mixtures were subjected to SDS-PAGE electrophoresis.
Coomassie stained gel showed the SCOT full length protein and the two fragments
generated after autolysis. Western blot analysis of the different mixtures, using the
anti-3NT antibody, showed that the 21 kDa carboxy terminal post-cleavage fragment
immunoreacted with the anti-3NT antibody. As expected, immunoblotting with the
anti-SCOT serum showed a positive reaction with the 37kDa amino-terminal
fragment, which contains the hydrophilic peptide recognized by the polyclonal
antibody (Fig. 7C).
Given the presence of a unique tyrosine residue in the carboxy region, it was
originally assumed that the nitrated residue was tyrosine 366.

47
Figure 7: Autolytic fragmentation of SCOT revealed the presence of nitrated
amino acid(s) in the 21 kDa carboxyl terminal fragment of the protein. SCOT
enzymatic catalysis involves the formation of a transient intermediate enzyme-CoA,
between the active site, glutamate 303 and the substrate, succinyl coenzyme A or
acetoacetyl-CoA. Under specific conditions, an unstable oxyproline can form,
rendering SCOT susceptible to a fragmentation that yields N-terminal and C-
terminal fragments (panel A). SCOT full length protein contains 11 tyrosine (Y) and
3 tryptophan (W) residues (panel B). The gray box represents the peptide recognized
by the anti-SCOT polyclonal antibody. The N and C-terminal peptides, of 37 and 21
kDa, respectively, generated after fragmentation are shown below. Panel C
represents the identification of the fragment containing the nitrated residue(s).
Purified SCOT from rat heart was incubated for 5 min at room temperature, and then
heated at 70ºC for one h. Lane contents are as follows: M- protein molecular weight
markers, C- nitrated bovine serum albumin, 1- purified SCOT from rat heart without
acetoacetyl-CoA, 2- purified SCOT from rat heart with acetoacetyl-CoA incubated at
room temperature, 3- purified SCOT treated with acetoacetyl-CoA for 5 min at room
temperature and heated at 70ºC for one hour. Arrows indicate the N-Terminal (NH
2
)
and C-terminal (COOH) fragments.

48

Figure 7
C
B
H
N
H
C C
R
O
N CH C
O
H
N
CH
2
H
N
H
C C
R
O
H
N
H
C C
O
H
N
CH
2
C
H
2
CO
S
CoA
C-Terminal fragment
N-Terminal fragment
and
Oxyproline
C
H
2
C
O
HN CH C
O
H
N
CH
2
C
H
2
C
O
Enzyme-CoA
α-peptide
bond
cleavage
Y Y Y Y Y Y Y Y Y Y W

N-Terminal fragment,
37 kDa

Y W W
C-Terminal fragment,
21 kDa
SCOT full length,
58 kDa

Y Y Y Y Y Y Y W Y Y Y Y W W

Glu 303
A
Anti-3NT
M C 1 2 3
Anti-SCOT
M C 1 2 3
NH
2

COOH
M C 1 2 3
Coomassie

49
B. Amino acid analysis by HPLC/electrochemical detection
To confirm the presence of nitrated residue(s) in the carboxy region of
SCOT, amino acid analysis by HPLC, coupled to electrochemical detection, was
carried out. To generate individual amino acids, prior to HPLC analysis, the purified
full length protein, the carboxy and amino-terminal fragments were digested with
pronase overnight. Tyrosine, nitrotyrosine, tryptophan, 4- and 5-nitrotryptophan
were used as calibration standards. Chromatograms thus obtained showed that 3-
nitrotyrosine, 4-and 5-nitrotryptophan could not be detected in SCOT full length, C-
terminal and N-terminal fragments (Fig. 8A). However, an unknown amino acid,
named here as X, was present in the SCOT full length protein and the carboxy
terminal fragment. X eluted at 21 min, prior to 4-nitro (23 min) and 5-nitro (24 min)
tryptophan, and displayed a characteristic UV absorption peak of nitrated aromatic
amino acids at 360 nm, which is similar to 3-nitrotyrosine and 5-nitrotryptophan
(Fig. 8B). Overall, the HPLC analysis indicated (i) the absence of any detectable
3NT, 4- or 5-nitrotryptophan, and (ii) the presence of an unknown amino acid, X,
located in the carboxy region of SCOT, which appeared to share structural
similarities with nitrated aromatic amino acids.

50
Figure 8: Detection of an unknown amino acid, X, in the SCOT protein. Panel A
shows the HPLC chromatograms obtained after pronase-induced hydrolysis of amino
acids of purified full length SCOT (trace b), C-terminal fragment (trace c), and N-
terminal fragment (trace d). Tyrosine (Y), nitrotyrosine (Y
3N
), tryptophan (W), 4-
and 5-nitrotryptophan (W
4N
, W
5N
) were used as calibration standards (trace a).
Nitrohydroxytryptophan (W
N,5OH
) was synthesized by mixing 5-hydroxytryptophan
(W
5OH
) with tetranitromethane (trace e). Vertical bars indicate intensities, i.e. 1000
nA for the chromatograms outside, and 50 nA inside dotted boxes. No nitrotyrosine
or nitrotryptophan was detectable in the SCOT protein, however, an unknown peak,
named X, with an elution time of 21 min, was observed. The UV spectra of nitrated
aromatic amino acids, 3-nitrotyrosine and 5-nitrotryptophan, and X are shown in
panel B.

51

Figure 8
A
B
0 10 20 30
Y Y
3N
W
W
4N
X
X
a
b
c
d
1000 nA
50 nA
Time (min)
e
W
5OH
W
5N
W
N,5OH
250 300 350 400 450 500
Wavelength (nm)
0
100
Absorbance
X
UV spectra
Y
3N
W
5N
W
N,5OH

52
C. Mass spectrometric characterization of the amino acid X
To obtain additional information on the nature of the amino acid X, matrix-
assisted laser desorption/ionization (MALDI) and electrospray ionsiation (ESI) mass
spectrometric (MS) studies were performed. MALDI and ESI MS allow accurate
determination of the masses of biomolecules. While MALDI is useful for the
analysis of mass of small molecules, such as amino acids, ESI is more frequently
employed for the analysis of proteins.
1. MALDI mass spectrometry
MALDI mass spectrum of the HPLC fraction containing X, displayed a
major protonated ion with a mass to charge (m/z) value of 250.3, and additional
peaks at m/z 212.3, 228.06 and 266.34 (Fig. 9A). Treatment of X with dithionite, an
agent that reduces nitro (-NO
2
) to amino (-NH
2
) groups, resulted in the
disappearance of the two peaks at m/z 250.3 and 266.34 on the MALDI spectrum,
while major peaks at m/z 173.92, 190.07 and 212.23, and a minor peak at m/z 236.38
were present (Fig. 9B). Peaks at m/z 212.3, 228.06 (A) and at 190.07 and 212.23 (B)
are the expected ions corresponding to the matrix compound, α-cyano-4-
hydroxycinnamic acid. The peak at m/z 173.92 (B) corresponds to dithionite. The
protonated ion at m/z 266.34 (A) was tentatively attributed to a modified tryptophan
(m/z 205), with a mass shift of +61 Da. This +61 Da mass addition was accounted
for the simultaneous presence of a nitro (-NO
2
=46 Da) and hydroxy (-OH=17 Da)
groups (46+17 - 2 hydrogens (2 Da), because of the substitution= 61), namely a
nitrohydroxytryptophan. The major peak at m/z 250.3, indicative of a loss of oxygen

53
(-16 Da), was attributed to the photodeoxygenation of the nitro-hydroxytryptophan to
a nitroso-(NO)-hydroxytryptophan derivative. Thus the loss of one, and sometimes
two oxygen (nitrene derivative) from the nitro group is characteristic of nitrobenzyl
groups under MALDI conditions (Nielsen and Pennington 1995). The presence of a
protonated ion at m/z 236.38 on the spectrum obtained after treatment of X with
dithionite matched the theoretical mass of an aminohydroxytryptophan. The
structures of such tryptophan derivatives are depicted in Fig. 10. For the purpose of
comparison, the MALDI spectra of 5-nitrotryptophan and 5-hydroxytryptophan were
acquired. The MALDI spectrum of 5-nitrotryptophan showed the molecular ion at
m/z 250.7, and a less abundant ion at m/z 234.7, corresponding to nitroso (-NO)-
tryptophan, but nitrene tryptophan was not detected (Fig. 9D). It should be noted that
the MALDI fragmentation pattern of nitro-containing compounds reflects the initial
compound concentration used: elevated concentrations result in major peaks for the
protonated molecular ion, as seen on Fig. 8D, while low concentrations yield
predominantly the fragmented ions, as can be observed with X, Fig. 9A. The MALDI
spectrum of 5-hydroxytryptophan shows a protonated molecular ion at m/z 221.6,
and a fragmented peak at m/z 204.6, corresponding to the loss of a hydroxyl group
from the carboxylic acid group (Fig. 9E). The peaks at m/z 212.7 (Fig. 9D) and
212.5 (Fig. 9E) correspond to the matrix.

54
2. In vitro synthesis of nitrohydroxytryptophan and
characterization by HPLC and MALDI analysis
To further ascertain the presence of such modified tryptophan in SCOT, in
vitro synthesis of an identical tryptophan derivative was attempted, by mixing (i) 5-
hydroxytryptophan with tetranitromethane, (ii) 5-hydroxytryptophan with
peroxynitrite and (iii) 5-nitrotryptophan with peroxynitrite. MALDI mass
spectrometric analysis of the resulting mixtures revealed that the reaction between 5-
hydroxytryptophan and tetranitromethane yielded nitrohydroxytryptophan: three
major peaks at m/z 266.35, 250.38, 234.4 and additional peaks at m/z 221.5 and
204.5 (Fig. 9C) were observed. The ions at m/z 266.35, 250.38 and 234.4 correspond
to the expected masses of nitro-hydroxytryptophan and its photodecomposition
products namely nitroso-hydroxytryptophan (loss of one oxygen) and nitrene-
hydroxytryptophan (loss of two oxygen). The ions at m/z 221.5 and 204.5 originated
from hydroxy-tryptophan (Fig. 9C). Such spectrum was similar to the one of X,
except for the intensity of the ions, which likely represents a concentration effect, as
mentioned earlier. HPLC amino acid analysis of the mixture revealed that the elution
time and the UV absorption of synthetic 5-hydroxy-nitrotryptophan were identical to
X (Fig. 8A, trace e, and 8B).
Thus, amino acid X was confirmed to be nitrohydroxytryptophan.

55
Figure 9: MALDI mass spectra of the amino acid X, and various tryptophan
derivatives. To determine the nature of the amino acid X, MALDI mass
spectrometric analysis of the HPLC fraction containing X (A) and X, treated with
dithionite, an agent which reduces nitro groups to amino derivatives (B) was
performed. For comparison, the MALDI mass spectra of nitrohydroxytryptophan,
synthesized from 5-hydroxytryptophan and tetranitromethane (5-hydroxy-Trp +
TNM (C), 5-nitrotryptophan (D), and 5-hydroxytryptophan (E) were obtained.

56

Figure 9

200 220 240 260 280 300
m/z
0
20
40
60
80
100
Relative Intensity (%)
250.3
266.34
228.06
212.3
X A
170 190 210 230 250 270
m/z
236.38
212.23
X + Dithionite
173.92
190.07
B
200 220 240 260 280 300
m/z
266.35
250.38
234.4
221.5
204.5
5-Hydroxy-Trp + TNM
C
200 220 240 260 280 300
m/z
0
20
40
60
80
100
Relative Intensity (%)
250.7
234.7
212.7
5-Nitro-Trp
D
200 220 240 260 280 300
m/z
221.6
204.6
212.5
5-Hydroxy-Trp
E

57

Figure 10: Structure of 6-nitro-5-hydroxy-tryptophan and its derivatives under
MALDI conditions and after reducing treatment. Amino acid X was identified as
nitrohydroxytryptophan. The nitro group is indicated on carbon 6, but other positions
can not be ruled out. Under MALDI conditions, the nitro group can undergo
photodecomposition, leading to the loss of one oxygen (nitroso-hydroxytryptophan)
or sometimes two oxygen (nitrene-hydroxytryptophan). After treatment with
dithionite, the nitro group is converted to an amino group. The corresponding
theoretical mass to charge (m/z) values are indicated.
3. SI-MS/MS analysis
To identify the specific modified tryptophan in the SCOT protein, liquid
chromatography coupled to ESI- tandem mass spectrometric analysis (LC-ESI-MS)
of the SCOT protein was carried out.
Purified full length SCOT, amino and carboxy terminal fragments, in solution
or excised from gels, were digested with trypsin, and the resulting peptides were
separated and analyzed by LC-ESI tandem mass spectrometer. Protein identification
was achieved using the MASCOT program. Eight unique tryptic peptides were
assigned to SCOT, corresponding to 32% sequence coverage of the protein. SCOT

58
tryptic peptides were highly abundant, while the amount of tryptic peptides
originating from mitochondrial proteins co-purifying with SCOT, namely aconitase,
L-3-hydroxyacyl-Coenzyme A dehydrogenase, protein expressed in non-metastatic
cells 2, creatine kinase and hemoglobin alpha 1 chain was limited (Table 1).
The search for posttranslational modifications (PTM) (nitro, +45 Da,
hydroxy, +16 Da, nitrohydroxy, +61 Da) was conducted using the TurboSEQUEST
program, which can match the observed tandem mass spectra of the tryptic peptides
to the predicted tandem MS from proteins carrying the specified PTM, and
establishes a cross correlation score (X
corr
), reflective of the accuracy of the fit. The
criteria chosen for positive matches are described in Material and Methods.
Tandem mass spectrometric analysis of the SCOT carboxy fragment, excised
from the gel, showed the presence of a tryptic peptide tyrosine 366-lysine 377,
Y
366
GDLANWMIPGK
377
, either unmodified (mass of the protonated parent
ion=1364.67, X
corr
2.39), or containing oxidized methionine (+16Da) (parent ion
mass=1380.67, X
corr
4.35), or with a mass addition of 61 Da (parent ion
mass=1425.67, X
corr
3.52) (Fig. 11). The fragmentation profile of the unmodified
peptide (Fig. 11A) displayed an incomplete series of y and b ions, including two
major ions, y3 and b9, and less abundant ions, namely y4, y5, y7, y8 and y9, and b4,
b5, b7, b8, b9 and b11. Such profile can be explained by the presence of a proline
(proline 375), which often results in a preferential fragmentation on the N-terminal
side of proline (Paizs and Suhai 2005), and therefore in the dominance of the
corresponding y3 and b9 ions, as observed in panel A. MS/MS spectrum of the

59
peptide containing oxidized methionine displayed the series of ions y2-11 and b2-10,
with y5 and b8 ions, indicative of the methionine sulfoxide (+16 Da) (Fig. 11B).

Protein
NCBI accession
number
Molecular
Mass (kDa)
Peptides
matched
§

Sequence
coverage
(%)
3-oxoacid CoA transferase
(SCOT)*
NP_001012221.1 57.5 8 (147) 32
Aconitase 2 NP_077374.2 86.1 12 (21) 19
L-3-hydroxyacyl-Coenzyme
A dehydrogenase
NP_476534.1 34.5 4 (6) 16
Protein expressed in non-
metastatic cells 2
NP_114021.2 17.4 3 (3) 22
Creatine kinase NP_036662.1 43.2 1 (1) 3
Hemoglobin alpha 1 chain NP_037228.1 15.5 1 (1) 8
Table 1. Results of mass spectrometric analysis of proteins identified in
enriched SCOT isolates from heart and kidney mitochondria.
§
indicates the
numbers of unique peptides found, and their abundance (in parenthesis).* the data
are based on combined results of MS/MS analysis of “in-solution” and “in-gel”
tryptic digests of SCOT.

60
Figure 11: Identification of tryptophan 372 as the site of nitration in SCOT.
MS/MS spectra of the tryptic peptide tyrosine 366-lysine 377,
Y
366
GDLANWMIPGK
377
, unmodified (panel A) and containing oxidized methionine
(M#) (panel B) are represented. The MS/MS spectrum of the purified C-Terminal
fragment and SCOT full length protein (panel C) shows the typical fragmentation
profile of the tryptic peptide containing the tryptophan 372 (W*) with characteristic
+61 Da mass increase corresponding to the presence of nitro and hydroxy groups.
The ion annotation is based on results presented in Sequest’s “display ion view”
window that were corroborated by the dta file.

61

Figure 11
0 100 200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
20
40
60
80
100
Relative Intensity (%)
y3
301.1
y4
414.5
y5
545.6
y6
792.3
y7
905.4
y8
976.4
y9
1089.3
y10
1204.4
b3
336.1
b8
1011.37
b4
448.9
b6
634.1
b5
520.11
b9
1124.3
b7
880.5
b2
220.6
y2
203.9
b10
1222.8
C
Y G D L A N W* M I P G K
b10
y2 y3
b9
y4
b8
y5
b7
y6
b6 b5
y7 y8
b4
y10
b2
y9
b3
0 100 200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
20
40
60
80
100
Relative Intensity (%)
y3
301.1
y4
414.1
y10
1161
y6
747.2
y7
861.2
y8
932.2
y9
1045.2 b3
336.0
b9
1081.1
b4
449.1
b6
633.9
b7
820.1
b8
967.3
b10
1177.9
b11
1234.8
b5
520.0
y11
1217.3
y5
561.2
b2
220.5
y2
203.9
B
Y G D L A N W M# I P G K
y11
b10
y3
b9
y4
b8
y5
b7 b5
y7 y8
b4 b2
y10 y9
b3
y6
b6
0 100 200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
20
40
60
80
100
Relative Intensity (%)
y3
301.0
y4
413.9
b4
449.2
b9
1064.2
y8
916.2
y5
545.0
b7
819.9
y9
1029.3
b8
952.1
y7
845.3
b5
520.0
b11
1219.3
A
Y G D L A N W M I P G K
b11
y3
b9
y4
b8
y5
b7 b5
y7 y8
b4
y9

62
The MS/MS spectrum of the tryptic peptide with a mass shift of + 61
displayed a major ion y6 at m/z 792.3 (Fig. 11C). This shift could be attributed to (i)
a nitrated tryptophan (+45 Da) and an oxidized methionine (+16 Da) or (ii) a
nitrohydroxytryptophan. The presence of the ions y5 at m/z 545 and b7 at m/z 880.5
indicated that the methionine was not oxidized, and that the additional +61 Da mass
was carried exclusively by the tryptophan. Thus tryptophan 372 was identified as the
site of the double modification, namely nitration and hydroxylation.
D. Localization of tryptophan 372 in the SCOT three dimensional
structure
In order to establish whether tryptophan 372 could be a functionally relevant
residue in SCOT, it was necessary to localize its position within the three
dimensional structure of SCOT. The specific location of tryptophan 372 was
determined using a visualization program, Visual Molecular Dynamics, with the pdb
file of crystallized SCOT from pig. A monomer of SCOT is shown Fig. 12A. In
addition to its key catalytic residue, glutamate 303, the active site of SCOT is
composed of several amino acids, namely asparagine 279, lysine 327, glutamate 241,
arginine 242, glutamine 99, asparagine 51, glycine 383 and 384, alanine 385,
tyrosine 76, and lysine 380, which help the binding of substrate, succinyl-CoA (Fig.
12B). Succinyl-CoA might bind to glutamate 303 in an extended conformation
(Rangarajan, Li et al. 2005), and in this particular conformation, the distance
between its phosphorus atom on the ribose and the sulfur atom was 25 Å (Fig. 12C).

63
Tryptophan 372 was found to be localized in a hydrophobic region, and 12 Å from
the active site residue, glutamate 303.
E. Summary of the findings
Taking advantage of SCOT auto-fragmentation characteristic, and using a
combination of analytical methods, namely HPLC amino acid analysis with
electrochemical detection, MALDI and LC-ESI mass spectrometric studies, the
specific posttranslational modification present in the SCOT protein was identified as
a nitrohydroxytryptophan. Tryptophan 372, located within 10 Å of the active site,
was found to be the site of this modification.

64
Figure 12: Ribbon diagram of SCOT monomer (A) showing amino acids
involved in catalysis and the proximity of Trp 372 (B) and structure of the
substrate, succinyl-CoA (C). SCOT active site is constituted by the catalytic
residue, glutamate 303 (Glu 303), and other residues (Asn 51, Tyr 76, Gln 99, Glu
241, Arg 242, Asn 279, Lys 327, Lys 380, Gly 383 and Gly 384) that facilitate the
binding of the substrate, succinyl-CoA. Tryptophan 372 was found to be located
within 10 Å of the active residue, Glu 303. This scheme was drawn using Visual
Molecular Dynamics, using PDB data file 1ooy of the crystal structure of SCOT
from pig heart. The side chains of the amino acid residues are shown (B) and the
positions of the α-carbon atoms and sulphur atom of succinyl-CoA (C) are depicted
as balls. Bars: 5 Å.

65

Figure 12
B
Succinyl Coenzyme A
W 372
N 279
E 241,
R 242
K 380
Y 76
K 327
G 383, G 384,
A 385
Q 99
N 51
5 Å
5 Å
C A
E 303

66
III. Effects of age and calorie restriction on SCOT nitrohydroxytryptophan
content, protein amount and catalytic activity
A. Age-associated variations in SCOT nitrohydroxytryptophan content
in mitochondria of various rat organs
The aims of this experiment were to determine: (i) the impact of age on
SCOT nitration levels, and (ii) whether additional nitrated proteins could be detected
among mitochondrial soluble proteins in the old rats. Accordingly, heart, kidney,
brain cortex, hind limb skeletal muscle, lung, spleen, testis and liver mitochondria
were isolated from 4- and 24-old rats. Soluble proteins were submitted to Western
blot analysis using an anti-3NT antibody. The immunoblot showed that SCOT
nitration content increased with age in heart and brain, decreased in kidney, lung and
spleen, and remained unchanged in skeletal muscle. No additional immunopositive
protein band could be detected among the soluble proteins from old organs (Fig. 13).
Thus the impact of age on SCOT nitration levels varied in different tissues. In
addition, SCOT remained the single major nitrated protein in mitochondria of both
young and old rat tissues.

67

Figure 13: Age-associated changes in SCOT nitration content in different
tissues of the rat. Soluble mitochondrial proteins, purified from different young (Y,
4 months-old) and old (O, 24 months-old) rat tissues were separated by SDS-PAGE
and after transfer of proteins to PVDF membranes, an immunoblot analysis was
performed with an anti-3NT antibody. Lanes 1-8 contain 10 µg of mitochondrial
soluble proteins from heart, kidney, brain cortex, hind limb skeletal muscle, lung,
spleen, testis and liver. Lanes M and C refer to the mixture of protein molecular
weight markers and untreated BSA, and nitrated bovine serum albumin, respectively.
Arrow on the right indicates the position of SCOT.
B. Impact of age on SCOT parameters in heart and kidney
mitochondria
The purpose of these experiments was to assess the impact of age-associated
changes in nitration observed qualitatively on the immunoblot on SCOT specific
catalytic activity. As stated before in another context, due to the intense reactivity
between the anti-3NT antibody and the SCOT protein in heart and kidney, the
studies on enzyme activity were restricted to these two tissues. Thus the following
SCOT parameters were quantitated in the heart and kidney mitochondria from young
and old rats: (i) SCOT nitrohydroxytryptophan content, (ii) protein amount and (iii)
specific catalytic activity, expressed in mmol/mol SCOT, pmol SCOT/mg protein
and mol acetoacetyl-CoA formed/min/SCOT protein.
M C 1
Y O
2
Y O
3
O Y
4
O Y
5
O Y
6
O Y
7
O Y
8
O Y
Anti-3NT

68
Mitochondrial soluble proteins, isolated from heart and kidney of 4-, 13-, 19-
and 24 months-old rats were electrophoresed, and gels were stained with Coomassie
or transferred to a membrane for immunodetection with the anti-3NT antibody or the
anti-SCOT serum. SCOT protein amount was quantitated by optical densitometry
(OD), and normalized to the OD of known concentrations of purified SCOT.
Measurement of nitrohydroxytryptophan content was made by a combination of (i)
HPLC amino acid analysis using 5-nitrotryptophan as a standard, and (ii) OD
analysis of nitrated bands, using nitrated bovine serum albumin as the calibration
standard. SCOT catalytic activity was measured as described in Materials and
Methods, and normalized to the total amount of SCOT protein present in the sample,
to obtain SCOT specific catalytic activity.
For heart and kidney tissues, Coomassie stained gel indicated equal loading
of proteins for each age (results not shown).
In heart mitochondria, a comparison between 4-, 13- , 19- and 24 months-old
animals indicated that SCOT nitrohydroxytryptophan content increased from 36 to
80 mmol/mol SCOT (p<0.0001, n=3), while SCOT protein amount remained
unchanged (56.3 ± 5 pmol SCOT/mg mitochondrial soluble proteins). Between 4-
and 24 months of age, SCOT specific catalytic activity rose by 30% (p<0.0001, n=9)
(Fig. 14).
In contrast, in kidney mitochondria, between 4 and 24 months of age, SCOT
protein amount declined by 55% (p<0.0001, n=3) while SCOT
nitrohydroxytryptophan content and SCOT specific catalytic activity remained

69
unchanged (36 ± 1.5 mmol/mol SCOT and 5.05 ± 0.27× 10
3
mol acetoacetyl-CoA
formed/min/mol SCOT protein, respectively) (Fig. 15).

70

Figure 14: Impact of age on SCOT nitrohydroxytryptophan content, protein
amounts and specific activity in rat heart mitochondria. Panel A: Western blot
analysis of mitochondrial soluble proteins isolated from heart of 4, 13, 19 and 24
months-old animals with the anti-3-nitrotyrosine antibody (Anti-3NT) or the anti-
SCOT serum (Anti-SCOT), showing age-associated increase in SCOT nitration
content, and stable average protein amounts. Blots are representative of 3
independent experiments. Panel B: SCOT content in nitrohydroxytryptophan,
average protein amount, and specific activity are expressed as percentage of data
measured in 4-month old animals (36 ± 2.2 mmol/mol SCOT, 56.3 ± 4.9 pmol/mg
matrix proteins and 5.15 ± 0.15 × 10
3
mol acetoacetyl-CoA formed/min/mol SCOT
proteins, respectively). SCOT activity was not measured in 13 months-old animals.
Anti-3NT
Anti-SCOT
A
B
4 13 19 24
Age (months)
4 13 19 24
Age (months)
0
50
100
150
200
Percent of 4 Months
Nitration
Activity
SCOT
*
*
*
*
*

71

Figure 15: Impact of age on SCOT nitro-hydroxy-tryptophan content, protein
amounts and specific activity in rat kidney mitochondria. Panel A: Immunoblot
of mitochondrial soluble proteins isolated from kidney of 4, 13, 19 and 24 months-
old animals with the anti-3-nitrotyrosine antibody (Anti-3NT) or the anti-SCOT
serum (Anti-SCOT), showing age-associated decline in anti-3NT and anti-SCOT
reactivity. Blots are representative of 3 independent experiments. Panel B: SCOT
content in nitrohydroxytryptophan, average protein amount, and specific activity are
expressed as percentage of results obtained in 4-months old animals (36 ± 1.5
mmol/mol SCOT, 36.2 ± 1.4 pmol/mg matrix proteins and 5.05 ± 0.27 × 10
3

acetoacetyl-CoA formed/min/mol SCOT proteins, respectively). SCOT activity was
not measured in 13 months-old animals.
Anti-3NT
Anti-SCOT
4 13 19 24 Age (months)
A
B
4 13 19 24
Age (months)
0
50
100
Percent of 4 Months
Activity
Nitration
SCOT
*
*

72
C. Impact of calorie restriction on SCOT nitrohydroxytryptophan
content, protein amount and specific activity
Calorie restriction (CR) has been shown to prolong the life span of some
strains of rats and mice, and to retard age-associated functional decrements. Thus the
effects of CR on SCOT nitration content, specific catalytic activity and protein levels
were determined in rats at 19 months of age.
Mitochondria were isolated from heart and kidneys of rats fed ad libitum
(AL) or 40% less food from the AL rats (CR) rats. Mitochondrial soluble proteins
were prepared and subjected to SDS-PAGE electrophoresis, followed by
immunoblotting using the anti-3NT antibody or the anti-SCOT serum.
In heart mitochondria, SCOT nitrohydroxytryptophan content, protein levels
and specific activity in CR animals remained unchanged when compared to AL
animals (60 ± 5 mmol/mol, 56 ± 5 pmol/mg, 6.25 ± 0.28× 10
3
mol acetoacetyl-CoA
formed/min/mol SCOT proteins, respectively).
In contrast, in kidney mitochondria of CR animals, SCOT protein amount
increased by 50% (p<0.0001, n=3), while SCOT nitrohydroxytryptophan content and
specific activity (36 ± 3 mmol/mol and 5.2 ± 0.5 x10
3
mol acetoacetyl-CoA
formed/min/mol SCOT proteins,

respectively) remained unchanged in comparison to
AL animals.
Thus, while CR did not seem to have any effect on SCOT parameters in heart
mitochondria, it reversed the effect of age in kidney mitochondria (Fig. 16).

73

Figure 16: Effect of calorie restriction on SCOT parameters in rat heart and
kidney of rat. Soluble proteins from heart and kidney mitochondria were isolated
from ad libitum-fed (AL) and calorically restricted (CR) rats at 19 months of age,
compared for the immunodensity of SCOT protein bands with anti-3NT and anti-
SCOT antibodies (Panel A) and specific catalytic activity of SCOT. Percent
differences between AL and CR groups in SCOT nitration content, protein amounts
and specific activity are presented in Panel B. In heart and kidney of AL rats,
respectively, the specific activities of SCOT were 6.25 ± 0.28 and 5.2 ± 0.5 x10
3
mol
acetoacetyl-CoA formed/min/mol SCOT protein, the nitrohydroxytryptophan
contents were 60 ± 5 and 36 ± 3 mmol/mol, and the amounts of SCOT protein were
56 ± 5 and 14 ± 1.5 pmol/mg. Values represent mean ± standard deviation of n=6 for
immunoblots and n=9 for activity assays; * indicates significant difference vs. AL
(P<0.0001).

Anti-SCOT
Anti-3NT
AL CR
Anti-3NT
Anti-SCOT
AL CR
Kidney Heart
A
B
AL CR
0
50
100
150
Percent of AL
AL CR
0
50
100
150
*
Nitration
SCOT
Activity

74
D. Summary of the effects of aging and calorie restriction on SCOT
parameters
SCOT parameters, measured in heart and kidney of aging and calorie
restricted rats, are indicated in Table 2. From these values, it was possible to
calculate the amount of nitrated SCOT relative to the total levels of SCOT protein at
young and old ages. For example, in heart of young rats, SCOT nitration content was
36 mmol/mol SCOT and SCOT protein amount was 56.3 pmol/mg matrix proteins;
Thus, in the heart of young animals, there was 56.3 x 0.036= 2.03 pmol nitrated
SCOT, and 56.3-2.03=54.27 pmol unmodified SCOT. These values were used to
determine the specific activity of nitrated and unmodified SCOT, as explained in the
next section.

75

Heart Young Old AL CR
SCOT protein total
amount
(Expressed in pmol/mg
matrix proteins)
56.3±5 56.3±5
Unchanged Unchanged
SCOT specific activity
(Expressed as mol
acetoacetyl-CoA
formed/min/mol SCOT)
5.2±0.15×10
3
6.7±0.25×10
3
6.25±0.35×10
3

30 % significant increase Unchanged
SCOT nitration content
(Expressed as mmol/mol
SCOT)
36±1.6 80±3.6 60±2
222 % significant increase Unchanged
Amount of nitrated SCOT
(pmol)
2.03 4.5 3.38
Amount of unmodified
SCOT (pmol)
54.27 51.8 52.92

Table 2: Summary of SCOT parameters measured in the heart and kidney of
young (4 months), old (24 months), ad-libitum fed and 40 % calorie restricted
(19 months) rats.

Kidney Young Old AL CR
SCOT protein total
amount
(Expressed in pmol/mg
matrix proteins)
36.2±1.4 16.3±1.8 14±1.1 21.1±1.7
55% significant decrease 50% significant increase
SCOT specific activity
(Expressed in mol
acetoacetyl-CoA formed
/min/mol SCOT)
5.05±0.27×10
3
5.2±0.2×10
3

Unchanged Unchanged
SCOT nitration content
(Expressed in mmol/mol
SCOT)
36±1.5 36±0.5
Unchanged Unchanged

76
E. Specific activity of nitrated SCOT
Based on the results obtained in heart, where SCOT nitration content and
specific catalytic activity increased with age, while the protein content remained
unchanged, and in the absence of any indication of the presence of other
modifications in the SCOT protein purified from old animals, it was hypothesized
that nitration of SCOT may enhance its activity.
The following equation was used to compare the activity of the fraction of
SCOT that is nitrated to the activity of the unmodified SCOT:
A
tot
=A
nitrated
+A
unmodified
,
where A
tot
, the total SCOT activity, is the sum of the activity of nitrated SCOT
(A
nitrated
) and of unmodified SCOT (A
unmodified
).
SCOT activity (A), by definition, corresponds to the specific activity (SA) multiplied
by the amount of SCOT protein.
Knowing the amounts of SCOT protein which are nitrated or remain unmodified (see
Table 2), the following two equations can be resolved:
SCOT activity in young animals:
5.15 x10
3
x 56.3 = SA
nitrated
x 2.03 + SA
unmodified
x 54.27
SCOT activity in old animals:
6.7 x10
3
x 56.3= SA
nitrated
x 4.5 + SA
unmodified
x 51.8
The specific activity of nitrated SCOT (SA
nitrated
=38.5±3.5 x10
3
mol acetoacetyl-
CoA formed/min/mol SCOT protein) was thus calculated to be ~10 times higher than

77
the specific activity of unmodified SCOT (SA
unmodified
=3.9±0.3 x10
3
mol acetoacetyl-
CoA formed/min/mol SCOT protein).
F. Summary of the findings
Age and calorie restriction had a differential effect on SCOT parameters in
heart and kidney.
In heart mitochondria, there was an increase of 222% (p<0.0001, n=3) in
SCOT nitrohydroxytryptophan content with age accompanied with a 30%
enhancement of its specific catalytic activity (p<0.0001, n=9), while the amount of
SCOT protein remained unchanged. It was estimated that nitrated SCOT was
roughly ten times more active than unmodified SCOT. Long term calorie restriction
(16 months) did not have an impact on any SCOT parameter.
In kidney, SCOT protein levels decreased by 55 % with age (p<0.0001, n=3),
while SCOT specific activity and nitration content remained stable. Calorie restricted
rats exhibited a 50% higher SCOT protein than the AL rats (p<0.0001, n=3), whereas
SCOT specific activity and nitration were unaffected.
IV. Assessment of functional consequences of SCOT nitration via in vitro
experiments
A. Impact of peroxynitrite-induced nitration on SCOT catalytic activity
Purpose of this experiment was to determine whether peroxynitrite-induced
nitration could replicate the effects of nitration observed in vivo. Peroxynitrite (PN)

78
might be generated in mitochondria, because of the high rates of superoxide
production and the presence of a hypothetical isoform of nitric oxide synthase.
Heart and kidney mitochondrial soluble proteins from young animals were
incubated with 0, 25, 50, 125, 250 and 500 µM PN, following which the protein
mixtures were either submitted to Western blot analysis with an anti-3NT antibody,
or used for measurement of SCOT catalytic activity.
Using the anti-3NT antibody, immunoblotting indicated that at PN
concentration < 125 µM, specific proteins, including SCOT, showed nitration,
whereas at higher concentrations, virtually all proteins became nitrated (Fig. 17A).
At 25 µM PN, SCOT catalytic activity was increased by 24 % in heart (from
4.91±0.16 to 6.07±0.1 x10
3
mol acetoacetyl-CoA formed/min/mol SCOT protein)
and by 18 % (from 4.85±0.18 to 5.72±0.15 x10
3
mol acetoacetyl-CoA
formed/min/mol SCOT protein) in kidney (p<0.01, n=5), while higher PN
concentrations led to a linear decrease in SCOT activity (Fig. 17B&C).
Concomitantly, 25 µM PN increased SCOT nitration content from 36±1.6 to 66±3
mmol/mol SCOT in heart, and from 36±1.9 to 60±4 mmol/mol SCOT in kidney
(p<0.01, n=3) (Fig. 17C). To estimate the specific activities of in vitro nitrated and
unmodified SCOT, aforementioned calculations were made.

79
Figure 17: Peroxynitrite-induced nitration of SCOT in kidney and heart and its
impact on nitration and activity. Mitochondrial soluble proteins from heart and
kidney of young animals were incubated for 15 min with increasing concentrations
of peroxynitrite, from 0 to 500 µM. Panel A: immunoblots of the treated proteins
from heart and kidney using the anti-3NT antibody. Lanes 1-6 contain samples
incubated with 0, 25, 50, 125, 250, 500 µM of peroxynitrite; lane 7 shows SCOT
incubated with degraded peroxynitrite (500 µM). Panel B: SCOT activity was
measured at different peroxynitrite concentrations, and expressed as percent of
untreated mitochondrial proteins. Panel C shows specific activity (left) expressed in
mol acetoacetyl-CoA formed/min/mol SCOT protein, and nitration content (right) in
mmol/mol SCOT protein upon exposure to 25 µM peroxynitrite.

80

Figure 17
C
0 100 200 300 400 500
Peroxynitrite (µM)
0
50
100
150
SCOT activity (%)
Kidney
Heart
B
Heart
1 2 3 4 5 6 7
Anti-3NT
A
Kidney
1 2 3 4 5 6 7
Anti-3NT
Heart Kidney
0
20
40
60
80
SCOT nitration
Control
25 µM PN
*
*
(mmol/mol SCOT)
Heart Kidney
0.00
2.50
5.00
7.50
SCOT acttivity
*
*
(x 10
3
)

81
SCOT activity in absence of peroxynitrite:
4.91 x 10
3
56.3= SA
nitrated
2.03 + SA
unmodified
54.27

SCOT activity in presence of 25 µM peroxynitrite:
6.07 x10
3
56.3= SA
nitrated
3.75 + SA
unmodified
52.55
The specific activity of nitrated SCOT (41.3 ±2.2 x10
3
mol acetoacetyl-CoA
formed/min/mol SCOT protein) was calculated to be 12 times higher than the
specific activity of unmodified SCOT (3.5±0.4 x10
3
mol acetoacetyl-CoA
formed/min/mol SCOT protein). Such results accord with the calculated values of
SCOT specific catalytic activity in heart mitochondria during aging.
B. Susceptibility of SCOT to proteolysis
To further evaluate the functional consequences of nitration, susceptibility of
SCOT to hydrolysis by proteases with distinct cleavage specificities, was examined.
It is well documented that the SCOT protein contains, besides the autolytic site
previously described, a very hydrophilic region which is susceptible to proteolytic
cleavage. Upon proteolysis, SCOT is nicked in two domains, the N-terminal domain
(~32 kDa) and the C-terminal domain (~26 kDa) (Lin and Bridger 1992). SCOT
sensitivity to trypsin and chymotrypsin-induced degradation was deemed to be of
crucial interest, since trypsin and chymotrypsin activities are components of the
proteasome.

82
1. Susceptibility to trypsin induced-degradation
Mitochondrial soluble proteins, incubated at 37ºC for 0, 30, 60, 90 and 120
min with trypsin at a protein to protease ratio of 50:1, were separated by SDS-PAGE,
and the gels were either stained with Coomassie Blue or electrotransferred to
membranes for immunodetection with anti-3NT or anti-SCOT antibodies.
The time course of degradation of mitochondrial proteins and nitrated SCOT
was evident in the Coomassie gel (Fig. 18A) and on the immunoblot with the anti-
3NT antibody (Fig. 18B). The optical density (OD) of randomly selected protein
bands on the Coomassie gels was measured as a function of time, and compared to
the OD of nitrated SCOT bands. Results indicated that nitrated SCOT was degraded
more quickly than other non nitrated mitochondrial proteins in both heart and kidney
tissues (Fig. 18C).
To investigate whether such observation was simply due to the fact that the
SCOT protein itself is sensitive to proteolysis, SCOT degradation was slowed down.
Thus mitochondrial proteins were also incubated with a relatively lower trypsin
concentration (a protein to protease ratio of 250:1). Comparison of OD between
unmodified and nitrated full length SCOT showed that nitrated SCOT was more
susceptible to degradation than the unmodified SCOT (Fig. 19A, B). This
observation was corroborated by the finding that the 26 kDa carboxy terminal
fragment, containing nitrohydroxytryptophan 372, was degraded faster than the
unmodified 32 kDa amino terminal fragment (Fig. 19C, insert). Thus, nitrated SCOT
is relatively more susceptible to degradation than other mitochondrial proteins. The
propensity for degradation was apparently linked to the presence of the nitrated

83
residue, as indicated by the faster degradation of the carboxy terminal fragment than
the amino terminal fragment.

84

Figure 18: Degradation of SCOT during proteolysis in vitro. Soluble
mitochondrial proteins from heart and kidney mitochondria were incubated with
trypsin for 180 min at a protein to protease ratio of 50:1, and separated by SDS-
PAGE, following which the gels were either stained with Coomassie (A) or
electrotransferred for immunodetection with an anti-3NT antibody (B). Samples
taken at 0, 30, 60, 90 and 120 min after incubation with trypsin are in lanes 2-6,
respectively; lane 1 contains untreated soluble proteins, and lane C, nitrated BSA.
Randomly selected protein bands a-f, indicated by arrows on the right of Coomassie
stained gels were quantitated by densitometry. SCOT full length (SCOT) and
carboxy-terminal domain (C-Term, ~26 kDa) are indicated on the immunoblot by
arrows on the right. The bottom graphs (C) show a comparison of time-dependent
degradation of SCOT (SCOT activity, large circles) with those of selected proteins a,
b, c, d, e and f (small circles) as percentages of the controls (0 min of trypsin
incubation).
Heart Kidney
a
b
c
d
e
f
C 1 2 3 4 5 6
Coomassie
A
b
a
c
d
e
f
C 1 2 3 4 5 6
Coomassie
0 50 100 150 200
Time (min)
0
25
50
75
100
Percent of 0 min
c
b
a
d
f
e
SCOT
C
0 50 100 150 200
Time (min)
c
b
a
d
e,f
SCOT
Anti-3NT
B
Anti-3NT
SCOT
C-Term

85
Figure 19: Degradation of carboxy and amino terminal fragments of SCOT
upon partial trypsin induced-proteolysis in vitro. Soluble mitochondrial proteins
from kidney mitochondria were incubated with trypsin for 120 min at a protein to
protease ratio of 250:1, and were then subjected to SDS-PAGE and immunoblotting
for SCOT and nitration content at various time intervals. Proteins incubated with
trypsin were removed at 0, 15, 30, 45, 90 and 120 min (lanes 2-7, respectively); lane
1 contains untreated proteins. C refers to the positive control, nitrated BSA. Arrows
on the right indicate the positions of full-length SCOT protein (SCOT), carboxy-
terminal (C-Term) and amino-terminal (N-Term) fragments. The time-dependent
disappearance of full-length SCOT band intensity was normalized to the controls (0
min) and presented as percent changes in the bottom graph. The insert shows
normalized optical densities (percent of SCOT immunointensity at 0 min) of amino-
terminal (N-Term) and carboxy-terminal (C-Term) fragments of SCOT, detected by
anti-3NT and anti-SCOT antibodies, respectively.

86

Figure 19
Anti-3NT
C 1 2 3 4 5 6 7
SCOT
C-Term (26 kDa)
Anti-SCOT
N-Term (32 kDa)
SCOT
0 30 60 90 120
Incubation time (min)
0
25
50
75
100
Normalized OD (%)
SCOT
Nitration
0 30 60 90 120
Incubation time (min)
0
25
50
75
100
Normalized OD (%)
C-Term
N-Term

87
2. Susceptibility to chymotrypsin-mediated proteolysis
As in the experiment described with trypsin, mitochondrial soluble proteins
were incubated for 0, 30, 60, 90 and 120 min with chymotrypsin at a protein to
protease ratio of 50:1, and the resulting mixtures were separated by SDS-PAGE,
following which the gels were either stained with Coomassie Blue or
electrotransferred to membranes for immunodetection with anti-3NT or anti-SCOT
antibodies.
Some mitochondrial soluble proteins were evidently degraded in the presence
of chymotrypsin, as indicated by the time-dependant progressive decrease in
Coomassie staining of some protein bands (Fig. 20A). In contrast, during the same
time frame, the entire pool of SCOT protein, nitrated and non-nitrated, remained
insensitive to chymotrypsin treatment (Fig. 20B&C).

88

Figure 20: Lack of degradation of the SCOT protein in response to
chymotrypsin treatment. Soluble mitochondrial proteins from kidney mitochondria
were incubated with chymotrypsin for 120 min at a protein to protease ratio of 50:1,
and separated by SDS-PAGE, following which the gels were either stained with
Coomassie (A) or electrotransferred for immunodetection with an anti-3NT antibody
(B) or the anti-SCOT serum (C). Samples taken at 0, 30, 60, 90 and 120 min after
incubation with chymotrypsin are in lanes 2-6, respectively; lane 1 contains
untreated soluble proteins taken at 0 min, lane 7, untreated soluble proteins incubated
at 37ºC for 120 min, and lane C, nitrated BSA. The arrows on the right indicate
SCOT position. M contains the molecular weight standards and non-nitrated BSA.
Coomassie
M 1 2 3 4 5 6 7 C
210
120
80
49
35
29
20
7
A
Anti-3NT
210
120
80
49
35
29
20
7
B
Anti-SCOT
210
120
80
49
35
29
20
7
C

89
3. Susceptibility to other proteases induced-degradation
To investigate whether the nitration of SCOT was associated with a relatively
higher/lesser sensitivity to degradation by proteases other than trypsin or
chymotrypsin, proteinase K, endoproteinase GluC, bromelain and thrombin-
mediated proteolysis of mitochondrial proteins was examined. The specific cleavage
sites for these enzymes are different: chymotrypsin preferentially cleaves at
tryptophan, tyrosine and phenylalanine; Proteinase K preferentially cleaves carboxyl
side of aliphatic, aromatic or hydrophobic residues; Endoproteinase GluC selectively
cleaves peptide bonds carboxy-terminal to glutamic acid residues, and, to a lesser
extent, at aspartic acid residues; Bromelain cuts at lysine, alanine and tyrosine
residues; Thrombin, which natural substrate is fibrinogen, preferentially cleaves at
arginine residues.
Kidney mitochondrial soluble proteins were incubated with proteases for 2 h
at a ratio protein to protease 1:50, separated by SDS-PAGE, and gels were either
stained with Coomassie blue or electrotransferred to membranes for
immunodetection with an anti-3NT antibody (Fig. 21).
The susceptibility of mitochondrial proteins to digestion by the various
proteases was analyzed qualitatively on the Coomassie stained gel. In comparison to
the control (lane 1), proteinase K (lane 4) digested all mitochondrial proteins into
amino acids. Trypsin, bromelain and chymotrypsin hydrolyzed most mitochondrial
proteins (lane 2, 6 and 3, respectively). In contrast, mitochondrial proteins seemed to

90
be more resistant to endoproteinase GluC-induced proteolysis (lane 5). Thrombin
had no apparent effect on mitochondrial proteins (lane 7).
As the majority of mitochondrial proteins, nitrated SCOT was completely
degraded upon treatment with trypsin, proteinase K and bromelain, and remained
intact upon exposure to thrombin and endoproteinase GluC; however, the entire pool
of SCOT protein, i.e. nitrated and unnitrated SCOT, was resistant to chymotrypsin-
induced cleavage.

Figure 21: SCOT susceptibility to degradation by proteases with different
cleavage specificities. Soluble proteins from kidney mitochondria were incubated
with various proteases for 2 h at a protein to protease ratio of 50:1, and then
subjected to SDS-PAGE and immunoblotting for 3NT content. Lanes 1-7 correspond
to soluble mitochondrial untreated (lane-1) or incubated with trypsin, chymotrypsin,
proteinase K, endoproteinase GluC, bromelain and thrombin (lane 2-7), respectively.
C refers to the positive control, nitrated BSA.
Anti-3NT
C 1 2 3 4 5 6 7
Coomassie
SCOT

91
C. Summary
In a series of in vitro experiments, the functional consequences of SCOT
nitration were assessed.
At low concentration of peroxynitrite (25 µM), there was a significant
increase in SCOT nitration content and specific activity, in both heart and kidney
tissues. In vitro nitrated SCOT activity was calculated to be ~12 times greater than
the unmodified SCOT, which accords with the in vivo data. Higher PN
concentrations caused a linear decrease in SCOT activity.
SCOT nitration in kidney was associated with an enhancement of
susceptibility to trypsin degradation in comparison to the non-nitrated mitochondrial
proteins. The carboxy fragment, containing nitrohydroxytryptophan, was more
sensitive to trypsin-induced degradation than the non modified, amino-terminal
fragment.
SCOT susceptibility to degradation by various proteases was comparable to
other mitochondrial proteins, with the exception of a resistance to chymotrypsin-
induced proteolysis.
V. Impact of age and calorie restriction on SCOT parameters in different mice
strains
The purposes of these experiments were to investigate: (i) whether SCOT or
other mitochondrial proteins were nitrated in three different strains of mice, namely
C57BL/6 (C57), DBA/2 (DBA) and monoamine oxidase A/B double knockout mice
(MAO-A/B DKO mice), (ii) if SCOT was also nitrated in mice, and (iii) whether

92
aging and calorie restriction affect SCOT protein amounts, nitration degree and
activity in C57 and DBA mice. The rationale for assessing SCOT parameters in these
strains is as follows: it has been observed that a 40 % reduction in calorie intake
increases lifespan of C57 mice by ~ 25-30%, while it has little effect on longevity of
DBA mice (Forster, Morris et al. 2003). Thus the hypothesis tested was that the
relative resistance of DBA mice to CR-mediated life span extension could stem from
a differential rate of accrual of protein oxidative damage. Monoamine oxidase A and
B are located in the outer mitochondrial membrane. They catalyze the deamination
of polyamines, and hydrogen peroxide (H
2
O
2
) is generated as a byproduct of this
activity. It was postulated that putative mitochondrial oxidative stress in these
knockout mice could be lowered in comparison to the control strain, C57, thereby
affecting SCOT nitration. Kidney was chosen for these studies, because of the
greater amount of available tissue in comparison to heart.
A. SCOT nitration in three different mice strains
To check whether SCOT and/or other proteins were targets of nitration in
mice, Western blot analysis of kidney mitochondrial soluble proteins, isolated from
young animals was performed using the anti-3NT and the anti-SCOT serum. To limit
nonspecific reactions with mouse heavy chains immunoglobulins, whose molecular
weight is similar to SCOT, a secondary anti-mouse directed against light chain
immunoglobulins was chosen for immunodetection of nitrated proteins in mice.
As observed in rat, the immunoblot using the anti-3NT antibody revealed that
only a single major immunopositive band was detected among mitochondrial soluble

93
proteins in the kidney from the three strains of mice. Using the anti-SCOT serum,
this protein band was confirmed to contain SCOT (Fig. 22).
In comparison to rat (Fig. 22, lane1), SCOT nitration and protein content
appeared slightly reduced in C57 and MAO-A/B DKO mice (lanes 2-3), while the
same parameters were relatively similar in DBA mice (lane 4).

Figure 22: Nitration of the protein SCOT in kidney mitochondria from three
different strains of mice. Kidney mitochondrial soluble proteins from young mice
(6 months-old) were separated by one-dimensional SDS-PAGE. Gels were
transferred to PVDF membranes for Western blot analysis with the anti-3-
nitrotyrosine antibody (anti-3NT) or the anti-SCOT serum. M refers to the protein
standards and 0.5 µg of control non-treated bovine serum albumin (BSA) (67 kDa).
Lane C contains 0.5 µg of peroxynitrite-treated BSA. Lanes 1-4 contain 10 µg of
kidney soluble mitochondrial proteins from rat, C57, MAO-A/B DKO and DBA
mice. The arrow on the right indicates the position (58 kDa) of SCOT.
B. Impact of age on SCOT parameters in C57 and DBA mice
Isolation of mitochondria, preparation of mitochondrial soluble proteins from
kidney, and Western blot analysis with the anti-3NT or the anti-SCOT antibodies
were performed as described in Materials and Methods.
In contrast to the age-related variations observed for SCOT parameters in rat
kidney mitochondria, SCOT nitration content, protein levels and catalytic activity
remained unchanged with age in both strains of mice. SCOT catalytic activity in
120
80
49
35
29
20
7
1 M C 2 3 4
Anti-3NT Anti-SCOT
1 M C 2 3 4

94
young and old DBA mice was 26% (p<0.0001, n=5) and 18% (p<0.05, n=6) higher
than in the young and old C57 mice, respectively. Such differences were accounted
for the relatively higher content in SCOT protein in DBA mice at 6 and 23 months of
age, as indicated by the immunoblots probed with the anti-SCOT serum (Fig. 23).

95

Figure 23: Impact of age on SCOT nitration, protein amount and catalytic
activity in C57 and DBA mice. Panel A: Immunoblot analysis of soluble
mitochondrial proteins isolated from the kidney of C57 or DBA mice at 6 and 23
months of age, showed no effect of age on SCOT nitration content (anti-3NT) or
protein amounts (anti-SCOT). Panel B: SCOT catalytic activity, expressed in nmol
acetoacetyl-CoA formed/min/mg matrix proteins, remained stable with age in both
strains. Note that SCOT catalytic activity in DBA mice is significantly higher than in
C57 mice at young (p<0.0001, n=5) and old ages (p<0.05, n=6). This phenomenon
was accounted for by a higher SCOT protein content in DBA mice, as can be
observed on the immunoblots with the anti-SCOT serum.
Age (months)
C57BL/6
Anti-SCOT
Anti-3NT
6 23
DBA/2
Anti-SCOT
Anti-3NT
6 23
B
A
C57BL/6 DBA
0
50
100
150
200
SCOT Activity (nmol/min/mg prot)
Young
Old
***
*

96
C. Impact of calorie restriction
Calorie restriction by 40%, started at 3 months of age, induced a significant
decrease in SCOT catalytic activity at young (6 months-old) and old age (23 months-
old) in both strains of mice. In comparison to young AL animals, SCOT catalytic
activity declined by 27 % (p<0.0001, n=6) and by 13 % (p<0.01, n=6) in calorie
restricted C57 and DBA strains, respectively. Such reduction in SCOT activity in
young CR animals was significantly higher in C57 animals than in CR DBA mice
(p<0.05, n=6). At 23 months of age, SCOT catalytic activity in CR animals was 29%
(p<0.0001, n=6) and 19% (p<0.0001, n=5) lower than in matched controls C57 and
DBA mice. The difference in the magnitude of the decrease between the two strains
was not significant (Fig. 24).

97

Figure 24: Impact of calorie restriction on SCOT catalytic activity in the kidney
of the young (6 months) and old (23 months) C57BL/6 and DBA/2 mice. In the
young mice, the impact of calorie restriction on SCOT catalytic activity was
significantly greater in C57 mice (27% significant decrease) than in DBA mice (13%
significant decrease). In contrast, in the old mice, the decrease in activity was
comparable in the two strains.

CR-associated variations in SCOT nitration content and total protein content
were measured in the old mice only, using western blot analysis with the anti-3NT
antibody or the anti-SCOT serum.
In comparison to the old AL-fed mice, SCOT protein levels were 40 %
(p<0.05, n=3) lower than in CR C57 mice, and by 25 % in DBA mice (p<0.05, n=3).
For both strains, the ratio of OD
nitrated SCOT
to OD
total SCOT
, indicative of the fraction of
protein SCOT that is nitrated, remained unchanged in calorie restricted animals when
compared to AL-fed animals (Fig. 25). Thus, at 23 months of age, i.e. after 21
months of CR regimen, SCOT nitration content remained unaffected in C57 and
DBA mice. Therefore, the decrease in catalytic activity in CR animals was attributed
C57BL/6 DBA/2
0
50
100
150
200
SCOT Activity (nmol/min/mg prot)
AL 6 months
CR 6 months
AL 23 months
CR 23 months
***
***
**
***
*

98
to the decrease in SCOT protein amount. Such decline in SCOT protein was greater
in C57, but did not differ significantly from DBA mice.
These results are in contrast to the data obtained in rats, where the CR
regimen elevated both SCOT protein content and catalytic activity.

Figure 25: Impact of calorie restriction on SCOT protein amount and relative
nitration content, estimated by the ratio OD
nitrated SCOT
to OD
total SCOT
, in
C57BL/6 and DBA/2 mice at 23 months of age. Panel A: Immunoblot analysis of
soluble mitochondrial proteins, isolated from the kidney of ad libitum (AL)-fed or
calorie restricted (CR) C57 or DBA mice, showing decline in both SCOT nitration
and protein content. Panel B: graphs showing the differences between the 2 strains in
percent of AL values of SCOT protein amount and degree of nitration, expressed by
the ratio OD
nitrated SCOT
to OD
total SCOT
(OD
3NT
/OD
SCOT
).
AL CR
Anti-SCOT
DBA/2
Anti-3NT
C57BL/6
Anti-SCOT
Anti-3NT
CR AL
A
B
C57BL/6 DBA/2
0
25
50
75
100
SCOT protein content (% AL)
*
*
C57BL/6 DBA/2
0
25
50
75
100
Ratio OD
3NT
/OD
SCOT
(% AL)
AL
CR

99
D. Summary
As in rat, SCOT was the major nitrated protein in the kidney mitochondria
from the three mice strains tested, namely C57, DBA and MAO-A/B DKO mice.
SCOT nitration levels and protein amounts were qualitatively similar in the
kidney mitochondria from MAO-A/B DKO mice and C57 mice.
Comparison of age- and CR-associated changes in SCOT parameters between
C57 and DBA mice showed that (i) SCOT catalytic activity at young and old ages in
DBA mice was significantly higher than in comparable C57 mice, most likely due to
a greater SCOT protein content in the former; (ii) CR decreased SCOT catalytic
activity in both strains at young and old ages, however, such decrease was
significantly higher at young age in the C57 mice, while at 23 months of age, the
decrease in the two strains was not significantly different, and that (iii) these changes
appeared to be linked to a decrease in SCOT protein content, since the fraction of
SCOT that was nitrated remained unchanged in AL and CR animals of both strains.
VI. Effect of age and calorie restriction on enoyl-CoA hydratase protein
amount and catalytic activity in rat kidney and liver mitochondria
To investigate whether age and calorie restriction affected enoyl-CoA
hydratase (ECH) functional parameters, ECH catalytic activity and protein amount
were measured in kidney and liver mitochondria, the two tissues where the protein
was found to be nitrated. Age-associated profile of nitration could not be determined
using the standard Western Blot analysis with the anti-3NT antibody, because ECH
nitration was only detectable after fractionation of mitochondrial proteins.

100
A. Effect of age on ECH activity and protein amounts in kidney and
liver
Isolation of liver and kidney mitochondria, preparation of mitochondrial
soluble proteins, and Western blot analysis with the anti-ECH serum were performed
as described in Materials and Methods.
In kidney mitochondria, with age, ECH protein amounts and catalytic activity
decreased by 45% (p<0.01, n=3) and by 34% (p<0.0001, n=6), respectively. In
contrast, in liver mitochondria, the same parameters increased by 33% (p<0.05, n=3)
and by 31 % (p<0.05, n=5) between 4 and 24 months of age (Fig. 26).
Thus in both tissues age-related changes in ECH catalytic activity reflected,
most likely, the age-associated variations in ECH protein levels.

101

Figure 26: Age-associated variations in ECH protein content and catalytic
activity in kidney and liver mitochondria. Panel A: Immunoblot analysis using the
anti-ECH polyclonal antibody showing the age-associated decrease and increase in
ECH protein levels in rat kidney and liver mitochondria, respectively. Panel B:
Graphs showing ECH catalytic activity and protein levels expressed in percent of the
values in the young animals.
B. Impact of calorie restriction
In kidney mitochondria of CR-fed rats, ECH catalytic activity increased by
36% (p<0.05, n=6), while protein amounts rose by 20%. However, this rise in ECH
protein content was not significantly different from ECH levels in AL-fed rats. In
liver mitochondria, there was no difference in ECH activity or protein levels between
AL and CR animals (Fig. 27).
4 24
Age (months)
0
50
100
150
*
Activity
*
Protein
4 24
Age (months)
0
25
50
75
100
Percent of 4 Months
***
**
B
A
Age (months)
Kidney
Anti-ECH
24 4
Liver
Anti-ECH
24 4

102

Figure 27: Impact of calorie restriction on ECH catalytic activity and protein
amounts. Panel A: Immunoblot analysis using the anti-ECH antibody showing the
relative, but not significant increase in ECH protein amounts in kidney mitochondria,
while no change was observed in liver mitochondria. Panel B: Graphs showing ECH
catalytic activity and protein levels expressed in percent of values measured in young
animals.
AL CR
0
50
100
Activity
Protein
Anti-ECH
Liver
AL CR
Anti-ECH
Kidney
AL CR
A
AL CR
0
50
100
150
Percent of AL
*
B

103
VII. Succinyl-CoA:3-oxo-acid CoA-transferase and enoyl-Coenzyme A
hydratase nitration in the kidney from species exhibiting life spans that
differ from the rat
A. SCOT nitration in kidney mitochondria of pig, dog, cow and rabbit
The purposes of these experiments were to (i) determine whether the SCOT
protein was nitrated in species exhibiting life spans that differed from rats, and if so,
(ii) to determine whether the modification, i.e. nitrohydroxytryptophan also occurred
in such species.
Mitochondria and soluble mitochondrial proteins were isolated from kidney
of young dog, cow, pig and rabbit according to the procedures described in Materials
and Methods.
Immunoblot using the anti-3NT showed that only one single immunopositive
protein band was present in the kidney mitochondria of dog and cow, but not in pig
and rabbit. The immunoreactive protein was confirmed to be SCOT using the anti-
SCOT serum. SCOT was almost equally expressed in the species tested. Thus, the
fraction of SCOT that is nitrated in the kidney mitochondria from dog and cow
appeared to be lower than in the rat (Fig. 28).

104

Figure 28: SCOT nitration in kidney mitochondria from pig, dog, cow and
rabbit. Western blot analysis showed that SCOT was the main nitrated protein in the
kidney mitochondria of dog and cow, but does not appear to be nitrated in pig and
rabbit. Soluble proteins (10 µg per lane) were separated by one dimensional
electrophoresis and immunodetection was performed using anti-3NT antibody or
anti-SCOT serum. Lane contents were as follows: 1-5, soluble mitochondrial
proteins isolated from kidney of rat, pig, dog, cow and rabbit. M refers to the protein
standards and 0.5 µg of control non-treated bovine serum albumin (BSA) (67 kDa).
C refers to peroxynitrite treated BSA.
B. Purification of the SCOT protein from kidney of different species
For mass spectrometric analysis, SCOT was purified from kidney
mitochondria of C57 mice, pig, dog, cow, and rabbit. Procedures for SCOT
enrichment were identical to these used in the rat. Briefly, after mitochondrial
isolation and soluble proteins preparation, HPLC chromatofocusing, i.e. separation
of the proteins according to their pI, was performed. The fractions collected were
separated by SDS-PAGE electrophoresis and gels were either stained with
Coomassie or transferred to a membrane for immunodetection with an anti-3NT
antibody (Fig. 29). Fractions containing immunopositive bands were pooled,
concentrated, and submitted to HPLC gel filtration, i.e. fractionation based on size.
Resulting eluates were separated by SDS-PAGE analysis for Western blot analysis
with the anti-3NT antibody (data not shown).
120
80
49
35
29
20
7
210
Anti-3NT
1 M C 2 3 4 5
Anti-SCOT
1 M C 2 3 4 5

105
Coomassie Blue stained gels showed the elution profile of kidney
mitochondrial soluble proteins of the various species, each fraction containing
proteins with close pI value.
Immunodetection of the fractions with the anti-3NT antibody revealed
several new characteristics. (i) Another immunopositive protein, of ~29 kDa, besides
nitrated SCOT, was present in C57 mice. This immunopositive band was specific, as
confirmed by a control with the secondary antibody only (data not shown). Given the
similar molecular weight to enoyl-CoA hydratase, it was postulated, but not fully
confirmed, that this band was indeed ECH (Fig. 29, C57 mice). (ii) Despite the lack
of immunoreaction in the Western blot analysis of soluble mitochondrial proteins, an
immunopositive band was detected in pig after chromatofocusing. This protein was
confirmed to be SCOT after probing the membrane with the anti-SCOT serum (data
not shown). It seemed that two isoenzymes of SCOT, exhibiting different pI, were
present, one highly nitrated and the other one less nitrated (Fig. 29, PIG). (iii)
Similarly, in cow and dog mitochondria, two nitrated isoenzymes of SCOT
displaying distinct pI were detected (Fig. 29, COW, and DOG). (iv) In rabbit, SCOT
was confirmed not to be nitrated (Fig. 29, RABBIT).

106
Figure 29: Purification of SCOT from kidney mitochondria of C57 mice, pig,
cow, dog and rabbit. Kidney mitochondrial soluble proteins from C57 mice, pig,
cow, dog, and rabbit were fractionated by HPLC chromatofocusing, and the fractions
were separated by SDS-PAGE. Gels were either stained with Coomassie Blue or
immunostained with an anti-3NT antibody. M refers to a mixture of prestained
standards and control BSA, as described in the legend of Fig. 1. Lane C contains the
positive control, i.e. 0.5 µg of peroxynitrite-treated BSA. S represents the initial
sample loaded onto the HPLC column, i.e. concentrated soluble mitochondrial
proteins from the species indicated. Lanes 5-53 (C57 mice, pig and cow), 5-77 (dog)
and 5-71 (rabbit) correspond to the chromatofocusing fraction numbers. The arrows
on the right indicate the position of the 58 kDa or 29 kDa immunopositive proteins.

107

Figure 29

Anti-3NT
210
120
80
49
35
29
20
7
M S 5 7 9 11 13 15 17 19 21 23 27 29 31 33 35 37 39 41 43 45 47 49 51 53 25 C
Coomassie
210
120
80
49
35
29
20
7
PIG
M S 5 7 9 11 13 15 17 19 21 23 27 29 31 33 35 37 39 41 43 45 47 49 51 53 25 C
Coomassie
210
120
80
49
35
29
20
7
Anti-3NT
210
120
80
49
35
29
20
7
C57 mice

108

Figure 29 (Continued)

210
120
80
49
35
29
20
7
Anti-3NT
120
80
49
35
29
20
7
M S 5 7 9 11 13 15 17 19 21 23 27 25 29 31 33 35 37 39 41 43 45 47 49 51 53 C
Coomassie
COW

109

Figure 29 (Continued)

C M S 5 7 9 11 13 15 17 19 21 23 27 29 31 33 35 37 39 41 43 45 47 49 51 53 25 55 57 61 69 63 65 67 71 59 73 75 77
120
80
49
35
29
20
7 Coomassie
120
80
49
35
29
20
7
Anti-3NT
DOG

110

Figure 29 (Continued)

C M S 5 7 9 11 13 15 17 19 21 23 27 29 31 33 35 37 39 41 43 45 47 49 51 53 25 55 57 61 69 63 65 67 71 59
210
120
80
49
35
29
20
7 Coomassie
210
120
80
49
35
29
20
7
Anti-3NT
RABBIT

111
C. Identification of the site of nitration in the SCOT protein from
various species
To investigate whether the nitrated residue, as that detected in rat SCOT, was
also present in the carboxy terminal fragment of the SCOT from various species,
SCOT auto-fragmentation was performed as described in Materials and Methods.
Briefly, purified SCOT from pig, or kidney mitochondrial proteins from C57 mice,
cow and dog were incubated in the absence or presence of the substrate, acetoacetyl-
CoA, for 5 min at room temperature and heated for 1 h at 70º C. The mixtures were
then separated by one-dimensional SDS-PAGE, and gels were either stained with
Coomassie Blue, or transferred to a membrane for immunodetection with the anti-
3NT antibody.
Immunodetection for nitration showed that the nitrated amino acid in SCOT
from pig, C57 mice, cow and dog was also located in the carboxyl fragment of the
protein (Fig. 30).

112

Figure 30: SCOT fragmentation in pig, C57 mice, cow and dog. SCOT from
various species was subjected to autolytic fragmentation, as described for the rat in
Fig. 5. Purified SCOT from pig kidney mitochondria, and kidney mitochondrial
soluble proteins (10 µg) from C57 mice, cow and dog were incubated in the absence
(lane 1) or in the presence (lane 2) of 1 mM acetoacetyl-CoA for 5 min at room
temperature, and heated for 1h at 70ºC, following which western blot analysis was
performed with the anti-3NT antibody. Lane C contains the positive control, i.e. 0.5
µg of peroxynitrite-treated BSA and lane M corresponds to the mixture of prestained
standards and control BSA, as described in the legend of Fig. 1. Arrows on the right
indicate SCOT full length protein (SCOT) and carboxy-terminal fragment (COOH).

ESI-tandem mass spectra were acquired for the purified carboxy-terminal
fragments of SCOT from C57 mice, cow and pig. A tryptic peptide, corresponding to
tyrosine 368-lysine 379, Y
368
DLANWMIPGK
379
, was found to contain the
characteristic +61 Da mass addition on tryptophan 374 as observed for rat SCOT
(Table 3).

Species Sequence Charge X
corr

C57BL/6 Mice 2 2.6
Cow 2 3.5
Pig
YGDLANW*MIPGK
2 1.8

Table 3: SCOT tryptic peptide containing the +61 Da mass increment in C57
mice, cow and pig. The charge state of the peptide and the highest cross correlation
score (X
corr
) obtained for that peptide are indicated. W* corresponds to tryptophan
containing nitro and hydroxy adducts (+61 Da)
210
120
80
49
35
29
20
7
C
M
1 2
C57
1 2
COW
1 2
DOG
1 2
PIG
Coomassie
C 1 2
C57
1 2
COW
1 2
DOG
1 2
PIG
Anti-3NT
SCOT
COOH

113
Chapter Four: Discussion
Main findings of the study
The protein succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT), involved
in the degradation of ketone bodies, was found to be a major target of nitration in
the mitochondria of various rat tissues, such as heart, kidney, brain, skeletal
muscle, lung and spleen, and also in the kidney mitochondria from three different
strains of mice, i.e. C57BL/6, DBA/2, monoamine oxidase A/B double knockout
mice, as well as from pig, cow and dog. Despite relatively similar protein
amounts in kidney mitochondria in different species, the nitration of SCOT was
the highest in rodents, followed by dog and cow, and least in the pig.
Tryptophan 372, located within 12 Å of the active site residue, was identified to
be the site of a novel posttranslational modification, namely simultaneous
nitration and oxidation. This modification was characterized in the SCOT protein
from rat heart and kidney from C57 mice, pig and cow.
In rat heart mitochondria, there was a 222% increase in nitrohydroxytryptophan
content with age, accompanied by a 30 % gain in specific catalytic activity, while
SCOT protein levels remained unchanged. Based on these results, it was
estimated that the specific activity of nitrated SCOT was 10 times higher than the
specific activity of unmodified SCOT.

114
In rat kidney mitochondria, between 4 and 24 months of age, SCOT protein
amounts declined by 55%, while SCOT nitrohydroxytryptophan content and
specific catalytic activity remained unchanged.
Calorie restriction did not affect any of the SCOT parameters in rat heart. In
contrast, SCOT protein amounts increased by 50% in the kidney of calorie
restricted rats, while SCOT nitrohydroxytryptophan content and specific activity
remained unchanged in comparison to AL animals.
After treatment of mitochondrial proteins with 25 µM peroxynitrite, there was,
respectively, a 24% and 18 % augmentation in SCOT catalytic activity, and a
83% and 66% elevation in SCOT nitrohydroxytryptophan content in heart and
kidney. It was thus estimated that in vitro nitrated SCOT was twelve times more
active than unmodified SCOT. However, at higher peroxynitrite concentrations,
there was a linear decrease in SCOT catalytic activity.
In comparison to other mitochondrial proteins, SCOT was relatively more
susceptible to trypsin-induced proteolysis. In addition, the carboxy-terminal
fragment, containing the nitrated tryptophan, was degraded more quickly than the
amino-terminal, non nitrated fragment. Conversely, SCOT was also more
resistant than other mitochondrial proteins to chymotrypsin-induced degradation.
A comparison between C57 and the DBA mice, whose lifespan is not
significantly prolonged by calorie restriction, indicated that age and calorie
restriction had a similar qualitative effect on SCOT parameters in kidney.
Between 6 and 23 months of age, SCOT nitration content, protein levels and
catalytic activity remained unchanged in both strains. In comparison to AL-fed

115
animals, three months of calorie restriction induced a 27 % drop in SCOT
catalytic activity in C57 mice, while SCOT activity in DBA mice decreased by
only 13%. However, after 23 months of calorie restriction, the decline in SCOT
catalytic activity in DBA mice was not significantly different from that in C57
mice: SCOT catalytic activity in CR animals represented 71% and 81% of the
activity measured in AL-fed C57 and DBA mice, respectively. At old age, the
drops in catalytic activity observed in the two strains of mice could be accounted
by a decline in SCOT protein content. The degree of nitration remained similar in
AL and CR animals.
Enoyl-CoA hydratase (ECH), an enzyme that catalyzes the second step of β-
oxidation of fatty acids, was also found to be nitrated in kidney and liver
mitochondria. The nitration of ECH was not visible after one dimensional
electrophoresis (1D-gel) followed by immunodetection with an anti-3NT
antibody, but was detectable if mitochondrial soluble proteins were fractionated
by chromatofocusing prior to 1D-gel western blot analysis. Thus its age-related
nitration profile could not be investigated. Measurements of ECH catalytic
activity and protein levels as a function of age and dietary regimen were assessed,
and indicated that: (i) in rat kidney mitochondria, ECH catalytic activity and
protein levels declined with age by 34 % and 45%, respectively. Conversely,
calorie restriction induced a 36% increase in ECH catalytic, accompanied by a
20% elevation in ECH protein content; however such increase in protein amount
in CR rats was not significantly different than in AL rats; (ii) in contrast, in liver
mitochondria, ECH protein amounts and activity augmented significantly by 33

116
% and 31 % with age. Calorie restriction did not impact either parameter. It is
possible that ECH was also a target of nitration in C57 mouse kidney, although
this could not be confirmed.

SCOT is the major nitrated protein among mitochondrial soluble proteins
Among other findings, results of the present study showed that, among
mitochondrial soluble proteins, SCOT was the main protein exhibiting nitration in
various tissues of the rat, the kidney of three different strains of mice (C57, DBA and
monoamine oxidase A/B double knock-out), and in mammalian species with
relatively longer maximum life spans (MLSP) than rodents, such as pig (MLSP=27
years), dog (MLSP=21 years) and cow (MLSP=30 years), but not rabbit (MLSP=18
years). Thus, SCOT nitration is a widely-occurring, but not universal phenomenon.
SCOT was initially reported to be nitrated in rat heart and kidney in response to
lipopolysaccharide administration (Marcondes, Turko et al. 2001), as well as in
diabetic mice models (Turko, Marcondes et al. 2001; Turko, Li et al. 2003). This
study demonstrates that SCOT nitration is not necessary limited to inflammatory or
diabetic conditions, but is also encountered in tissues of young, healthy animals,
where the enzyme is expressed.
In contrast to the present finding of SCOT being the primary target of
nitration among mitochondrial soluble proteins, Kanski et al. (2005), using two
dimensional gel electrophoresis (2D-gel) combined with Western blot analysis and
immunoprecipitation with an anti-3NT antibody, reported 13 mitochondrial proteins
to be nitrated in rat heart, including the soluble proteins creatine kinase and

117
aconitase. They also reported nitration of some proteins in the cytoplasm. Applying
similar methodologies, the same group found other nitrated mitochondrial proteins in
the rat skeletal muscle (Kanski, Alterman et al. 2003), and cerebellum (Gokulrangan,
Zaidi et al. 2007). In none of these studies, SCOT was identified as a specific target
of nitration. In this study, the possibility that nitrated proteins were lost due to
reduction of the nitro group (-NO
2
) to amino (-NH
2
) group during sample
preparation, or in the course of the one-dimensional electrophoresis (1D-gel) and
western blot analysis, was excluded on the basis of a series of control experiments.
For instance, standard controls for establishing the specificity of the anti-3NT
antibody, (i.e. omission of the anti-3NT antibody, preincubation of the primary
antibody with 3-nitrotyrosine or tryptophan derivatives, and reduction of the
membrane with dithionite), omission of butylated hydroxytoluene from the
antioxidant buffer, and lowering of dithiothreitol concentrations in sample buffer
supported the validity and reliability of the methods used in the present study. In
contrast, if conditions (buffers and antibodies concentrations) identical to those in the
aforementioned reports were used, additional immunopositive bands were indeed
detected, but they lacked specificity towards the anti-3NT antibody (data not shown).
The 2D-gel has certain well known limitations, one being that the most abundant
proteins are more readily detectable than the medium and low abundant proteins
(Gygi, Corthals et al. 2000). In the study by Kanski, Behring et al. (2005), putative
nitrated proteins, such as F1-ATPase A chain, 3-oxoacyl-coenzyme A thiolase,
NADH DH1 β-subcomplex, electron transfer flavoprotein are quite highly expressed
in heart mitochondria (Johnson, Harris et al. 2007). It is likely that SCOT, being a

118
low abundant protein, could have remained undetected by 2D-gel analysis. In
addition, except for the electron transfer flavoprotein, mass spectrometric
confirmation of nitration was not provided by Kanski, Behring et al. In this study,
SCOT was demonstrated to be nitrated using not only immunoblot, but also HPLC-
electrochemical detection, MALDI and ESI tandem mass spectrometric analysis.
Aconitase and creatine kinase, the two enzymes that co-purified with SCOT, did not
show nitration after western blot or tandem mass spectrometric analysis. This finding
also contrasts with the results of Kanski, Behring et al. (2005). Nevertheless, it is
possible that the lack of cross-reaction with the anti-3NT antibody could be due to
low nitration content of these proteins. Furthermore, nitrated peptides might have
remained undetected during MS/MS, as the sequence coverage for these two
proteins, 19% for aconitase, and 3% for creatine kinase, was very low. To overcome
these short comings of the 2D-gels, the same group, in their latest study, fractionated
rat heart homogenates by solution phase isoelectric focusing (IEF), followed by
nanoelectrospray ionization-tandem mass spectrometry analysis (NSI-MS/MS), and
compared the results to one-dimensional electrophoresis-western blot analysis (our
approach) (Hong, Gokulrangan et al. 2007). It was striking to note that (i) their 1D-
gel immunoblot profile was similar to ours, with one major protein band nitrated
around 52 kDa, and (ii) the molecular weight of proteins found to be potentially
nitrated using IEF coupled to NSI-MS/MS analysis did not correspond to the
molecular weight of the bands observed on the 1D-gel western blot analysis. Authors
argued that different techniques yield different results, but it seems that IEF still
favors the detection of highly expressed proteins, since sarcomeric proteins (e.g.

119
myosin or tropomyosin), known to be very abundant in cardiac cells (Kislinger,
Gramolini et al. 2005), were found to be the major putative targets of nitration. It
should also be noted that the tandem mass spectra presented were extremely noisy
due to the fact that a mixture of proteins, rather than a purified protein, was analyzed.
Such an approach tends to increase the probability of false positive identification.

Tryptophan 372 is the site of a novel posttranslational modification, namely
nitration and oxidation, in the protein SCOT
Tandem mass spectrometric analysis of the purified SCOT protein from rat
heart and kidney, C57BL/6 mouse, pig and cow kidney showed that tryptophan 372
was the site of a novel posttranslational modification, namely simultaneous nitration
and oxidation. Neither hydroxy- or nitro adducts of tryptophan, nor nitrotyrosine
were detected. In contrast, methionine oxidation, observed for non-modified tryptic
peptides, was quite likely introduced during “in-gel” sample handling for MS/MS
analysis (Mann, Hendrickson et al. 2001; Taylor, Fahy et al. 2003). While studies
related to in vitro or in vivo nitration of tyrosine residues in a variety of mammalian
proteins abound (for a review, see Greenacre and Ischiropoulos 2001), the
occurrence of nitrotryptophan is far less documented. Tryptophan has been shown to
be a specific target of nitration upon in vitro exposure to nitrating agents, such as
peroxynitrite or the peroxidase/hydrogen peroxide/nitrite system (Herold,
Shivashankar et al. 2002; Thiagarajan, Lakshmanan et al. 2004). Recently, Ikeda,
Yukihiro Hiraoka et al. (2007) developed an antibody against 6-nitrotryptophan, and
identified several tryptophan nitrated proteins following in vitro exposure to

120
peroxynitrite in a model system for neuronal inflammation. Other authors have
reported the presence of nitrotryptophan-containing proteins in the liver of mice,
treated with acetaminophen, an analgesic which can cause hepatoxicity, in part via
RNS-mediated damage. Nevertheless, the nitrated proteins were not individually
identified, and no nitrotryptophan-containing proteins were observed in the liver of
untreated mice (Ishii, Ogara et al. 2007). Thus far, with the exception of the
identification of a bacterial peptide (Kers, Wach et al. 2004), nitrotryptophan
containing proteins have not been found in mammalian cells in vivo. In this context,
SCOT is the first protein that has been demonstrated to contain a nitro and hydroxy
groups on a single tryptophan residue. Evidence that this particular modification was
present in the protein SCOT was further demonstrated by the synthesis of an
identical tryptophan derivative. Thus, nitrohydroxytryptophan, which is not
commercially available, was successfully synthesized by incubating 5-
hydroxytryptophan with tetranitromethane. The resulting compound displayed
biochemical properties identical to the amino acid found in SCOT, i.e. HPLC
retention time, UV absorption, as well as characteristic ions with the expected m/z
upon MALDI analysis. This modification seems to be conserved across at least 4
different species, whose maximum life span ranges from 3.5 (C57BL/6 mice) to 30
years (cow). Even though the sequence for SCOT in rabbit has not been reported, it
is hypothesized that the absence of nitration could be due to a variation in protein
sequence.

121
Functional relevance of nitro-hydroxy adducts to tryptophan in the protein
SCOT
Our data indicate that the elevation of SCOT nitration content with age in rat
heart, or after exposure to a low dose of peroxynitrite, correlates with a significant
gain in catalytic activity. Studies examining the functional impact of oxidative
modifications have shown that tyrosine nitration and tryptophan oxidation are often
associated with a loss of catalytic activity of proteins (Viner, Ferrington et al. 1999;
Sharov, Galeva et al. 2006; Lemma-Gray, Weintraub et al. 2007). Enzyme activation
upon peroxynitrite exposure, and through tyrosine nitration, was nevertheless
demonstrated for microsomal glutathione S-transferase 1 (Ji, Neverova et al. 2006).
In contrast, the functional relevance of tryptophan nitration is virtually unknown. In
the present study, the fraction of SCOT that is nitrated was calculated to increase
from 3.6 % to 8 % with age in rat heart, and from 3.6 % to 6.6% (in rat heart) and to
6% (in rat kidney) after treatment with 25 µM peroxynitrite. Assuming that the
associated increase of enzyme activity in vivo and in vitro was due to the accrual of
nitrated SCOT, the catalytic activity of nitrated SCOT was estimated to be ~ 10 fold
greater than the activity of the unmodified protein. Protein activation associated with
post-synthetic modifications, such as phosphorylation, can elevate enzyme activity
from ~ 2 (Lo, Antoun et al. 2004) to ~14 fold (Pitson, Moretti et al. 2003). Thus, the
degree of activation of SCOT upon nitration falls in the range of other activating
processes associated with posttranslational modifications.
An obvious question raised by this study is how the presence of a nitro and
hydroxy groups on tryptophan 372 may enhance SCOT catalysis. Structural studies

122
have indicated that SCOT catalytic active site was composed of a key residue,
glutamate 303, to which the substrate, succinyl-CoA, binds covalently, as well as of
several amino acids (Asn 51, Tyr 76, Gln 99, Glu 241, Arg 242, Asn 279, Lys 327,
Lys 380, Gly 383, Gly 384, Ala 385) which favor the binding of the substrate and co-
substrate, acetoacetate (Bateman, Brownie et al. 2002; Coros, Swenson et al. 2004).
Using the molecular visualization software VMD (Humphrey, Dalke et al. 1996),
tryptophan 372 was found to be located 12 Å from glutamate 303, in a very close
distance to most amino acids constituting the binding pocket of the substrate and co-
substrate. Even though the crystal structure for SCOT with its bound substrate has
not yet been resolved, studies on enzymes from the same family have indicated that
succinyl-CoA might bind to glutamate 303 in an extended conformation
(Rangarajan, Li et al. 2005). More specifically, the pantetheine part of succinyl-CoA
has been shown to form van der Waals contacts with hydrophobic residues of the
binding pocket, and this interaction was reported to favor the binding of the co-
substrate and accelerate the transfer of the CoA moiety to acetoacetate (Fierke and
Jencks 1986; Whitty, Fierke et al. 1995). In the extended conformation, the distance
between the phosphate group on the ribose, and the sulfur atom of succinyl-CoA was
calculated to be 20 Å. Therefore, the nitro and hydroxy adducts on tryptophan 372,
which enhance its hydrophobicity, might influence the orientation and/or binding of
the pantetheine moiety of succinyl-CoA, and eventually accelerate SCOT catalysis.
Such proximity of Trp 372 to the active site residues of the enzyme further supports
the hypothesis that the increase of nitration of Trp 372 with age is correlated with
enhanced SCOT activity in heart mitochondria.

123
A practical implication of the present study is that the anti-3NT antibody not
only cross-reacts with 3-nitrotyrosine containing proteins, but also with the
nitrohydroxytryptophan residue present in SCOT. This immunoreaction probably
occurs because of the structural similarities between the benzyl rings of tyrosine, and
tryptophan carrying nitro- and hydroxy-groups. It should be noted that
immunocompetition experiments with free 5-nitrotryptophan or 5-
hydroxytryptophan, in contrast to free 3NT, did not abolish the immunoreactivity
with the anti-3NT antibody in Western blot analysis. This implies that the presence
of both modifications on the benzyl ring (nitro- and hydroxy-) of tryptophan is
required for such interaction to occur.

Impact of age on SCOT protein amounts, nitration degree and catalytic activity
in the Fischer rat 344 and C57 and DBA mice
An obvious question that arises concerns the plausible causes of variations in
SCOT protein levels and nitration content observed with age in rat heart and kidney.
It is known that factors governing the steady-state amount of any protein, especially
that exhibiting oxidative or nitrative modifications, include the rates of synthesis of
the mature non-modified protein, oxidation/nitration, and degradation (i.e. turnover)
(Stadtman 1992; Ischiropoulos 1998). Since nitration is a post-translational
modification, the age-associated elevation in the fraction of SCOT protein that
contains nitrohydroxytryptophan adducts in rat heart, might reflect an increased rate
of nitration/oxidation (and/or decreased rate of denitration), and/or a decline in
proteolysis with age. It is presently unclear whether an age-associated increase in

124
nitration rates causes the increase in nitration of SCOT, as the mechanism(s)
responsible for the nitration of SCOT remain to be elucidated. One plausible
nitrative/oxidative mechanism implicates peroxynitrite, which arises from the
recombination of NO
.
and superoxide. NO
.
levels have been observed to decrease in
the endothelium of aorta and the heart tissue with age (Tschudi, Barton et al. 1996;
Adler, Messina et al. 2003). As superoxide generation in various tissues, including
heart, is known to increase with age (Sohal, Arnold et al. 1990; Sohal, Ku et al.
1994), it was hypothesized that the decrease in NO
.
levels was due to the inactivation
of NO
.
by superoxide, leading to an increased formation of peroxynitrite with age
(van der Loo, Labugger et al. 2000). Accordingly, the rise in the nitration content of
SCOT is a plausible consequence of this event. Considerable progress has been made
in the identification of proteolytic systems involved in the turnover of mitochondrial
matrix proteins. Thus ATP-dependant proteases, such as Lon-like proteases,
participate in the degradation of oxidatively modified or misfolded mitochondrial
soluble proteins (Wagner, Arlt et al. 1994; Bota and Davies 2002). In addition, the
proteasome and lysosomes contribute to the degradation of mitochondrial proteins,
albeit in a less specific manner (Allan and Welman 1980; Sullivan, Dragicevic et al.
2004). It has been shown that expression levels of Lon protease increase with age in
rat heart mitochondria, without a corresponding change in catalytic activity,
suggesting that Lon protease specific activity declines with age (Delaval, Perichon et
al. 2004). Even though denitrase activity towards 3NT-containing proteins has been
reported in rat lung and spleen homogenates (Kamisaki, Wada et al. 1998), no
particular denitrase protein has been isolated. In this context, it is unclear whether

125
such an enzyme might be present in rat heart mitochondria, and could catalyze the
denitration of a nitrohydroxytryptophan-containing protein. Thus an age-associated
decline in the removal of nitrated proteins is also a plausible cause for the observed
age-related increase in nitrated SCOT in the rat heart.
In contrast, the age-related decline in the amounts of SCOT protein observed
in rat kidney mitochondria might reflect an accelerated turnover of SCOT, and/or a
decrease in SCOT synthesis, either at a transcriptional or a translational level, and/or
a decline in the number of nephrons or mitochondria. A loss of nephrons, as well as a
reduction in the number of mitochondria have been commonly observed during
aging in rats and human (Goyal 1982; Sato and Tauchi 1982; Takaki, Jimi et al.
2004; Long, Mu et al. 2005). Proteomic studies in old Fischer rats and human have
shown that despite a reduction in cell numbers most of the genes remain either
equally expressed, or are relatively up-regulated in kidney of aged animals (Melk,
Mansfield et al. 2005; Sung, Jung et al. 2005). In this study, Western blot analysis of
young and old kidney mitochondrial fractions indicated that the protein content of
lipoamide dehydrogenase, a component of various mitochondrial enzymatic
complexes, e.g. pyruvate dehydrogenase, did not exhibit any age-associated variation
in the kidney (data not shown). In contrast, only a few genes were reported to be
downregulated in the aforementioned studies, and the gene encoding SCOT was not
among them. This implies that the diminution in SCOT protein levels can not be
accounted for by a loss in nephrons, mitochondria, or a reduced expression of the
SCOT gene, but rather it stems from posttranslational events. It is thus hypothesized
that an enhanced proteolytic degradation of SCOT might occur with age in the

126
kidney, as has been reported in the skeletal muscle of diabetic animals (Fenselau and
Wallis 1976). It is well documented that the SCOT protein itself is very susceptible
to proteolytic degradation, due to the presence of a highly hydrophilic hinge in the
sequence of the protein. This cleavage yields an N-terminal fragment of ~32 kDa and
a C-terminal fragment of 26 kDa. Nevertheless, immunoblot analysis using the anti-
SCOT and the anti-3NT antibodies did not indicate the presence of such fragments in
kidney fractions from the old animals. The hypothesis that the nitration might
accelerate SCOT turnover, and thus contribute to the age-associated decline of
SCOT protein amounts in kidney, was also tested. Nitrated proteins have been shown
to be more susceptible to proteasome-mediated degradation (Grune, Blasig et al.
1998; Souza, Choi et al. 2000). The proteasome exhibits multiple peptidase activity,
including trypsin, chymotrypsin and caspase-like activity, but has not been
characterized in mitochondria. Nevertheless, since the substrate specificity and
cleavage sites of mitochondrial matrix proteases, such as Lon, have not been
elucidated yet, the sensitivity of SCOT to degradation was evaluated using proteases
exhibiting distinct cleavage properties. Five serine proteases, i.e. trypsin,
chymotrypsin, endoproteinase GluC, proteinase K and thrombin, and one cysteine
protease, i.e. bromelain, were tested. Results of the present study indicate that the
nitrated full-length SCOT is more susceptible to trypsin-mediated proteolysis in vitro
than other mitochondrial soluble proteins, and also that the carboxy-terminal
fragment of SCOT, containing nitrated Trp 372, is more susceptible to proteolysis
than the non-nitrated amino-terminal fragment. Prediction of positions of trypsin
cleavage sites in the primary sequence of mature SCOT, using the PeptideCutter

127
algorithm (http://us.expasy.org/tools/peptidecutter/), indicated 24 and 14 sites (100%
probability) for the amino- and carboxy-terminal fragments of SCOT, respectively.
Therefore, the possibility that the preferential degradation of the nitrated fragment of
SCOT, compared to the non-nitrated fragment, is simply due to the existence of a
greater number of cleavage sites in the carboxy-terminal fragment can be excluded.
Thus the presence of nitrohydroxytryptophan correlates with an acceleration of
SCOT degradation by trypsin. Conversely, the entire pool of SCOT protein, i.e.
nitrated and unnitrated, was insensitive to chymotrypsin treatment. This finding was
unexpected because (i) the PeptideCutter algorithm indicated that SCOT sequence
contained 29 cleavage sites (100% probability) for chymotrypsin, and (ii) it has been
reported that SCOT, isolated from rat skeletal muscle, could be digested by both
trypsin and chymotrypsin (Fenselau and Wallis 1976). Nevertheless, in the latter
study, the proteolysis was performed with purified SCOT, whereas a mixture of
mitochondrial proteins was used in our experiment. Thus conditions used by us are
closer to the in vivo situation, suggesting that chymotrypsin specificity might be
greater for other mitochondrial soluble proteins than for SCOT. It is noteworthy that
it has been convincingly demonstrated that nitration can reduce chymotrypsin-
mediated proteolysis (Souza, Choi et al. 2000). Present data do not confirm such a
phenomenon for nitrated SCOT. Nitrated SCOT was also shown to be sensitive to
proteinase K and bromelain-mediated proteolysis, but not to endoproteinase GluC or
thrombin. Overall, the results indicate that SCOT is differentially susceptible to
trypsin and chymotrypsin-mediated proteolysis, but otherwise tends to exhibit
similar protease sensitivity in comparison to other mitochondrial proteins. The

128
results also suggest that the nitration of tryptophan 372 might enhance SCOT
susceptibility to trypsin-mediated proteolysis, and that such a property could
contribute to the age-related decline in SCOT protein amount. It is known that
mitochondrial protein expression profiles vary considerably among rat tissues
(Johnson, Harris et al. 2007). Thus it is plausible that each tissue will express
activities of various proteases in mitochondria, explaining why oxidized proteins
might accumulate in some tissues, while being preferentially degraded in other
tissues.
In contrast to rat kidney, where a decline in SCOT protein amounts occurred
in old animals, age did not have an impact on SCOT protein levels, degree of
nitration and catalytic activity in both C57 and DBA mice strains. This suggests that
the effect of age on ketolysis is species specific, but is similar in closely related
strains.

Relevance of the present findings to the aging process
Under physiological conditions, organs such as heart and kidney rely on
several different metabolic pathways for the production of energy, including the
oxidation of fatty acids, glucose, and to a lesser but still significant extent, the
degradation of the two main ketone bodies, acetoacetate and β-hydroxybutyrate
(Wijkhuisen, Djouadi et al. 1997; Kodde, van der Stok et al. 2007). The latter can
become the preferred energetic substrates under certain circumstances, such as a
prolonged starvation, or after intense exercise, which lead to an increase in ketone
body production by the liver, and subsequently in their concentration in the plasma.

129
In this context, the age-related elevation in SCOT specific catalytic activity in heart
mitochondria reflects a rise in the consumption of ketone bodies as energetic
substrates. It is presently unclear which substrates are most preferentially utilized by
the heart in the aged animals. It has been reported that there is an age-associated
decrease in the utilization of fatty acids, accompanied by an increase in the
consumption of carbohydrates for ATP synthesis in rat heart (Abu-Erreish, Neely et
al. 1977; McMillin, Taffet et al. 1993), while another group has reported an age-
associated increase in fatty acid oxidation, no change in glucose oxidation and a
dramatic decrease in lactate utilization (Sample, Cleland et al. 2006).
Notwithstanding, all of these studies point to a shift in energetic metabolic pathways
in heart during aging, for which elevation in SCOT catalytic activity may be partially
responsible. The functional consequences of this age-associated shift in fuel
preference in heart remain nevertheless uncertain. Some authors argue that this
remodeling in energetic pathways might contribute to the functional deterioration of
the heart during aging (Sample, Cleland et al. 2006). For instance, an increase in the
oxidation of fatty acids might be deleterious, because it consumes more mol of
oxygen per mol ATP produced, in comparison to glucose oxidation, and therefore
reduces the efficiency of myocardial contraction, a phenomenon referred to as
oxygen wasting effect of fatty acids (Hutter, Piper et al. 1985). Others (Lee, Allison
et al. 2002) plead that it might represent an adaptive and compensatory response to
age-associated disturbances in ATP producing pathways observed in the heart of
aged animals, such as decreases in the activities of the electron transport chain
(Lesnefsky, Gudz et al. 2001), aconitase and ATP synthase (Yarian, Rebrin et al.

130
2005). As ketone bodies are thought to be a relatively more efficient fuel than
glucose or fatty acids (Veech 2004), elevation in SCOT catalytic activity might be
interpreted as an adaptation, rather than an adverse event per se. In contrast, the
decrease in SCOT protein content observed with age in kidney mitochondria
suggests a reduced capacity to utilize ketone bodies, even though SCOT specific
catalytic activity remained intact. A plausible consequence of this alteration is an
increase in the content of ketone bodies in the kidney, and, since ketone bodies are
strong acidic compounds, a decrease in the intramitochondrial pH, which can affect
the activity of some enzymes. It is also known that the capacity of kidney to excrete
acids upon an acid load is impaired in aged rats (Prasad, Kinsella et al. 1988). This
suggests that an age-associated increase in the concentration of ketone bodies might
lead to an enhancement of their reabsorption, and eventually an acidification of the
blood pH, a phenomenon referred to as metabolic acidosis. In humans, it has been
shown that a metabolic acidosis progressively develops in the plasma with age, with
detrimental consequences such as osteoporosis and loss of muscle mass (Frassetto,
Morris et al. 1996). Thus, in aging Fischer rats, an elevation in the concentration of
ketones in the kidney could also lead to a metabolic acidosis, potentially triggering
other age-related impairments.
As age did not have any impact on SCOT catalytic activity, protein amounts
and degree of nitration in the kidney mitochondria of C57, DBA or monoamine
oxidase A/B double knock-out mice (data not shown for the latter strain), this
implies that the ability for ketolysis remains intact in these three strains, suggesting
that the impact of age is species-specific.

131
Impact of calorie restriction
It has been demonstrated that calorie restriction (CR), without malnutrition,
delays the onset of age-related physiological changes, and can extend the lifespan of
many species (Sohal and Weindruch 1996). In the present study, the impact of long
term CR regimen (21months) on protein nitration was investigated in Fischer rats,
C57BL/6 (C57) and DBA/2 (DBA) mice. The effect of a short term CR (3 months)
was also examined in the two aforementioned strains of mice at young age. In
contrast to Fischer rats and C57 mice, the life span of DBA mice is not significantly
prolonged after long term CR. The goal of these investigations was to test whether
differential levels of oxidative stress, measured by the degree of SCOT nitration in
the study, might contribute to the extension of life span by CR in Fischer rats and
C57 mice, or to the absence of such beneficial effect in DBA mice.
Results of this study indicate that the impact of long term CR in Fischer rats
was tissue specific. Thus, while CR did not impact SCOT protein amounts, nitration
content or specific catalytic activity in heart mitochondria, the SCOT protein amount
in kidney from CR animals was similar to the level in the young AL-fed rat.
Therefore, in Fischer rats, CR seems to have a protective effect on SCOT protein in
the kidney, but not in the heart mitochondria.
In contrast, CR induced a decrease in SCOT protein levels and catalytic
activity, but no change in the degree of SCOT nitration in kidney of C57 and DBA
mice at young and old age. This suggests that the specific activity of SCOT in CR
animals remained unchanged in comparison to AL animals, and that CR did not
affect the mechanism of SCOT nitration in either strain of mice. A plausible

132
explanation for the decline in SCOT protein content might be the reduction in kidney
mass induced by CR at young and old ages in both strains (data not shown).
Alternatively, since CR has been shown to increase the production of ketone bodies
in mouse liver ~4 times (Hagopian, Ramsey et al. 2003), it is possible that elevated
concentrations of substrates trigger SCOT autolytic fragmentation, which could
explain the decline observed in SCOT protein content in kidney in response to CR.
Indeed, Howard, Zieske et al. (1986) have proposed that such mechanism might
regulate the lifetime of the enzyme in vivo. The sole differential impact of CR in
these two strains was observed at young age, where the magnitude of the decrease in
SCOT protein content was relatively greater in C57 than in DBA mice. However, the
impact of a long term CR regimen with regard to the diminution in SCOT protein,
was similar in the two strains.
These findings indicate that the impact of CR on SCOT protein content is not
only tissue, but also species specific. This finding is not surprising, as the impact of
age on this parameter also varied in different tissues and species. Overall, it can be
concluded that neither the extension of life span in Fischer rats and C57 mice, nor
the lack of prolongation of life span in DBA mice observed upon a long term CR
regimen can be attributed to a difference in SCOT protein oxidative/nitrative levels,
since the degree of nitration of SCOT remained unchanged in the tissues of both AL
and CR of all models studied.

133
Enoyl CoA hydratase: a target of nitration in kidney and liver mitochondria of
Fischer rat
Short chain enoyl-Coenzyme A hydratase, (ECH, EC 4.2.1.17) which
catalyzes the second step of fatty acid β-oxidation, was found to be another target of
nitration in kidney and liver mitochondria from young Fischer rats. Even though
Western blot analysis of various young rat tissues using the anti-ECH serum showed
that this protein was relatively highly expressed in heart, we did not find evidence
that ECH was also nitrated in this organ. It is possible that the ECH isoenzyme in
heart has a different pI than in other tissues such as kidney, and therefore it did not
co-purify with the SCOT protein after chromatofocusing. Nevertheless, ECH has
been reported to be nitrated in rat heart mitochondria, and its degree of nitration was
found to increase as a function of age (Kanski, Behring et al. 2005).

Impact of age on ECH in kidney and liver
Results of the present study indicate that ECH protein levels and catalytic
activity decreased with age in kidney mitochondria, suggesting that ECH specific
catalytic activity remained intact during aging. It is known that the four reactions in
the β-oxidation of fatty acids occur at similar rates, therefore any decrease in the
catalytic activity or in the protein content of one these four enzymes will eventually
lead to a slowdown of the β-oxidation (Fong and Schulz 1978). Together with the
decline in SCOT protein amounts with age, the decline in ECH protein levels might
be indicative of a disturbance in the metabolic energetic pathways in the kidney in
the old Fischer rats. Indeed, it is well documented that Fischer 344 rats are relatively

134
more susceptible to develop age-related kidney pathologies in comparison to other
rat strains or rodents (Weindruch and Masoro 1991), and that CR virtually eliminates
the occurrence of nephropathy in this strain (Masoro, Iwasaki et al. 1989). Even
though in our study kidney did not display obvious lesions at 24 months of age, it is
possible that these changes preclude further functional renal impairments. Long-term
CR had a relative protective effect on ECH parameters, since ECH catalytic activity
in old CR animals was not significantly different from that in the young AL animals.
It is presently unclear whether the elevation in ECH catalytic activity is the sole
consequence of an elevation in ECH protein content, since the rise in protein level
did not reach statistical significance. In this context, it is possible that nitration or
other posttranslational events could play a role in such increase.
In contrast, in liver, a mitotic tissue, the age-associated increase in ECH
catalytic activity reflects a higher rate of oxidation of short chain saturated fatty
acids, and again demonstrates that the impact of age is tissue specific. Calorie
restriction did not induce any changes in ECH parameters. Since the age-associated
rise in ECH protein content was accompanied by a similar gain in activity, this
suggests that the nitration of this protein in this particular tissue might not have any
relevant functional impact. It is known that aging is accompanied by an increase in
visceral fat in rats, which leads to elevated levels of free fatty acids in the plasma
(Engler, Engler et al. 1998). Such a phenomenon has been ascribed in part to an age-
related decline in the oxidation of long chain fatty acids, such as palmitate, which are
the most abundant of the free fatty acids (Park, Kim et al. 2006). In this context, the
elevation in the catalytic activity of mitochondrial short chain ECH with age might

135
represent a compensatory response to these age-related impairments in the disposal
of fatty acids. Altogether, data indicate significant changes in energy producing
pathways in the heart, kidney and liver of Fischer rats during aging.

136
Chapter Five: Conclusions
To conclude, this study has identified succinyl-CoA:3-oxoacid CoA
transferase (SCOT) and enoyl-Coenzyme A hydratase (ECH) as selective and major
targets of nitration in mitochondria during aging in Fischer rats. These two proteins
are involved in critical energy producing pathways, namely the degradation of
ketone bodies, and the oxidation of fatty acids.
Using mass spectrometry in combination with HPLC-electrochemical
detection, a novel posttranslational modification, namely nitrohydroxytryptophan,
was identified in the protein SCOT. Tryptophan 372, located in close proximity to
key catalytic residues, was found to be the specific site of nitration and oxidation.
Identical adducts, i.e. nitro and hydroxy groups, to this functionally relevant
tryptophan were also present in the SCOT protein isolated from species such as
C57BL/6 mice, pig and cow, but not in rabbit, suggesting that the nitrative/oxidative
mechanisms targeting this particular tryptophan in the SCOT protein are quite
conserved across mammalian species, but probably not universal. Heretofore,
nitration of proteins was thought to be restricted mainly to tyrosine residues. This
study demonstrated, for the first time, that nitration of a tryptophan residue also
occurred in vivo.
The age-related increase in nitrohydroxytryptophan content was correlated
with an enhancement of SCOT specific catalytic activity during aging in rat heart,
since SCOT protein amounts remained unaltered with age. Both in vivo and in vitro

137
data indicated that nitration of SCOT may enhance its catalytic activity by ~10 fold.
However, in rat kidney mitochondria, aging was associated with a decline in SCOT
protein amount, while nitration content and specific catalytic activity remained
unchanged. CR prevented the age-related loss in SCOT protein content in rat kidney
mitochondria, but had no effect in rat heart mitochondria. The presence of
nitrohydroxytryptophan in the protein SCOT was also correlated with an enhanced
susceptibility to undergo in vitro-induced proteolysis. Thus, considered together, our
data indicate that SCOT catalytic activity in Fischer rats is affected by both its
degree of nitration and the protein levels.
The age-associated decline in enoyl-Coenzyme A hydratase (ECH) protein
levels and catalytic activity in kidney mitochondria of Fischer rats, indicate a marked
attenuation of energy pathways in kidney mitochondria of Fischer rats during aging.
Together with the loss in SCOT protein levels, these events might preclude the
common age-associated kidney lesions observed in this strain of rats. Even though
the levels of nitration were not determined, age and CR -related changes in ECH
catalytic activity seem to be related to its levels in expression in liver and kidney,
except in kidney mitochondria of CR animals, where the significant increase in ECH
catalytic activity was not associated with a corresponding significant increase in
ECH protein content. A possible involvement of nitration in the rise of ECH catalytic
activity remains to be demonstrated.
Comparison of two strains of mice that differ in their response to CR, that is
C57BL/6 exhibits life span extension, while DBA/2 does not, indicated that the
impact of age and long term CR on SCOT nitration content, catalytic activity and

138
protein levels in kidney mitochondria were qualitatively similar in these two strains.
This suggests that the biological mechanisms underlying the prolongation of life
span in C57BL/6 mice upon CR do not affect the SCOT parameters. Such
comparative investigation also showed that the effect of age and CR on ketolysis in
kidney mitochondria was different in Fischer rats and the two aforementioned strains
of mice.
The age-related increase in the nitrohydroxytryptophan content of SCOT and
the concomitant enhancement in specific catalytic activity in rat heart mitochondria
confirm that, as predicted by the oxidative stress hypothesis, an increase in protein
nitrative/oxidative damage occurs selectively. Such event might represent an
adaptation rather than a deleterious event per se in the heart of old rats. However, the
impact of age on SCOT nitration is tissue-specific. Thus in kidney, there was no age-
related change in SCOT degree of nitration. Whether the nitration of SCOT
contributes to the age-associated decline in protein content in kidney in vivo, as
suggested by the enhanced sensitivity to trypsin-mediated proteolysis in vitro,
remains to be determined. This study also showed that a long term-CR regimen does
not affect nitrative/oxidative mechanism(s) of the SCOT protein. Overall, results
clearly demonstrate that the impact of CR is tissue and species specific.

139
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Abstract (if available)

Abstract The main goal of this study was to test the hypothesis that specific proteins undergo an age-related increase in nitration, which results in their functional alteration, and that calorie restriction (CR), a regimen which prolongs the life span of many rodents, can attenuate or postpone such age-associated changes. Succinyl-CoA transferase (SCOT) and enoyl-CoA hydratase (ECH), two enzymes involved in energy production, were detected immunochemically with an anti-3-nitrotyrosine antibody to be targets of nitration in mitochondria from Fischer rat tissues. Mass spectrometric studies revealed that, rather than tyrosine, tryptophan 372, located in the vicinity of key catalytic residues, was the site of a novel posttranslational modification, namely nitrohydroxytryptophan in the SCOT protein. This amino acid alteration was also detected in mice, pig and cow, but not rabbit, suggesting that oxidative/nitrative mechanisms targeting this particular tryptophan residue are relatively well conserved, but not universal, among mammals. In rat heart mitochondria, an increase in nitrohydroxytryptophan content was associated with an elevation in SCOT specific catalytic activity during aging, while SCOT protein amounts remained unchanged.

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Asset Metadata

Creator Brégère, Catherine (author)

Core Title Targets of protein nitration during aging

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology

Publication Date 02/04/2008

Defense Date 11/16/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag aging,mitochondria,nitration,OAI-PMH Harvest

Language English

Advisor Sohal, Rajindar S. (committee chair), Kaplowitz, Neil (committee member), Shen, Wei-Chiang (committee member), Shih, Jean C. (committee member)

Creator Email bregere@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1006

Unique identifier UC1200429

Identifier etd-bregere-20080204 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-43355 (legacy record id),usctheses-m1006 (legacy record id)

Legacy Identifier etd-bregere-20080204.pdf

Dmrecord 43355

Document Type Dissertation

Rights Brégère, Catherine

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu